INTRODUCTION

Top Abstract Introduction Methods Results Discussion References

THE TRANSPORT CAPACITY for hexoses in the small intestine of many species can be changed to meet the dietary load over a period of a few days. This allows the animal to take advantage of the altered energy content of the food and at the same time ensure that hexose transporter protein expression is sufficient to allow for complete absorption of hexoses from the lumen to the bloodstream. Three major transport proteins are known to be involved in this process: SGLT-1 is a sodium-dependent secondary active transporter found in the brush-border membrane (BBM) and moves glucose and galactose against a concentration gradient into the enterocytes (10). Glucose transporter isoform 5 (GLUT-5) is also found in the BBM and mediates the entry of fructose, although it does not appear to be coupled to an ion gradient to energize the uptake of substrate (3). GLUT-2 is expressed in the basolateral membrane (BLM) and mediates the exit of all three hexoses out of the cell into the bloodstream (4). Expression of all three of these transport proteins can be altered to match the dietary hexose content such that SGLT-1 and GLUT-2 will increase in response to an elevated glucose intake (22, 23), while fructose will induce GLUT-5 and GLUT-2 protein in the plasma membranes of the enterocytes (5, 12). Details of the signals and mechanisms of this control mechanism are not known, but control appears to be exerted at both the transcriptional and translational level (14, 16, 17).

In addition to this ability to alter the amount of transporter protein in the plasma membrane of the enterocytes over a period of several days, there is also compelling evidence that the activity of at least one of these transport proteins, GLUT-2, can be changed within 30 min to ~2 h (6). This response is again related to the presence of glucose in the intestinal lumen but appears to involve the release of two peptide hormones from intestinal endocrine cells (25). One of these is gastric inhibitory polypeptide (GIP), which is synthesized and released by K cells in the duodenum and jejunum (26, 28). The other is glucagon-like peptide-2 (GLP-2), which is synthesized along with GLP-1 from the proglucagon gene in ileal L cells (2, 27). Vascular infusion of either of these peptides induces a large change in the glucose transport capacity in the intestinal BLM as characterized with isolated membrane vesicles (7). At least part of this response appears to involve protein movement between the trans-Golgi and the plasma membrane, but whether this movement is of GLUT-2 itself or some regulatory protein is not yet established (25).

In contrast, there is very little evidence to suggest that the activity of SGLT-1 or GLUT-5 in the BBM can be altered rapidly. Epidermal growth factor (EGF) has been implicated as a luminal and vascular signal for the upregulation of SGLT-1, but not GLUT-2 (9). A 1-h exposure in vivo to EGF increases the maximal rate of transport (V_max) for sodium-dependent glucose transport in BBM vesicles by 40%, and this effect can be blocked by tyrphostin, suggesting a role for tyrosine kinases in this response. Similarly, there is a preliminary report that luminal glucose can induce an increase in glucose transport across isolated BBM vesicles by 60-90% at low concentrations of substrate with no effect at high concentrations (13). This has been interpreted to mean that luminal glucose can increase the affinity of SGLT-1 for glucose without changing the V_max. There is also evidence that there may be a regulatory subunit for SGLT-1. Veyhl et al. (29) have cloned a 66.8-kDa protein, which can be cross-linked to the transport protein, and coexpression in Xenopus oocytes of the two proteins can alter the rate of glucose uptake. However, neither the signal nor the pathway for modulating the activity of SGLT-1 are known. These observations strongly suggest that certain physiological conditions may promote an increased transport activity for SGLT-1. Therefore, we decided to determine if GLP-2, which can upregulate GLUT-2 activity, could also change the sodium-dependent transport of glucose across the BBM via SGLT-1. Demonstration of such an effect would provide compelling evidence that this hormone promotes the transepithelial absorption of glucose and galactose and possibly fructose in the small intestine.

	METHODS

Top Abstract Introduction Methods Results Discussion References

In vivo intestinal perfusion. Rats were anesthetized with 6.5 mg pentobarbital sodium/100 g body wt ip, and the intestine was exposed by laparotomy. Cannulas were fitted into the proximal and distal ends of a 30-cm length of jejunum, and the intestine was returned to the body cavity. After being flushed with warmed Krebs, the lumen was perfused at a flow rate of 1.6 ml/min for up to 4 h with Krebs containing either wortmannin (80 µM) or brefeldin A (30 µM) when required.

Vascular infusion of peptides. While the animal was anesthetized, a cannula was inserted into the jugular vein and a saline solution containing the peptide hormone was infused at a flow rate of 3 ml/h.

Preparation of BBM vesicles. The lumen of a 30-cm segment of jejunum was flushed with ice-cold phosphate-buffered saline containing 0.1 mM phenylmethylsulfonyl fluoride (PMSF) and opened along the antimesenteric border. The mucosa was scraped off with a microscope slide and placed in 65 ml of ice-cold mannitol tris(hydroxymethyl)aminomethane (Tris) buffer [solution A: (in mM) 300 mannitol, 5 ethylene glycol-bis(-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), 12 Tris · HCl, pH 7.4, and 0.1 PMSF]. The tissue was homogenized in a Polytron homogenizer for 2 min before addition of magnesium chloride to a final concentration of 12 mM. After the solution was stirred on ice for 15 min, the solution was centrifuged at 4,500 revolutions per minute (rpm) for 15 min to remove debris. The supernatant was further centrifuged at 16,000 rpm for 30 min, and the pellet was homogenized in a mannitol/Tris buffer [solution B: (in mM) 150 mannitol, 2.5 EGTA, 6 Tris · HCl, pH 7.4, and 0.05 PMSF] with a glass homogenizer before further addition of magnesium chloride. After stirring on ice, the centrifugation was repeated as before, and the pellet was then washed with solution C (300 mM mannitol, 5 mM Tris · HCl, pH 7.4) before repelleting at 16,000 rpm. This vesicle preparation was diluted in solution C to an appropriate protein concentration, usually 8 mg/ml.

Uptake measurements. Vesicles were kept on ice until use on the same day as they were prepared. Ten microliters of vesicle suspension were spotted onto the side of a polycarbonate tube, and 20 µl of uptake medium were pipetted onto the bottom of the same tube. Uptakes were performed with either an inwardly directed sodium or potassium gradient. This was achieved by using an uptake medium containing either 125 mM sodium or potassium thiocyanate. When the uptake medium mixed with the vesicle suspension, a cation gradient of 83.3 mM was produced. Thiocyanate was used as the anion to reduce membrane potential effects because of its lipophilic nature. The tube was held on a vortexer, which was activated by a foot switch. The uptake was initiated by starting the vortexer and was terminated by the rapid injection of 1.1 ml of ice-cold stop solution (100 mM NaCl, 2 mM Tris · HCl, pH 7.4, 1.0 mM phloridzin). Sodium was included in the stop solution to prevent loss of substrate from the vesicles during the subsequent washes. The solution was then filtered through 0.45-µm pore size cellulose filters (MSI, Westboro, MA) on an Amicon filtration apparatus. The filters were washed twice with ice-cold stop solution and placed in liquid scintillation vials. Five milliliters of EcoLite (ICN) scintillation fluid was added, and the samples were counted on an Beckman LS 6500 liquid scintillation counter.

Phoridzin binding assay. BBMs were prepared from control rats and those infused with GLP-2 for 30, 60, and 120 min as before. Membranes were stored under liquid nitrogen until use. Ten microliters of BBM (6-10 mg protein/ml) were then incubated with [³H]phloridzin (90 µl) over a concentration range of 0.05-2.5 µM for 30 min at 22°C. The membranes were separated from the incubation medium by filtration on 0.45-µm cellulose filters (MSI, Westboro, MA) and washed twice with 5 ml stop solution. Filters were then placed in scintillation vials and treated as for the filtration experiments (see above). Total binding was measured using sodium thiocyanate buffer, and nonspecific binding was determined using potassium buffer and 5 mM excess cold phloridzin.

Western blotting. Membranes (15 µg) from each cell fraction were solubilized in Laemmli sample buffer and run on a 10% sodium dodecyl sulfate-polyacrylamide gel using a Mini-Protean II cell (Bio-Rad). The proteins were immobilized onto nitrocellulose membrane (Bio-Rad) by electrotransfer for 75 min at 4°C using the Mini Trans-Blot Cell (Bio-Rad). The membranes were stained for total protein with Ponceau S to ascertain that equivalent amounts of protein were loaded and transferred from each lane. Blocking of the membrane was carried out in 3% nonfat milk in TPBS (0.05% Tween 20, phosphate-buffered saline, pH 7.4) for 1 h and then incubated with 1:1,000 rabbit polyclonal antibody to rat SGLT-1 (gift from Dr. Soroya Shirazi Beechey, University of Wales) in 3% nonfat dry milk in TPBS overnight at 4°C. The membrane was washed three times in 3% nonfat dry milk/TPBS for 15 min, 1 h, and 15 min, respectively. The nitrocellulose membrane was then incubated with a secondary antibody, anti-rabbit immunoglobulin G coupled to horseradish peroxidase diluted 1:2,000 in 3% nonfat dry milk/TPBS for 1 h. Three subsequent washes followed as described above. Finally, the membrane was treated with the enhanced chemoluminescence detection solution (Amersham Life Sciences, Oakville, Canada) before autoradiography for 30 s using Kodak XAR-5 film with an intensifying screen. Two distinct bands were detected by this method with apparent molecular masses of 97 and 71 KDa.

	RESULTS

Top Abstract Introduction Methods Results Discussion References

Glucose uptake into the isolated BBM vesicles was greatly accelerated by the imposition of an inwardly directed 83.3 mM sodium gradient. The anion used was thiocyanate, which is highly permeant and thus helps to voltage clamp the vesicles and reduce the membrane potential induced by the inward flux of sodium. In the presence of an equivalent potassium gradient, glucose uptake was much slower, but under both conditions the glucose content was the same after 1 h (Fig. 1). The sodium gradient failed to produce an overshoot of glucose within the vesicles, which were prepared from rats infused with saline alone (control). This would suggest that under these conditions there is insufficient transporter activity to generate the actual overshoot. Previous reports have also shown that rat BBM preparations show low transporter activity compared with those from other species such as rabbit. However, in vesicles prepared from rats infused for 2 h with a solution containing 800 pM GLP-2, the glucose uptake in the presence of a sodium gradient was much faster and a significant overshoot was achieved. The glucose uptake after GLP-2 infusion in the presence of a potassium gradient (equivalent to diffusion) was identical to control vesicles prepared from saline-infused animals.

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Effect of glucagon-like peptide 2 (GLP-2) infusion in vivo on the time course of glucose uptake into jejunal brush-border membrane (BBM) vesicles. Rats were infused via a jugular cannula with 800 pM GLP-2 saline, or saline alone, at a flow rate of 3 ml/h for 2 h. Membrane vesicles were then prepared from jejunal scrapings, and the time course of 100 µM D-glucose uptake was followed in the presence of an 83.3 mM sodium (closed symbols) or potassium (open symbols) gradient. Uptake into vesicles from control animals (, ) or those infused with GLP-2 (, ) was measured in the presence of a sodium or potassium gradient.

Effect of GLP-2 on glucose transport kinetics. Glucose uptake was measured over a range of concentrations from 33 µM to 6.7 mM in the presence of either a sodium or a potassium gradient. Three-second incubations were used to allow for the measurement of close to the initial rate of uptake (cf. Fig. 1). In the presence of a potassium gradient, the glucose uptake was linearly related to substrate concentration; however, in the presence of a sodium gradient the uptake was curvilinear and could be resolved into a linear component, with a slope the same as that seen in the absence of sodium and a Michaelis-Menten-type curve (Fig. 2). This type of analysis was applicable to vesicles prepared from both control and GLP-2-infused animals, although the vesicles from the peptide-infused animals exhibited a greater capacity for sodium-dependent glucose transport. Nonlinear regression analysis of the uptake in the presence of sodium showed that the K_m for uptake was lower in vesicles from control compared with GLP-2-infused animals [0.13 ± 0.01 mM (n = 4) and 0.20 ± 0.06 mM (n = 4), respectively, although there was a high degree of variability].

View larger version (13K): [in this window] [in a new window]   Fig. 2.   Effect of in vivo GLP-2 infusion for 2 h on sodium-dependent glucose uptake in jejunal BBM vesicles. Data points represent the mean from 4 experiments ± SE, for sodium-dependent glucose uptake in the presence of an 83.3 mM sodium gradient, outside to inside. Values were obtained by subtracting uptake in the presence of an 83.3 mM potassium gradient from the total uptake in the presence of the sodium gradient. Rats were infused with 800 pM GLP-2 in saline () or saline alone () at a flow rate of 3 ml/h for 2 h before BBM vesicles were prepared from jejunal mucosal scrapings. The curves were fitted assuming a single Michaelis-Menten component by nonlinear regression analysis.

To determine how long it took for the upregulation of glucose transport to be induced by GLP-2 infusion, the kinetic experiments were repeated using 30- and 60-min in vivo infusions prior to the preparation of BBM vesicles. Both infusion periods produced an increased glucose transport in the vesicles of the type seen with the 2-h infusion. The 1-h infusion gave an almost identical increase compared with 2 h, with a V_max of 1,118 ± 159 pmol · mg protein¹ · s¹ and a K_m of 0.15 ± 0.02 mM. However, the 30-min infusion induced less of an increase, with a V_max of 833 ± 84 pmol · mg protein¹ · s¹ and a K_m of 0.30 ± 0.08 mM. The changes in V_max are summarized in Fig. 3 and show that upregulation is apparently maximal after 60 min, although all three time periods produced a significant increase.

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Time course of effect of vascular infusion of GLP-2 in vivo on the maximal rate of sodium-dependent glucose uptake into jejunal BBM vesicles (Vmax). The kinetics of uptake were measured as described for Fig. 2 using vesicles prepared from rats infused with saline for 120 min (Cont) or GLP-2 for 30, 60, or 120 min. In all cases, the intestinal lumen was perfused with mannitol (100 mM)-saline at the same time. The bars represent the mean ± SE maximal rate of sodium-dependent glucose uptake (pmol · mg vesicle protein1 · s1) from 4 individual experiments, each using 3 rats. * Significant change from control (P < 0.05). BFA indicates the inclusion of 30 µM brefeldin A in the luminal perfusate while GLP-2 was infused into the vascular bed for 120 min. Wort, inclusion of 80 µM wortmannin in the luminal perfusate during the 2-h vascular infusion of 800 pM GLP-2.

Mechanism of upregulation. There are several ways in which the maximal rate of transport of SGLT-1 in the BBM could be increased. The simplest way is that more protein could be inserted into the membrane, increasing the number of active sites available for hexose translocation. We attempted to measure the abundance of SGLT-1 in the BBM vesicles by using phloridzin binding and semiquantitative Western blotting. Specific phloridzin binding to the BBMs was considered to be the saturable component left after subtraction of the binding seen in the presence of excess cold phloridzin (5 mM). So the data were analyzed using nonlinear regression analysis and assuming a single binding site. In no case did analysis using two binding sites improve the fit. Figure 4 shows the data for a series of binding experiments performed with BBM vesicles prepared from control jejuna and those from animals infused with GLP-2 for 1 h. The fitted curves were calculated to have a maximal binding capacity (B_max) for the control and GLP-2 infusion of 2,074 and 6,640 pmol/mg vesicle protein, respectively. Analysis of the vesicles from animals infused for 30 or 120 min with GLP-2 were also made, and relative values for the B_max values are summarized in Fig. 5.

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Sodium-dependent phloridzin binding to rat jejunal BBM vesicles prepared from animals infused in vivo with GLP-2 (800 pM) for 1 h () or saline alone (control; ). Data points represent the net sodium-dependent binding, for 4 separate experiments, calculated by subtracting from the total binding the nonspecific binding measured in the presence of 5 mM excess cold phloridzin. Error bars indicate SE, and the curves were fitted by nonlinear regression analysis assuming a single ligand binding site.

View larger version (32K): [in this window] [in a new window]   Fig. 5.   Time course of effect of GLP-2 infusion in vivo on the maximal specific binding of phloridzin to jejunal BBM vesicles. Each bar represents mean maximal binding from 4 separate experiments ± SE, after 30, 60, and 120 min of GLP-2 infusion or 120-min saline infusion (control). * P < 0.05 vs. control.

In the Western blotting experiments, equivalent amounts of protein were loaded from vesicle samples taken from the kinetic experiments and which had been stored under liquid nitrogen. A typical blot is shown in Fig. 6, indicating that BBM from saline-infused animals (control) had two major bands recognized by the anti-SGLT-1 antibody of an apparent molecular mass of 97 and 71 kDa. After infusion with GLP-2, changes occurred in the density of both bands, so in each case both bands were scanned and their densities integrated for the expression of any changes in SGLT-1 immunoreactivity. Figure 6 shows the mean densities for control and GLP-2-infused vesicles, and it is clear that although there was no apparent significant change in the two bands after 30 min of GLP-2 infusion, there was a significant increase after 60 and 120 min. These data suggest that there is significantly more SGLT-1 immunolike reactivity in the BBM after 1 and 2 h of GLP-2 infusion.

View larger version (24K): [in this window] [in a new window]   Fig. 6.   Effect of GLP-2 infusion in vivo on abundance of the sodium-dependent glucose transporter 1 (SGLT-1) protein in isolated BBM vesicles. This Western blot was probed with a specific anti-SGLT-1 antibody, which was visualized using the enhanced chemiluminescence system. In each case, 2 bands were detected at positions equivalent to molecular masses of 97 and 71 kDa (A). Lanes are for BBMs prepared from control rats in which the lumen was perfused for 120 min with mannitol saline and the jugular vein was infused with saline (C) or GLP-2 for 30, 60, or 120 min (30', 60', and 120', respectively) or infused with GLP-2 for 120 min while 30 µM brefeldin A (BFA) was included in the luminal perfusate. The final lane is for 120-min GLP-2 infusion with 80 µm wortmannin (WRT) in the luminal perfusate. B: summed relative densities of the 2 bands from 4 separate experiments from control infused animals or those infused with 800 pM GLP-2 for 30, 60, or 120 min. BFA indicates inclusion of 30 µM brefeldin A in luminal perfusate and Wort indicates inclusion of 80 µM wortmannin, while GLP-2 was infused for 120 min. Error bars indicate SE. * Significant difference from control (P < 0.05).

Brefeldin A is a fairly specific inhibitor of protein translocation from the trans-Golgi apparatus to the plasma membrane (1, 11, 15). Inclusion of this compound at a concentration of 30 µM in the luminal perfusate completely blocked the 2-h GLP-2-induced increase in both the maximal rate of sodium-dependent transport (Fig. 2) and in the change in immunoreactive bands on the Western blots (Fig. 6).

Signaling pathway for upregulation. Some hormones, like insulin, which upregulates glucose transport in muscle and adipocytes by stimulating the translocation of GLUT-4 into the plasma membrane, mediate their effects through phosphatidylinositol 3-kinase (PI 3-kinase) (18, 24). This kinase can be specifically and irreversibly inhibited by the fungal metabolite wortmannin (19, 30). Therefore, we tried the effects of perfusing wortmannin (80 µM) through the intestinal lumen for 120 min while infusing GLP-2 into the vascular bed at the same time. Kinetic analysis of glucose transport into BBM vesicles prepared from jejuna perfused with wortmannin in the luminal solution at a concentration of 10 mM during the GLP-2 infusion showed a very significant reduction in the V_max compared with infusion of GLP-2 alone [546 ± 98 and 1,121 ± 194 pmol · mg protein · s¹, respectively (P = 0.002)]. This suggests that the pathway, which mediates the effect of GLP-2, involves the PI 3-kinase, which has been implicated in a number of trafficking processes (20).

	DISCUSSION

Top Abstract Introduction Methods Results Discussion References

The absorption of hexoses from the intestinal lumen to the bloodstream after a meal requires entry of the substrates across the BBM, passage across the cytosol, and exit across the basolateral membrane. The previous data showing that GIP and GLP-2 could increase the transport activity for glucose and fructose in the BLM suggested a possible role for these peptide hormones in controlling hexose absorption. However, in the absence of data regarding entry across the BBM it was not clear if this effect only related to making substrates available from the bloodstream for enterocyte metabolism. Previous work with pancreatic glucagon suggested that hyperglucagonemia would promote increased glucose uptake in isolated membrane vesicles prepared from the small intestine (8). Exposure to elevated plasma glucagon for several days promoted transport across both the BLM and the BBM, whereas short-term exposure only increased BBM transport. Debnam and Sharp (8) proposed that the short-term changes were related to an altered membrane potential across the enterocyte apical membrane, which increased the driving force for SGLT-1. We have now been able to show that GLP-2, which is structurally related to glucagon, also induces an increase in sodium-dependent glucose transport across the BBM of the jejunum. This effect takes ~1 h, a time course that parallels that previously reported for regulatory changes in GLUT-2 activity in the BLM and so presents a strong argument for this being a control mechanism that upregulates hexose transepithelial absorption in the small intestine. An increase in the V_max of two- to threefold for glucose transport in both the BBM and BLM means that the throughput of hexoses could be greatly increased without a significant change in the intracellular content of the substrates, thus minimizing osmotic effects on the enterocytes. Similar cross-talk between the BBM and BLM has long been proposed for ion transport across epithelia, including the small intestine (21).

The mechanism responsible for this increased transport in the BBM appears to involve the insertion of additional SGLT-1 protein rather than a change in turnover rate of transporters already present or nonspecific effects on the membrane potential or permeability. When all three sets of data for transport, phloridzin binding, and Western blotting are normalized and put together, as in Fig. 7, it is clear that after 60 and 120 min of GLP-2 infusion the two- to threefold increase in transport V_max is paralleled by an increase in phloridzin binding and SGLT-1 immunoreactivity. All of this would argue strongly in favor of insertion of new SGLT-1 protein into the BBM in response to elevated plasma levels of GLP-2. The fact that luminal brefeldin A can block the change in transport and the increase in SGLT-1 protein abundance suggests that the additional transporter protein comes from a compartment related to the trans-Golgi apparatus. This fungal metabolite has been shown to cause marked changes in subcellular morphology and prevent many proteins from being transferred to the plasma membrane from the Golgi (15). A block occurs that results in the return of many proteins to the endoplasmic reticulum via tubular structures, which are not normally present.

View larger version (35K): [in this window] [in a new window]   Fig. 7.   Comparison of changes in glucose transport kinetics, phloridzin binding, and SGLT-1 protein abundance in the jejunal BBM induced by GLP-2. Data from Figs. 3, 5, and 6 were normalized to a percentage of control values for 30, 60, and 120 min of in vivo GLP-2 infusion. Bars represent average of the mean and the error bars the SE. BFA indicates presence of 30 µM brefeldin A and Wort indicates presence of 80 µM wortmannin in the luminal perfusate during vascular infusion of GLP-2 for 120 min.

However, after only 30 min of GLP-2 infusion we do see a change in transport, which is not reflected in a change of SGLT-1 abundance. At this point, the alteration may well be of the kind proposed by Debnam and Sharp (8) in which permeability of the membrane to sodium could decrease, hyperpolarizing the membrane and changing the electrical driving force for hexose transport. These very short-term changes in transport activity clearly need additional study.

The Western blots appear to detect two forms of the SGLT-1 protein under control conditions, one of molecular mass 97 kDa and one of molecular mass 71 kDa. Expression in the rabbit intestine has been most thoroughly studied and indicates a major peak spanning a range of molecular masses from 68 to 77 kDa. However, several previously published Western analyses of jejunal SGLT-1 in a number of other species indicate that more than one form of the protein can be detected in the BBM. The assumption is that these are simply differently glycosylated forms of the same protein. Analysis of our Western blots suggests that the proportion of the two bands may change with the infusion of GLP-2; however, there was no consistent pattern of a change in the expression of one over the other.

Finally, the inclusion of wortmannin in the luminal perfusate blocked the stimulatory effect of GLP-2. This suggests that the PI 3-kinase is involved in the signaling between the peptide hormone and the ultimate movement of SGLT-1 into the BBM. Wortmannin has been shown to specifically inhibit PI 3-kinase activity in a number of cell systems and, interestingly, activity of this kinase has been shown to be necessary for a number of endocytic and receptor recycling pathways (19, 30). There is also evidence in some cell types that PI 3-kinase may play a role in the regulation of glucose transport (24), which fits very well with our observations that regulation of hexose transport in the small intestine can be blocked by a specific inhibitor of this kinase.

Perspectives