EDITORIAL FOCUS
Heterogeneity of pig intestinal D-glucose transport systems

Nabil Halaihel, Daniele Gerbaud, Monique Vasseur, and Francisco Alvarado

Institut National de la Santé et de la Recherche Médicale, Unité 510, Faculté de Pharmacie, Université de Paris XI, 92296 Châtenay-Malabry, France


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
APPENDIX

Heterogeneity of intestinal D-glucose transport is demonstrated using pig jejunal brush-border membrane vesicles in the presence of 100/0 (out/in) mM gradients each of NaCl, NaSCN, and KSCN. Two D-glucose transport systems are kinetically distinguished: high-affinity, low-capacity system 1, which is equivalent to the symporter SGLT1; and low-affinity, high-capacity system 2, which is not a member of the SGLT family but is a D-glucose and D-mannose transporter exhibiting no preference for Na+ over K+. A nonsaturable D-glucose uptake component has also been detected; uptake of this component takes place at rates 10 times the rate of components characterizing the classical diffusion marker L-glucose. It is also shown that, in this kinetic work, 1) use of D-glucose-contaminated D-sorbitol as an osmotic replacement cannot cause the spurious appearance of nonexistent transport systems and 2) a large range (>= 50 mM) of substrate concentrations is required to correctly fit substrate saturation curves to distinguish between low-affinity transport systems and physical diffusion.

pig intestinal transport; D-mannose transport; SGLT1


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
APPENDIX

D-GLUCOSE TRANSPORT across the intestinal brush-border membrane (BBM) involves a sugar-Na+ cotransporter [system 1 (S1)], the specificity of which was established in the 1960s. The key structural requirements for sugar interaction with S1 are a pyranose ring and an -OH group in the D-glucose configuration at C-2. Accordingly, D-mannose, D-fructose, D-mannitol, and D-sorbitol are prototypes of sugars behaving essentially as inert toward S1 (4, 12). In 1987, S1 was cloned by Wright and colleagues (35) and is known today as SGLT1. We assume that S1 represents the functional expression of SGLT1 in the in toto enterocyte.

A heterogeneity of D-glucose transport systems in the BBM has been independently suggested by many workers using different animal species (13, 15, 18, 20, 34, 35). In 1986, using guinea pig BBM vesicles, we demonstrated the existence of a new transport system, which we called system 2 (S2); it was different from the Na+-dependent S1 and from the equilibratory, cytochalasin B-sensitive basolateral membrane carrier (7, 8). More recently, the basolateral carrier has been identified as GLUT-2, a member of the GLUT family (29). In 1990, it was proposed that the newly discovered GLUT-5 might be our S2 (19), but soon thereafter the same group proposed that GLUT-5 is, rather, a specific D-fructose symporter (9).

In the present work, we use pig jejunal BBM vesicles to show that S2 is a low-affinity, high-capacity, D-glucose and D-mannose transporter, distinct from any previously known intestinal transport activity, including SGLT1, GLUT-2, and GLUT-5. It appears to be unique, because it uses D-glucose and D-mannose as substrates but is not inhibited by D-fructose; it is not affected by inhibitors of the basolateral transporter, such as theophylline; it does not discriminate between Na+ and K+; and it is not rheogenic and is not an Na+-hexose symporter.

The existence of S2 has been questioned by workers who affirm that a single system, S1, suffices to explain all the known features of D-glucose uptake across the BBM (11, 17, 21-23). To explain this lack of accord between laboratories, in the present work we have reinvestigated the heterogeneity problem, with special emphasis on the experimental procedures variously used, including the following questions: 1) Can use of D-sorbitol as an osmotic replacement in kinetic studies cause the spurious appearance of nonexistent transport systems (22)? 2) What is the minimum range of substrate concentrations needed to fit correctly a given saturation curve?


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
APPENDIX

Animals

Young adult, Large White pigs, weighing ~100 kg, raised at the Institut National de la Recherche Agronomique (INRA; Jouy-en-Josas, France) were used. When these pigs were no longer available, commercially supplied animals were used with similar results. The animals were killed in the slaughterhouse of the INRA. Parts of the jejunum were immediately removed, rinsed with saline at room temperature, everted, randomized, and distributed into plastic bags; the bags were sealed and placed in dry ice for transfer to the laboratory for storage at -80°C.

Materials

L-[14C]glucose, D-[U-14C]glucose, and D-[1-14C]mannose were obtained from New England Nuclear, Amersham, or the Centre d'Etudes Nucleaires (Saclay, France). Sugar and radiochemical purity were verified as described previously (7). D-Mannose was recrystallized from water-ethanol. All chemical reagents were of analytic grade.

Preparation of Microvillous Membrane Vesicles

Vesicles were prepared according to the method of Hauser et al. (16). Unless stated otherwise, the final vesicle pellets were resuspended in a buffer composed of 10 mM HEPES, 7 mM maleate, and 7 mM n-butylamine (HMBA buffer), pH 7.4. Other buffers used with equivalent results consisted of 20 mM HEPES and 10 mM Tris, pH 7.4 (HEPES-Tris buffer), and 20 mM HEPES and 40 mM citric acid adjusted with Tris base to pH 7.4 (HEPES-citrate-Tris buffer). Vesicles were adjusted to ~15 mg protein/ml and stored in 200-µl batches in liquid nitrogen until use. All buffers contained 0.02% LiN3 as a bacteriostatic agent. Protein content was measured with the Bio-Rad assay kit, with gamma -globulin as the standard. Compared with the homogenate, the final vesicle preparations were enriched 16-18 times in the BBM, i.e., sucrase (alpha -D-glucohydrolase, EC 3.2.1.48). The absence of contamination by basolateral membrane vesicles was checked by enzymatic and functional tests (30).

Uptake Assays

Substrate uptake was quantitated using a rapid filtration technique (7). Initial uptake rates were determined at 35°C as single time points after it was established in separate experiments that uptakes were linear within the time interval used: 2.6 s for D-glucose or 10 s for D-mannose and L-glucose. Short-time incubations were performed by using a thermostated, electronically controlled apparatus constructed in our laboratory. In kinetic experiments where the extravesicular substrate concentration was varied from 0.1 to 250 mM, appropriate amounts of an inert sugar (D-sorbitol or D-arabinose, see RESULTS) were added to maintain isosmotic conditions (e.g., out = in = 540 mosM). Incubations were stopped with 2.5 ml of ice-cold stop solution containing 350 mM KCl and 25 mM MgSO4 in HMBA.

Expression of Results and Statistical Analyses

Results are expressed (5) as absolute uptakes (pmol/mg membrane protein), absolute velocities (pmol · s-1 · mg membrane protein-1), or relative velocities (nl · s-1 · mg membrane protein-1). The data shown are usually the means from several experiments performed with at least two different membrane preparations, each at least in triplicate. Data were compared by using a global F test (26).

Fitting of the Kinetic Results by Least-Squares Nonlinear Regression Analysis

Uncorrected, absolute initial velocity data as a function of substrate concentration were fitted by iteration to an equation involving the sum of one nonsaturable, diffusion-like component and one or two Michaelian, saturable transport terms
<IT>v</IT><SUB>total</SUB> = ({<IT>V</IT><SUB>m1</SUB>/(<IT>K</IT><SUB>T1</SUB> + [S])} + {<IT>V</IT><SUB>m2</SUB>/(<IT>K</IT><SUB>T2</SUB> + [S])} + <IT>K</IT><SUB>d(app)</SUB>) [S] (1)
where Vm and KT are the capacity and affinity parameters of classical Michaelis-Menten kinetics, respectively, the subscripts 1 and 2 identify two distinct transport systems coexisting in the same membrane surface, [S] is substrate concentration, and Kd(app) is an apparent diffusion constant (7).

To perform the fits, the procedure of Fletcher and Powell as modified by van Melle and Robinson (31) was used. By use of the commercial program Stata (Integral Software, Paris, France), the nonlinear regression functions were fitted in a single run to each data set by minimizing the sum of squares of errors. Comparison between "lack of fit" and "pure error" components yielded F values that provide a quantitative assessment of the goodness of fit. P = NS (not significant) means that data points do not differ statistically from the theoretical fit of the equation under study; i.e., the results can be accepted as valid. P = S (significant) warrants rejection (31).

Statistical comparison between different fits was done by applying the F' test of van Melle and Robinson (31). All calculations were done with Apple MacIntosh microcomputers.


    RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
APPENDIX

Initial D-glucose uptake rates were measured in the presence of 100/0 (out/in) mM gradients of NaCl or NaSCN, modified or not modified by superimposing a KCl gradient, with or without valinomycin, to clamp the membrane potential as desired. The lowest rate of glucose entry was observed when the membrane potential was clamped to zero (Fig. 1, curve 1). This rate increased by ~12-fold when an inside-negative potential of 59 mV was superimposed (curve 3), clearly reflecting the action of the rheogenic S1 (SGLT1). An intermediate 4.8-fold increase was observed when zero-trans NaCl in the absence of K+ was used (curve 2). The strongest activation (43-fold, curve 4) was observed when a zero-trans NaSCN gradient in the absence of K+ was used. Under all conditions, uptake was linear for >= 2.4 s (10 s for L-glucose or D-mannose, not illustrated), so these incubation times were used to measure the initial velocities necessary for kinetic analysis. No attempt was made to fix the membrane potential. To distinguish between rheogenic and electroneutral transport, gradients of NaSCN or NaCl were used. Even when, with NaCl, the actual membrane potential was not zero, we chose to use zero-trans NaCl rather than to clamp the membrane potential to zero, because we did not wish to unnecessarily complicate the experimental conditions. For instance, we know that a high intravesicular K+ concentration can strongly inhibit certain transport systems (32), and this was a condition we did not wish to use in the present work, which is aimed at detecting still poorly understood systems, other than S1.


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Fig. 1.   Time course of D-glucose uptake into pig brush-border membrane (BBM) vesicles: ion and electrical gradient effects. Uptake of 0.1 mM D-glucose was measured under standard conditions that included incubations at 35°C in HEPES-Tris buffer in presence of 100/0 (out/in) mM NaCl + 100/100 (out/in) mM KCl + valinomycin (10 µg/mg membrane protein) for curve 1; 100/0 (out/in) mM NaCl for curve 2; 100/0 (out/in) mM NaCl + 10/100 (out/in) mM KCl + valinomycin (10 µg/mg membrane protein) for curve 3; and 100/0 (out/in) mM NaSCN for curve 4 (n = 3 determinations/point; SD is smaller than symbols). Curves 1 and 2 were linear for up to 6 s; curves 3 and 4 were linear for up to 3 s. At 90 min, all curves converged to give an identical equilibrium value of 40 ± 9 (n = 12) pmol/mg membrane protein.

Because of the apparent homogeneity of the pig jejunum, we have been able to work for >3 yr with the same material that, furthermore, proved to be perfectly stable at -80°C (results not illustrated). To avoid problems related to individual variability, we used a restrained group of pigs obtained from a reliable source (INRA animals; see METHODS). Although other animals were used in some of the experiments, our entire set of results indicates that, kinetically, all the pig BBM vesicle preparations studied are semiquantitatively identical, as illustrated in Fig. 3.

Evidence for D-Glucose Uptake Heterogeneity in Pig Jejunal BBM Vesicles

Initial uptake rates were measured at 0.1 or 10 mM D-glucose to ascertain whether there was evidence of D-glucose transport heterogeneity in pig BBM vesicles according to criteria previously established by using guinea pig intestine (7, 8). D-Mannose and L-glucose were used as controls of the supposedly nontransported hexoses.

As summarized in Table 1, D-glucose uptake differed depending on the substrate concentration used. At 0.1 mM D-glucose, transport was rather homogeneous, as if a single transport system existed or predominated under such conditions. The uptake was 1) stereospecific (D-glucose >> L-glucose); 2) "Na+ dependent" (Na+ >> K+, independently of the anion used), 3) rheogenic (NaSCN > NaCl), 4) strongly inhibited by D-glucose and phlorizin, and 5) only slightly affected by D-fructose or by the specific inhibitors of the "Na+-independent" glucose carrier of the basolateral membrane, i.e., theophylline, phloretin, and cytochalasin B. In conclusion, similar to the guinea pig (8), most of the D-glucose uptake at low D-glucose concentration appears to be mediated by SGLT1. However, several anomalies strongly suggest the existence of heterogeneity. Thus 1) D-mannose was taken up at a rate about six times faster than L-glucose, and 2) in the absence of Na+ (KSCN media), D-glucose was taken up at a rate one order of magnitude faster than L-glucose.

                              
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Table 1.   Hexose uptake by pig jejunal BBM vesicles: effect of monovalent ions, key inhibitors, and sugar analogs

Further evidence for transport heterogeneity stems from work with 10 mM D-glucose as substrate. Total uptake remained stereospecific (D-glucose >> L-glucose), but the Na+ dependence was not so clear-cut, since uptake in the presence of K+ was as high as 60% of that in presence of Na+, irrespective of the anion. Furthermore, inhibition by D-glucose was only partial, similar to that caused by phloretin or cytochalasin B. Theophylline remained essentially inert, confirming the absence of contamination by basolateral membrane vesicles (30). Finally, it is noteworthy that D-mannose inhibited, whereas D-fructose had no effect or slightly activated, D-glucose uptake, a result reminiscent of those observed earlier with intact tissue preparations (1). This absence of inhibition by high D-fructose concentrations proves the nonparticipation of GLUT-2 (10, 14) or GLUT-5 (9) in the D-glucose uptakes described here.

Kinetic Evidence for Intestinal D-Glucose Transport Heterogeneity

Because, taken as a whole, the results in Table 1 cannot be expected from the sole operation of SGLT1, we conclude that D-glucose transport across the pig jejunal BBM is heterogeneous. Nevertheless, to enlarge the evidence, a kinetic study was undertaken by using D-glucose saturation curves obtained in the presence of 100/0 (out/in) mM gradients of appropriate salts. Confirming earlier work in guinea pigs (7, 8), total D-glucose uptake could be decomposed by nonlinear regression analysis into the three components forming Eq. 1 (Table 2). However, it should be emphasized that the numerical results obtained in this way depend strongly on the procedure used for diffusion correction. Because of its importance, this question will be dealt with first. The analysis that follows is applicable to all the data in Table 2, but only the NaSCN results are used here to illustrate the procedures involved.

                              
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Table 2.   Monovalent ion effects on kinetics of D-glucose transport

Restricted fits: L-glucose diffusion correction procedure. Following current practice, diffusion correction was performed first by fixing the Kd parameter to the value [Kd(app) = 2.7 nl · s-1 · mg membrane protein-1] estimated from the results in Fig. 2, with L-glucose as a marker.1 This classical procedure permitted decomposing the D-glucose uptake data into two distinct, Michaelian systems (fit B2, Table 2). The first component exhibits a high affinity but a low capacity and can be equated to the well-known Na+-D-glucose symporter, S1 (SGLT1). The second appears to be a low-affinity, high-capacity transport system, identified here as an "apparent" S2. However, this apparent S2 is not homogeneous (see Full fits). Rather, it is a kinetic composite of the "true" S2 plus an additional, very-low-affinity third component, the meaning of which is discussed below.


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Fig. 2.   Monovalent ion effects on kinetics of D-glucose uptake. D-Glucose saturation curves were performed under standard conditions with 100/0 (out/in) mM salt gradients of NaSCN (), NaCl (), or KSCN (). Bottom curves (triangle ), L-glucose uptake under identical conditions and with NaSCN as salt. Solid lines, theoretical fits of uncorrected v(total) = f[S] data [where v(total) is total transport velocity] computed by using Eq. 1 and kinetic parameters listed in Table 2. Although to perform fits, entire substrate concentration range (0.1-250 mM) was used, only 0.1-10 mM hexose is shown in A.

Full fits. All the parameters in Eq. 1 are set free. Following Robinson and van Melle (24), we made no presumption as to the actual value of Kd(app), and to fit the data, we allowed all five kinetic parameters in Eq. 1 to float, hence, the name "free" or "full" fits. The results again indicated the existence of two distinct saturable transport systems, but new, quite meaningful quantitative differences between fits became evident. One of these concerns the apparent S2, where each Vm2 and KT2 dropped significantly by ~10-fold (cf. fits B1 and B2, Table 2). However, even after this drop, Vm2 remained more than four times larger than Vm1, in agreement with an earlier definition of S2 as a "high-capacity" system (7). In sharp contrast, the kinetic parameters characterizing S1 remained little affected by this correction (fits B1 and B2, Table 2).

Another salient feature of the full fit results is the strong increase in the diffusion parameter from the artificially fixed L-glucose value (2.7 nl · s-1 · mg membrane protein-1) to Kd(app) = 22. Clearly, this greater than eightfold increase in Kd(app) cannot be explained in terms of an effect on physical diffusion, a parameter quite unlikely to change under the present conditions. Rather, these Kd results strongly indicate the existence of a very-low-affinity uptake component exhibiting no sign of saturation, even at the high D-glucose concentrations used in these experiments. If transport is defined as any uptake taking place above the diffusion level (7), then it would appear that the D-glucose Kd(app) contains an additional, specific transport component.

Lack of Fit of the D-Glucose Uptake Results in Terms of a Single Transport System Plus Diffusion

The preceding results differ sharply from those of other workers, who failed to find any evidence of D-glucose transport heterogeneity in intestinal BBM vesicle experiments (11, 17, 21-23). To ascertain the basis of this disagreement, we have critically reevaluated the results in Table 2, aiming in particular to uncover possible differences in the experimental procedures and/or mathematical analyses used in the various laboratories. Accordingly, we investigated whether the results in Table 2 can be best explained in terms of Eq. 1 or in terms of a restricted equation (Eq. 1R) involving only one saturable transport system plus diffusion (23)
<IT>v</IT><SUB>total</SUB> = {[<IT>V</IT><SUB>m1</SUB>/(<IT>K</IT><SUB>T1</SUB> + [S])] + <IT>K</IT><SUB>d(app)</SUB>}[S] (1R)
In practical terms, Eq. 1R is the same as Eq. 1, with the restriction that S2 is eliminated by fixing Vm2 to zero before any fit is performed (e.g., fits B3 and B4, Table 2). To interpret the results, we will apply the F test, where any fit yielding P = S can be rejected.

First, Kd(app) was fixed to 2.7 (fit B3, Table 2). This fit can be dismissed outright, simply on the basis of its exceedingly high F = 109. Second, similar to fit B1, Kd(app) was allowed to float (fit B4). The result improved considerably, the F value dropping by ~20-fold. However, the numerical result again warrants rejection because of its significant F = 5.6. We conclude that Eq. 1R cannot explain the results, independently of whether Kd(app) is fixed or allowed to float.

Range of [S] Needed to Analyze Correctly a Given Saturation Curve

Because at high [S] the experimental error is highest, the following question was considered: Would it be possible to improve the fits by deleting the points at high D-glucose concentration? This question inevitably led to another that, to the best of our knowledge, has not been considered in detail: What is the range of [S] needed to analyze correctly a given saturation curve, particularly if low-affinity systems are suspected to be involved? It is generally admitted that [S] must be between at least three times below and three times above the expected Michaelis-Menten constant (Km) (33). The fact that certain transport Km (KT) values are as high as 10-2 M, even much higher, would demand use of a [S] range well above those normally used in most laboratories, which rarely reaches 50 mM. An exception to this rule, where [S] as high as 300 mM were used, is considered in Concluding Remarks.

Consequently, the D-glucose uptake data in Table 2 have been reanalyzed by performing a series of fits using 1) Eq. 1 or 1R and 2) the entire [S] range available (0.1-250 mM) or a series of restricted data sets where maximum [S] ([S]max) was decreased stepwise, down to 4 mM. This analysis (Table 3) has the interest of exploiting as a continuum the entire set of available data, with [S]max as the independent variable. One important conclusion to be drawn from this analysis is that elimination of any data at high [S] values does not cause the fits to Eq. 1R to improve. Regardless of any purging, all the data for [S]max between 250 and 75 mM (Table 3) yielded P = S, confirming that Eq. 1R is unsuitable.

                              
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Table 3.   Fit of a D-glucose saturation curve as a function of range of substrate concentrations used: fits to a one- vs. two-transport systems + diffusion equation

With further consideration of the restricted fits (Eq. 1R and Table 3), the results can be divided into two groups. At [S]max between 4 and 50 mM, P = NS for all fits, indicating that Eq. 1R can suitably fit the data. However, at [S]max > 50 mM, P = S for all fits, meaning that Eq. 1R no longer applies. Such a result means that, at [S]max > 50 mM, there is strong evidence for the existence of low-affinity transport systems, which was not apparent when low [S]max values were used. Clearly, this is because, at low [S]max, the results are insufficient to permit a distinction between S2 and Kd.

In contrast, as concerns the full fits, all the data between 10 and 250 mM fit Eq. 1, and P = NS for all. Furthermore, all these fits improve significantly as [S]max increases, as indicated by the progressive drop of the corresponding F values (Table 3). This meaningful fact is easy to explain: as [S]max increases, the possibility of reliably distinguishing between S2 and Kd also increases. Although evidence for the separate existence of each of these two components can be seen at [S]max as low as 10 mM, it is only at [S]max >=  50 mM that the relevant parameters approach their respective limiting values, making the numerical results (Vm2 ~1,500, KT2 ~35, and Kd ~20) statistically more valid. Such evidence strongly supports our earlier proposal that, in practice, use of high [S]max (>= 50 mM) is mandatory to obtain statistically reliable results when low-affinity systems are studied (7).

Monovalent Ion Effects on the Kinetics of D-Glucose Uptake: Full Fits

D-Glucose saturation curves were performed in the presence of appropriate cation gradients and used to obtain full fits to Eq. 1 (Table 2). The results reveal the existence of two (Na+ media) saturable systems or one (KSCN medium) saturable system plus a strong apparent diffusion term with Kd(app) values ranging from ~20 nl · s-1 · mg membrane protein-1 in NaSCN or NaCl to 33 nl · s-1 · mg membrane protein-1 in KSCN. To understand better the meaning of these results, the relevant data have been illustrated by using a low D-glucose range (0.1-10 mM, Fig. 2A) or the entire concentration range (Fig. 2B).

The data in Fig. 2A indicate a pattern of ionic specificity (NaSCN > NaCl > KSCN), fully agreeing with the well-established notion that intestinal D-glucose transport is Na+ dependent (Na+ >> K+) and rheogenic (NaSCN > NaCl). However, it is evident that sugar uptake in the absence of Na+ surely does not occur only by diffusion, because even though the uptake under these conditions seems linear to the naked eye, it is not linear and occurs at rates higher than those to be expected from simple diffusion. Compare, for instance, the rate of D-glucose uptake in KSCN with that of L-glucose (Fig. 2, bottom curve). The discrepancy between these results and those to be expected from present beliefs appears even more evident when the entire range of D-glucose concentrations is considered (Fig. 2B). Here, the pattern of ionic specificity appears to be inverted, such that KSCN > NaCl = NaSCN, although the difference between D- and L-glucose persists. Further contradicting the notion that uptake in the presence of K+ occurs only by diffusion (27), the KSCN results clearly exhibit hyperbolic, rather than linear, kinetics. Although they can be forced to fit a straight line, such a result can be rejected because of its significant value (P = S; fit C4, Table 2). Taken as a whole, these results prove that the use of Na+-free, K+-substituted buffers to define "Na+-dependent transport" (27) is unsatisfactory.

The best explanation for the preceding set of results is that, at low or high D-glucose concentrations, different transport systems predominate. At low concentrations, SGLT1 clearly prevails, explaining the clear-cut Na+ dependence and rheogenicity evinced by the results in Fig. 2A. At high D-glucose concentrations, to the contrary, some other, low-affinity system(s) seems to take the upper hand, explaining the reversed order of ionic specificity (Fig. 2B). This interpretation is strongly supported by the numerical data in Table 2, which warrant the following conclusions. First, in presence of Na+, there is strong evidence for the existence of the Na+-dependent, rheogenic SGLT1, a system that becomes undetectable when Na+ is removed (fit C1, Table 2, KSCN medium). Second, in sharp contrast, there is evidence for the existence of a low-affinity S2 under all the conditions studied, independently of the absence or presence of Na+. Taken as a whole, the results indicate that this system is not rheogenic (Cl- > SCN-) and exhibits no preference for Na+ over K+. In sharp contrast to SGLT1, Na+ is not an obligatory activator of S2 (2).

General Validity of the Evidence for D-Glucose Transport Heterogeneity in Pig Vesicles

To illustrate the general validity of our results, two different vesicle lots, three buffers, and two osmotic replacements are directly compared in Fig. 3 and Table 4. Even if small quantitative differences between animal sources (vesicle lots) are apparent, all pig BBM vesicle preparations studied so far are semiquantitatively similar. They all reveal the presence of three uptake components exhibiting the kinetic characteristics of S1 and S2, respectively, plus a strong "diffusion." Even more important, in support of previous work, our entire experience indicates that these three components vary independently, as would be expected if they represent distinct physical entities (6).


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Fig. 3.   D-Glucose kinetics in NaSCN: comparison between animal and vesicle lots, buffers, and sugar used as osmotic replacement. Saturation curves were performed and fitted as described in Fig. 2 with BBM vesicle lots, buffers, and osmotic replacements as follows: lot 1 in HEPES-maleate-n-butylamine buffer and D-sorbitol and lot 2 in HEPES-citrate-Tris and D-sorbitol, HEPES-Tris and D-sorbitol, or HEPES-Tris and D-arabinose. Each curve was performed and fitted separately but, according to F' analysis, curves could be pooled into 2 distinct groups, i.e., BBM vesicle lots 1 (top curve, ) and 2 (bottom curve, ), involving kinetic parameters listed in Table 4.


                              
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Table 4.   Kinetic parameters of curves for D-glucose kinetics in NaSCN

With two different BBM vesicle lots from the INRA series (pooled as lot 1, top curve, Fig. 3), the results are quantitatively indistinguishable. The same is true for two different preparations from the commercial pig series (vesicle lot 2, bottom curve, Fig. 3), despite the fact that D-sorbitol or D-arabinose, respectively, was used as osmotic replacement. The significance of this particular finding is discussed in the APPENDIX.

According to an F' test, vesicle lots 1 and 2 are different, but this difference is small, as indicated by the borderline value of the F' test and the fact that, to the naked eye, the two sets of curves are very close to one another. In particular, at the lowest [S] values used, the two sets of curves are indistinguishable, indicating in particular that S1 is essentially the same for either set. In summary, the only significant difference between these two vesicle lots concerns the Kd(app), the value of which is ~24% lower for lot 2. Finally, the kinetics of D-glucose uptake are very little affected by the nature of the buffer, since essentially identical results were found when HMBA, HEPES-Tris, or HEPES-citrate-Tris buffer was used.

Is D-Mannose a Substrate Specific for S2?

Kinetics of D-mannose transport. We have seen (Table 1) that D-glucose uptake is inhibited by D-mannose. In turn, this hexose is itself transported by pig BBM vesicles (Fig. 4, Table 5). Kinetic analysis indicates that total D-mannose uptake involves a single transport system plus a diffusional component with Kd(app) for D-mannose = 7.8 nl · s-1 · mg membrane protein-1, which is about twice the value of L-glucose (2.7 nl · s-1 · mg membrane protein-1) and about one-third the value characterizing D-glucose in NaSCN (21.5 nl · s-1 · mg membrane protein-1, fit B1, Table 2).


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Fig. 4.   D-Mannose uptake kinetics. D-Mannose uptake was assayed for 10 s at 35°C in HEPES-maleate-n-butylamine buffer with a zero-trans, 100 mM gradient of NaSCN () or KSCN (). Osmotic replacement was D-sorbitol. Results are illustrated as uncorrected absolute velocities (means ± SD). Solid curves, theoretical values calculated by using Eq. 1R, since Michaelis-Menten capacity parameter (Vm1) was undetectable. Diffusion constant (Kd) was fixed to average value of entire set (Kd = 7.8) before each iteration was performed. Pooled results in Na+ or K+ have been illustrated as 2 separate curves, even when all relevant results were not significant (NS; Table 5). Bottom curve, L-glucose uptake from Fig. 2.


                              
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Table 5.   Estimated D-mannose transport parameters

Significantly, the D-mannose transport kinetics are essentially identical in the presence of NaSCN or KSCN. The general validity of this key observation has been verified by means of a detailed F' analysis involving four separate sets of D-mannose uptake curves, all of which gave statistically homogeneous results (Fig. 4). These results, together with others (not shown) indicating that D-mannose uptake is also the same in the presence of KSCN or KCl, permit the conclusion that D-mannose transport across the intestinal BBM is not activated by Na+ and does not involve any rheogenic Na+-D-mannose symport.

From the above results, interesting analogies between specific D-mannose transport and D-glucose transport through S2 (which is also the same in the presence of Na+ or K+) emerge. This can be best appreciated by comparing the kinetic parameters for D-mannose transport (Table 5) with those of D-glucose transport via S2 (Table 2). Compared by pairs, and even when they are not identical for each of the two hexoses, all these parameters are of the same order of magnitude and appear roughly to vary in parallel, suggesting that they involve the same transport system. Thus, for instance, the maximum velocity (Vmax) ratio of NaSCN to KSCN is 1.4 for D-glucose (via S2) and 2.0 for D-mannose. The equivalent KT ratios are 0.8 and 0.9 for D-glucose and D-mannose, respectively. Furthermore, independently of the cation used, the Vmax values for D-glucose are double those for D-mannose, and the KT values are higher for D-mannose than for D-glucose (although they always are of the same order of magnitude). It is unlikely that these parallelisms are fortuitous, which strongly indicates that the D-mannose transport activity described here involves S2.

It therefore appears that the key difference distinguishing S2 from S1 (SGLT1) lies not just in their relative affinities for D-glucose (low- vs. high-affinity systems), but in their respective, qualitative responses to Na+. Na+ was previously defined as an obligatory activator of S1 (2), explaining why D-glucose uptake via S1 is practically nil in the absence of Na+ (fit C1, Table 2). However, this striking difference does not apply to S2, characterized by not distinguishing between Na+ and K+. Taken as a whole, therefore, our results indicate that S2 is responsible for the "Na+-independent," electroneutral transport of each D-mannose and D-glucose across the intestinal BBM. However, the term Na+-independent is used here to mean that the activity does not require Na+ as an obligatory activator.

Site of inhibition of D-mannose on D-glucose transport. Because D-glucose uptake by pig BBM vesicles involves three distinct uptake components, it was essential to identify the exact site of action of D-mannose. As mentioned previously (4, 12), because of its low affinity, D-mannose should not be expected to inhibit S1, except very weakly. This is confirmed by the results in Fig. 5 and Table 6, where D-mannose is shown to have no consistent effect on Vm1 or KT1, nor does the inhibition take place at the level of Kd. Thus, at high D-mannose concentrations, S2 disappears (Vm2 becomes undetectable), whereas Kd is not affected and Kd(app) remains constant at its control value of 20 nl · s-1 · mg membrane protein-1. Moreover, this Kd(app) is exactly the same as that observed when D-glucose uptake is inhibited by itself: 21.5 nl · s-1 · mg membrane protein-1 (fit B1, Table 2).


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Fig. 5.   D-Mannose specifically and fully inhibits system 2. D-Glucose saturation curves were carried out in presence of 100/0 (out/in) mM NaSCN gradient. Effects of 200 mM (open circle ) or 340 mM () D-mannose are directly compared with controls without D-mannose (reproduced from Fig. 2, ). For clarity, theoretical curves that fit data in presence of D-mannose (Table 6) are not illustrated, but SD values corresponding to each of these curves are given. Curve a [sum of transport velocities (vt)], theoretical, total D-glucose uptake rate, defined as vt = v1 + v2 + v3 (where v1, v2, and v3 are transport velocities of individual components and v3 is diffusional component in Eq. 1). Curve c, theoretical curve for system 2 (v2). Curve b, theoretical curve for v't = v1 + v3, which results when v2 is fully inhibited by excess D-mannose.


                              
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Table 6.   Parameters for curves to fit D-mannose data

If total D-glucose uptake is the sum of three distinct components, such that vt = v1 + v2 + v3 (with vt the sum of transport velocities and v1, v2, and v3 the transport velocities of individual components, where v3 = Kd[S], see Eq. 1), then it can be predicted that, by fully inhibiting S2 with an excess of D-mannose (v2 will tend to zero), the total D-glucose uptake rate should fall to the limiting value v't = v1 + v3. This prediction is confirmed by results such as those illustrated in Fig. 5, where full S2 inhibition was achieved by using high D-mannose concentrations, more than seven times its apparent KT. The general applicability of this meaningful result has been verified by using several other BBM vesicle preparations. We conclude that D-mannose is indeed a substrate specific for S2.

In summary, S1 and S2 can be inhibited by excess D-glucose, whereas S2 is inhibited only by D-mannose. However, one point remains unclear. Even when D-glucose and D-mannose are each transported by a single Michaelian transport system that we have identified as S2, the inhibitory effect of D-mannose on D-glucose uptake does not appear to involve any simple, straightforward case of fully competitive inhibition, as would have been expected. As shown by the data in Fig. 5, the inhibition is clearly noncompetitive, although a "mixed" effect cannot be excluded. We have no explanation for these findings, but it seems worth mentioning here that similar instances of fully noncompetitive effects between closely related analogs have been described, e.g., the noncompetitive inhibition of intestinal L-methionine transport by D-methionine (3). However, explanation of this riddle would require additional work that would exceed the limits set for this study.

Concluding Remarks

Our results confirm the heterogeneity of D-glucose transport systems in pig intestinal BBM vesicles. Two distinct D-glucose transport systems have been identified: the well-known, high-affinity S1 (SGLT1), and a low-affinity, high-capacity second system, S2, with KT2 on the order of 10-2 M for D-glucose. D-Mannose is shown to be a substrate specific for S2, with a KT2 similar to that characterizing D-glucose. S2 is not specifically activated by Na+, is not rheogenic, does not catalyze D-hexose-Na+ cotransport, and therefore is not a member of the SGLT family.

A third, extremely-low-affinity uptake component has also been detected. Even when it follows diffusion-like kinetics, this activity can be distinguished from simple diffusion, because it exhibits a Kd(app) one order of magnitude higher than that of the classical diffusion marker L-glucose. Moreover, preliminary results (not shown) indicate that this activity is blocked by 0.1 mM HgCl2, a specific protein reagent that would not be expected to affect physical diffusion. Although operation of a stereospecific transport system may explain these results, the simplest explanation is that D-glucose enters by "leakage" through some transporter specific for some other substrate.

The D-mannose transport activity shown here to take place via S2 appears to involve a distinct transport system that, to the best of our knowledge, has not been described previously. In theory, it can take care of the animal's need for D-mannose, a physiologically important sugar. Silverman and Ho (25) described a D-mannose-Na+ symporter in renal BBM vesicles, but, in all probability, this has nothing to do with the intestinal D-mannose transport activity described here as S2. First, there is a difference of two orders of magnitude in the relative affinity of either system for D-mannose. Second, Na+ is not a specific activator of S2, so this system cannot be expected to catalyze Na+-sugar cotransport.

Concerning its physiological role and because of its high capacity, S2 can be expected to participate normally in the handling of high sugar loads during feeding. One interesting property of S2 is that its activity can be modulated according to the animal's physiological and nutritional state (6).

Why have several laboratories failed to detect S2? This is an old question for which no answer has been found. Several explanations are imaginable. First, there is the diffusion-correction problem. It is evident that inappropriate correction procedures, particularly that of calling "diffusion" any uptake observed in the absence of activators such as Na+ or in presence of an excess of inhibitors such as phlorizin or the substrate itself (18, 21), may inadvertently lead to overlooking specific transport systems that, for this reason, will never be found.

Are the kinetic analyses based on true initial velocity determinations? Malo and Berteloot (23) use what they call the "dynamic approach," based on utilizing the "fast-sampling, rapid filtration apparatus," which they developed. In contrast, in our laboratory we use the classical one-time-point measurement procedure. This methodological difference cannot explain the dissimilarity in results, because both approaches are known to be quite comparable, provided nonlinear regression analysis is used (23).

Finally, we propose here that the key difference between our results and those of others (23) is the preparation of the radiolabeled substrate mixtures before the initial velocity measurements are performed. The problem comes from the wide range of [S] values needed to perform each saturation curve, which can range from 0.1 to 300 mM, a 3,000-fold difference. There are two possible ways to handle this situation. First, our procedure is to use stepwise increases in the radiolabeled marker, so that the drop in specific activities when [S] is increased is compensated by the addition of more "hot" marker.

The second approach is to use a single quantity of radiolabeled marker, with the hope that the dilutions experienced by this marker might be compensated by mathematical means. However, when this approach is used, the marker is rapidly exhausted, so the effective specific activities drop sharply, rapidly approaching zero at [S] as low as 18 mM. For this reason, the saturation curves cannot be used for regular kinetic analyses based on use of v f[S] plots (23).

To circumvent this serious problem, the authors proposed transformation of the regular saturation curves into "displacement curves," where, it is claimed, the substrate can in theory be treated not as a substrate but, rather, as a competitive inhibitor. An equation was therefore derived (Eq. 3 in Ref. 23) that was thought to be suitable for nonlinear regression analysis of the relevant data, with [S] as the independent variable and Vmax and Km as the dependent variables. This argument is fallacious, however, because this equation is just a simple transformation of the Michaelis equation, where the v = f[S] function has been given the following form: vr = v/[S] = f[S] (7). The problem resides not in the equation, but in the experimental data. It cannot be expected that, by giving it a different form, a given equation could extract an answer from data that contain no information. No amount of mathematical manipulation can compensate for the fact that, at [S] > 18 mM, the effective specific activities and v are zero in these experiments (23).

We conclude that use of a single amount of radioactive substrate to work within a scale as large as that mentioned above (0.1-300 mM) is unsuitable for the detection of low-affinity transporters. Indeed, the results discussed here (23) can neither prove nor disprove the existence of S2.

The following question should be raised: What is the molecular nature of S2? It seems reasonable to suggest that it represents the functional expression of a family of transporters that, because of its lack of homology to any known transport family, has remained hidden. In support of this view, we have seen that S2 does not catalyze Na+-D-glucose cotransport and is probably not a member of the SGLT family, which could explain why all attempts at cloning S2 by using SGLT probes have been unsuccessful (17, 35). More work is required to establish the molecular nature of S2.


    APPENDIX
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
APPENDIX

Can use of D-sorbitol as an osmotic replacement give rise to kinetics spuriously indicating the presence of nonexistent transporters? Certain laboratories are unable to confirm the existence of the low-affinity D-glucose transport system, S2 (11, 21, 23). In a detailed study comparing experimental conditions and data processing, use of D-sorbitol as an osmotic replacement has been identified as the key factor responsible for this disagreement (22). This proposal is based on two distinct postulates.

First, there would be a direct effect whereby D-sorbitol, but not D-mannitol, would inhibit D-glucose uptake with an apparent inhibition constant of 64 mM. However, this observation contradicts a large body of earlier work showing that D-sorbitol and D-mannitol are essentially inert toward S1, which is the basis for the long-standing classification of these two polyalcohols among the so-called nontransported, practically inert sugar analogs (for reviews see Refs. 4 and 12). Such a conclusion has been confirmed by Malo (22), who, after having tested a purified (rather than a commercial) D-sorbitol preparation, concluded that D-sorbitol does not affect D-glucose transport per se in BBM vesicles.

Second, as shown by Malo (22), practically all commercial D-sorbitol preparations are contaminated by D-glucose at ~0.04% by weight. Because in kinetic experiments D-glucose and D-sorbitol need to be varied inversely, D-sorbitol concentration (hence, D-glucose contamination) will be highest at low D-glucose concentration, such that the substrate saturation curves will be deformed. Therefore, it is predicted that there should be a certain degree of transport inhibition at the lowest D-glucose concentrations, but this inhibition should progressively disappear as D-glucose concentration increases. Consequently, it has been proposed that a "release from inhibition" will take place as D-glucose concentration increases, which will manifest itself kinetically by the spurious appearance of transport systems that really do not exist (22). However, a clear-cut indication that this hypothesis is untenable is demonstrated by the following facts. If S2 were an artifact due to the use of contaminated D-sorbitol, it necessarily follows that S2 should become undetectable as soon as the D-sorbitol was replaced by some other, noncontaminated sugar. Use of D-mannitol instead of D-sorbitol has been proposed for this purpose (22). However, Thomas et al. (28), who used D-mannitol rather than D-sorbitol as the osmotic replacement, demonstrated in duckling intestine the existence of a low-affinity system quantitatively equivalent to the S2 described earlier by Brot-Laroche et al. (7) using guinea pig intestine and D-sorbitol. Furthermore, in the present study we have shown an equivalent result: S2 was clearly the same when the kinetics were performed by using commercial (contaminated) D-sorbitol or D-glucose-free D-arabinose as the osmotic replacement (Fig. 3). We conclude that, in practice, commercial D-sorbitol does not significantly modify the kinetics, because the contaminating D-glucose levels in regular D-sorbitol preparations are too low to have any quantitatively significant effect (see below).

To the experimental evidence just presented we now add a theoretical analysis demonstrating that the release from inhibition hypothesis is unsound. We make the following definitions. To maintain isosmotic conditions in kinetic experiments, the total sugar concentration ([T]) is kept constant by using D-sorbitol (or some other inert compound), such that
[T] = [G] + [R] (A1)
where [G] is the D-glucose concentration and [R] is the concentration of the "inert" replacement. Subsequently, we will distinguish between D-glucose the substrate (G) and D-glucose the inhibitor (I). By assuming that R is contaminated with a constant proportion of D-glucose
[I] = <IT>y</IT>[R] = <IT>y</IT>([T] − [G]) (A2)
where y represents a contamination factor (0 < y < 1) and [I] is the net concentration of the contaminant. Because the initial transport rate (v) follows simple Michaelis-Menten kinetics and, by definition, I is a fully competitive inhibitor, we can further assume that the inhibition constant Ki(D-glucose) should equal KT(D-glucose), the substrate dissociation constant. Here both constants are simplified to KT. Therefore
<IT>v</IT> = <FR><NU><IT>V</IT><SUB>max</SUB>[G]</NU><DE>[G] + <IT>K</IT><SUB>T</SUB>(1 + [I]/<IT>K</IT><SUB>T</SUB>)</DE></FR> (A3)
From these premises we obtain
<IT>v</IT> = <FR><NU><IT>V</IT><SUB>max</SUB>[G]/(1 − <IT>y</IT>)</NU><DE>[G] + (<IT>K</IT><SUB>T</SUB> + <IT>y</IT>[T])/(1 − <IT>y</IT>)</DE></FR> (A4)
which represents a simple Michaelis-type equation, with the following apparent kinetic parameters
<IT>V</IT><SUB>app</SUB> = <IT>V</IT><SUB>max</SUB>/(1 − <IT>y</IT>) (A5)

<IT>K</IT><SUB>T(app)</SUB> = (<IT>K</IT><SUB>T</SUB> + <IT>y</IT>[T])/(1 − <IT>y</IT>) (A6)
This means that as y increases, apparent velocity [V(app)] and apparent KT [KT(app)] will increase. Table 7 illustrates the variation of each of these two parameters as y is increased in increments of 10, beginning from the contamination detected by Malo (22) in commercial D-sorbitol (y = 0.04%) and ending at the unlikely value to hold when the "inert" replacement contamination reaches 100% (y = 1).

                              
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Table 7.   Variation in Vapp and KT(app) as y increases

From Table 7 it can be deduced that, at the quoted level of contamination in "regular" experiments (y = 0.04%), the predictable effects on the kinetics are negligible, particularly as concerns Vapp. Although it can be argued that the effect on KT(app) is not entirely negligible, the reality is that it is so low that it is practically impossible to detect with available methodology. In any case, at any value of y, the presence of a D-glucose contaminant will always cause inhibition, as illustrated in Fig. 6. Furthermore, the Eadie-Hofstee plots (Fig. 6B) are not curvilinear. There is no evidence for the suggested "progressive inhibition release" (22).


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Fig. 6.   Simulation of D-glucose uptake kinetics to be expected when osmotic replacement (D-sorbitol or any other sugar) is contaminated with D-glucose. Theoretical curves were computed by using Eq. A4, with total sugar concentration ([T]) = 250 mM, maximum velocity (Vmax) = 100%, affinity constant (KT) = 1 mM, and contamination by D-glucose as follows: 0% (), 0.04% (), 0.4% (black-diamond ), 4% (star ), 40% (black-triangle), and 99.9% (triangle ). A: v = f[G] plots, where [G] is D-glucose concentration. B: results in A plotted according to Eadie-Hofstee transformation.

We conclude that 1) contamination of D-sorbitol by D-glucose cannot possibly explain the appearance of fallacious low-affinity systems with Vmax values several times higher that those characterizing S1, such as those exhibited by S2, and 2) S2 is not an artifact but represents an authentic transport entity.


    ACKNOWLEDGEMENTS

This work was supported in part by the Association Française de Lutte contre la Mucoviscidose, the Institut National de la Santé et de la Recherche Médicale, the Fondation pour la Recherche Médicale, Paris, and INCO Programme of the European Economic Community Grant ERB 3514 PL 950019.


    FOOTNOTES

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

1 This Kd value is practically identical to that estimated by using guinea pig BBM vesicles [Kd(app) = 2.5 nl · s-1 · mg membrane protein-1 (8)].

Address for reprint requests and other correspondence: M. Vasseur, Unité 510, INSERM, Faculté de Pharmacie, Université de Paris XI, 5, rue J.-B. Clément, 92296 Châtenay-Malabry, France (E-mail: monique.vasseur{at}cep.u-psud.fr).

Received 21 May 1999; accepted in final form 3 August 1999.


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ABSTRACT
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METHODS
RESULTS AND DISCUSSION
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APPENDIX

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Am J Physiol Cell Physiol 277(6):C1130-C1141
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