Institut National de la Santé et de la Recherche Médicale, Unité 510, Faculté de Pharmacie, Université de Paris XI, 92296 Châtenay-Malabry, France
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ABSTRACT |
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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
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INTRODUCTION |
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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?
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METHODS |
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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 atMaterials
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, withUptake 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 · sFitting 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
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(1) |
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.
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RESULTS AND DISCUSSION |
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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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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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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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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 · s1 · 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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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 · sLack 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)
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(1R) |
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 10Consequently, 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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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 · sThe 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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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 · s1 · 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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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 · s1 · 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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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 10A 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.
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APPENDIX |
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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
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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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 · s1 · 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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