Effect of diet on glucose transporter site density along the intestinal crypt-villus axis RONALD0 P. FERRARIS, SOPHIE A. VILLENAS, BRUCE A. HIRAYAMA, AND JARED DIAMOND Department of Physiology, University of California Medical School, Los Angeles, California Ferraris, Ronald0 P., Sophie A. Villenas, Bruce A. Hirayama, and Jared Diamond. Effect of diet on glucose transporter site density along the intestinal crypt-villus axis. Am. J. Physiol. 262 (Gastrointest. Liver Physiol. 25): G1060G1068, 1992.-High-carbohydrate diets stimulate intestinal brush-border glucose uptake and increase the number of glucose-protectable phlorizin binding sites, but it has been unknown where along the crypt-villus axis these effects are expressed. We attacked this problem by three methods. First, by measuring phlorizin binding to isolated mouse enterocytes fractionated along the crypt-villus axis by the Weiser method, we identified a high-affinity binding site predominating from villus tip to midvillus and a site of possibly lower affinity predominating in the crypts. A high-carbohydrate diet increased by severalfold the density of the villus sites and probably also of the crypt sites, without changing their binding constants. Second, autoradiography revealed increased glucose-protectable phlorizin binding along the whole crypt-villus axis on a high-carbohydrate diet. Finally, a polyclonal antibody against the Na+glucose cotransporter recognized a protein in the brush-border membrane of villus cells. Hence, substrate-dependent upregulation of intestinal glucose transport involves increased numbers of transporters along the crypt-villus axis. mouse; Weiser method; phlorizin; autoradiography; immunocytochemistry; dietary carbohydrate
diets stimulate brush-border glucose uptake in mouse, rat, and sheep intestine (9, 33, 35). This effect of dietary substrate is a specific one on the maximal velocity ( Vmax) of the glucose transporter, without change in mucosal mass or glucose passive permeability. We previously showed that this change in glucose transporter Vmax arises from proportionate changes in glucose transporter site density, as measured in intact intestinal sleeves by specific binding of phlorizin, a competitive inhibitor of the brush-border glucose transporter (13). In that study, we detected two types of binding sites, differing in their affinity constants for phlorizin (12). Density of the Na+-glucose cotransporter protein, detected by immunoreactivity, also changes in parallel with changes in the transporter Vmax measured in brush-border membrane vesicles (35). These observations beg the question how this substrate-dependent regulation of the glucose transporter relates to the well-known crypt-villus axis of intestinal mucosal organization. Crypt and villus enterocytes differ in structure and function, correlated with continual mitosis in the crypts, enterocyte migration along the villus, and eventual sloughing-off of mature enterocytes at the villus tips. Some of the resulting questions could be answered by studies of transporter distribution along the crypt-villus axis in the steady state. For example, where along the axis is each type of transport site expressed? Do both site types coexist in the same cell or does one type become transformed into the other as enterocytes migrate from crypt to villus? Where along HIGH-CARBOHYDRATE
G1060
0193-1857/92
$2.00
Copyright
0
90024-l 751
the axis does the diet-dependent difference in site density take effect? The present article, the second in a series (see Ref. 16 for the preceding article), examines these questions involving steady-state transporter distribution. We shall use three methods for localizing transporters along the crypt-villus axis in mice on a high-carbohydrate (HC) or no-carbohydrate (NC) diet: phlorizin binding to isolated enterocytes fractionated by the Weiser method, phlorizin binding detected autoradiographically in tissue sections, and immunocytochemistry. The following article (14) examines transporter distribution during transients after a sudden change in dietary carbohydrate level to deduce where the regulatory signal is perceived. MATERIALS Animals
AND METHODS
and Diets
Adult male white Swiss Webster mice were initially maintained on Wayne Lab-Blox chow and were then switched for 2 wk to either the HC or NC diet used in Refs. 14 and 16. We found previously that 2 wk is ample time for glucose transporter activity of mouse intestine to adapt to dietary carbohydrate level (9). During those 2 wk, we recorded initial body weight and then monitored food consumption and body weight 2-3 times per week. As we had found previously (13), food consumption differed slightly between the HC and NC diets (P < 0.025, 2tailed t test), but body weight gain (P > 0.2) and intestinal length (P > 0.1) did not. For HC and NC mice, respectively, daily food consumption was 5.4 t 0.3 (n = 18) and 4.2 t 0.2 g/mouse, final body weight was 43.7 t 1.0 (n = 18) and 41.6 t 1.6 (n = 17) g, and intestinal length was 54.8 & 0.5 (n = 18) and 52.5 t 1.2 (n = 17) cm. Experiment
1: [3H]phlorizin
binding
to isolated
enterocytes
We fractionated enterocytes along the crypt-villus axis by the Weiser procedure already described, with the following minor modifications. Jejunums were pooled from three mice, not two mice. We used 18 mice (6 experiments with 3 mice each) per diet. Following Walters and Weiser (47), we incorporated 5 mM fructose and 5 mM glutamine in the citrate solution, 0.025 mg/ml trypsin inhibitor in the incubation phosphate-buffered solution with EDTA (PBS), and we used 10 consecutive incubation steps lasting 18, 12, IO, 8, 4, 4, 7, IO, 12, and 20 min, respectively (a total of 105 min). The resulting enzyme profiles proved similar to those described previously (16). We pooled the resulting IO cell fractions into five pairs (fractions 1 and 2,3 and 4,5 and 6, 7 and 8, and 9 and IO), respectively, representing cells from the villus tip, upper villus, midvillus, lower villus, and crypt (see Fig. 1 of Ref. 16). Before use, cells were kept in PBS-P solution [PBS modified for phlorizin binding (in mM): 105 NaCl, 2.7 KCl, 7.6 Na,HPO,, 2.1 KH2P04, pH 7.01 plus 50 mM mannitol and 1 mg/ml bovine serum albumin (BSA). To measure phlorizin binding by those isolated enterocyte fractions, we modified the method of Restrepo and Kimmich (32). A lo-p1 cell aliquot containing 80-100 pg cell protein was preincubated for 1 min at 37°C in a 2.2-ml microcentrifuge tube.
1992 the
American
Physiological
Society
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VILLUS
AND
CRYPT
We then added 110 ~1 of PBS-P solution at 37°C containing [“Hlphlorizin, 50 mM glucose or fructose (see below for rationale), and 1 mg/ml BSA. The mixture was vortexed for 5 s and rinsed after 2 s (i.e., total incubation time 7 s) with 2 ml ice-cold PBS-P solution plus 50 mM mannitol and 1 mg/ml BSA, spun down for IO s in a Beckman microfuge at 10,000 g, and the supernatant was discarded. The cell pellet was subjected again to the same rinse, dissolved in 200 ~1 of Protosol (New England Nuclear), and transferred to a scintillation vial for liquid scintillation counting of the [3H]phlorizin. We found that the first rinse of fructose-incubated cells removed 94 t 1% (n = 3) of the initial radioactivity, the second rinse 4.5 t 1.5%, and a third rinse 0.25). These time dependences of specific, nonspecific, and total binding in isolated enterocytes are very similar to those in intact intestinal sleeves, except that the hyperbolic phase of nonspecific and total binding and the initial rise of specific binding are slower in intact sleeves, due -presumably to their thicker unstirred layers (12). A priori, one might wonder whether calculating specific phlorizin binding as the difference between two measured larger numbers (total and nonspecific binding) would be sufficiently accurate. In practice, our values for all six time points were the same within experimental error, which was modest. The coefficient of variation was lowest at 7 s (16%), because shorter incubations were more difficult to time accurately, but longer incubations yielded larger nonspecific binding corrections. Hence, we chose an incubation time of 7 s to minimize nonspecific binding while ensuring completeness of specific binding. l
Delayed removal of cells does not affect specificphlorizin binding. The Weiser method removes villus tip cells first
and crypt cells last. We shall show (Table 1) that villus tip and crypt cells differ in their kinetics of specific phlorizin binding. Does that difference reflect a corresponding difference in native properties of villus tip and crypt cells or is it an artifact of the longer incubation of crypt cells l n 0
total binding nonspecific binding specific binding
Fig. 1. Binding of 0.015 FM phlorizin to isolated mid- and upp ler villus enterocytes, as a function of incubation time (means -c- SE, n = 4). Curves fitted through experimental points by least squares are a straight line for specific binding and a straight line plus a hyperbolic component for nonspecific and total binding.
TRANSPORTERS
during the Weiser cell fractionation procedure? We addressed this question by the same method that we used to address the corresponding question for enzyme activities (16): we decreased mechanical agitation from 90 to 30 Hz, thereby delaying release of villus cells from 30 to 75 min, nearly as long as the normal release time of crypt cells. Nonlinear ligand binding analysis with proportional weighting showed that this delay did not significantly change the dissociation constant (&; 0.260 t 0.049 PM for control cells vs. 0.276 t 0.075 PM for delayed cells, P > 0.8 by 2-tailed t test) nor the site density (23.2 t 3.4 vs. 17.0 t 3.4 pmol/mg protein, P > 0.1) of specific binding sites for phlorizin. Linear regression with simple weighting of Scatchard plot transforms of the data yielded the same conclusion: neither Kd [0.323 t 0.065 vs. 0.422 t 0.132 PM (P > 0.4)] nor site density [29.2 t 4.2 vs. 26.6 t 5.4 pmol/mg protein (P > 0.5)] differed between control and delayed cells. Hence a more than twofold increase in incubation time did not affect phlorizin binding kinetics, and the apparent difference in kinetics between villus tip cells and crypt cells is presumably real. AP activity. The steep decrease of AP activity (Fig. 2) from villus tip to crypt (P < 0.0001 by a 2-way ANOVA), the slightly (on average, 23%) higher AP activity on the NC than HC diet (P < 0.02), and the absolute values of activity were very similar to our previous results for AP activity in mouse jejunum (see Fig. 2, B and C, of Ref. 16). Effect binding.
of crypt-villus
position
on specific phlorizin
In four of our five cell fractions along the cryptvillus axis, specific phlorizin binding to the glucose transporter involved a single site (Table 1), as judged by linear Scatchard plots (Fig. 3) and by the criteria of Akaike (1) and Schwarz (34). The single sites in the three terminal positions (villus tip, upper and midvillus) shared similar Kd values, whereas the single site in the crypt was of much lower affinity (Kd 3-5 times higher) (Table 1). Data for the remaining cell fraction, the lower villus fraction that mixes some crypt and villus cells (16), were best fit by a two-site model, with Kd values of the high-affinity and low-affinity site similar to the Kd value of the single site in terminal positions and in the crypts, respectively. Because of large SEs on binding parameters of the lowaffinity site in the crypts, the difference between its Kd and that of the high-affinity site in terminal positions did not achieve statistical significance in the present study (P > 0.05 by 2-tailed t-test), but it did achieve significance in our subsequent study with larger sample sizes (P < 0.02, Ref. 11). Site density of the high-affinity site decreased twofold from villus tip to midvillus and fivefold from villus tip to lower villus in mice of both diet groups @-tailed t test, P < 0.05 in each case), but that site’s Kd did not change among those four terminal regions (P > 0.1). Effect of diet on specific phlorixin binding. Linear regression of Scatchard transforms and nonlinear regression both yielded the same conclusion: compared with the NC diet, the HC diet increased site density of specific phlorizin binding by 1.8- to 6.3-fold without affecting Kd
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VILLUS
Table 1. Effect of diet and crypt-villus
AND
position
Tip UPPer Mid Lower Site Site CrVpt
Kinetic constants are simple weighting (fitted the latter set is given in lower villus, whose data mice over the experimental two sites, the dissociation site 2 (note large SEs in
binding sites No-Carbohydrate
Diet Density, pmol/mg protein
Kci, PM
1 2
Gl063
TRANSPORTERS
on Kd and density of phlorizin
High-Carbohydrate Villus Segment
CRYPT
Diet
PM
Density, pmol/mg protein
19.223.0 12.5t3.7 10.5k2.0
&,
0.51t0.11 0.47t0.11 0.34&O. 10
37.9t6.3 33.8t5.8 18.4t4.2
0.36t0.07 0.36&O. 14 0.2620.07
0.38t0.18 0.70t0.18 1.4t0.9
4.7t2.6 22.4t3.8 27.0t14.2
0.23t0.19 0.3t39 1.2t0.7
4.1t3.8 68t99 4.3t2.4
given as estimates t SE of the estimate. Phlorizin binding data were analyzed by linear regression of Scatchard plots with lines of Fig. 3) or else by nonlinear regression with proportionate weighting. Both methods yielded very similar parameters; this table. By the criteria of Akaike (1) and Schwarz (34), a l-site model yielded the best fit for all villus segments except were best fitted by a 2-site model (site 1, high-affinity site; site 2, low-affinity site; site 2 nonsaturable in no-carbohydrate range of concentrations). For lower villus of no-carbohydrate mouse, our data sufficed to establish the coexistence of constant (&) and density values for site 1, and the density/& ratio for site 2, but not density and & separately for that case). m 0
1 t i p ---
2 ------___ VILLUS
no carbohydrate high carbohydrate
3 -----
4 _____
0 no carbohydrate 0 high carbohydrate
5 c ry
p t
SEGMENT
activity of alkaline phosphatase in jejunal cells fractionFig. 2. Specific ated along crypt-villus axis, from mice fed a HC or NC diet. Fraction 1, villus tip cells; fraction 5, crypt cells. Each bar gives mean t SE (n = 6). Within a diet, bars sharing same letter do not differ significantly (P > 0.05).
at any position (Table 1). The high-affinity site continued to characterize the villus tip, upper villus, and midvillus, with densities increased 1.9-, Z.7-, and I.&fold, respectively, over values in NC mice (P = 0.005, 0.004, and 0.052 by l-tailed t test). The low-affinity site continued to characterize the crypts, with 6.3-fold increased density (borderline significance, P = 0.06, due to large SEs). Experiment pH]phlorizin
2: Binding
Detected Autoradiographically
As summarized in Fig. 4, two-way ANOVA showed large effects both of diet and of villus position on total binding (P = 0.0002 and 0.003, respectively) and on specific binding (P c 0.0001 and P = 0.006, respectively). In total binding studies, HC mice greatly exceeded NC mice in number of silver grains per villus [251 t 28 (n = 8) vs. 100 t 17 (n = 6) respectively, P < O.OOl]. Because, however, nonspecific binding was low and independent of diet [16 t 2 (n = 8) vs. 21 t 3 (n = 6), P > 0.051, specific binding per villus was also three times higher in HC than in NC mice [234 t 28 (n = 8) vs. 80 t 14 (n = 6) respectively, P < O.OOl]. This factor of three for increased specific binding derived from autoradiography is in good agreement with the factor derived from liquid scintilla-
10
20
I 30
amount of phlorizin bound (pmoles/mg protein) Fig. 3. Effect of dietary carbohydrate on Scatchard plots of [3H]phlorizin binding by isolated enterocytes from villus tip (A) or crypt (B). Each point is mean rt SE (n = 6). Lines are least-squares linear fits of Scatchard-transformed data with simple weighting.
tion counting of everted sleeves of the same mice (HC mice 3.1 times higher than NC mice). Because we used a low phlorizin concentration, most specific binding was to the high-affinity sites located in the terminal three positions of the villus, regardless of diet. Because in addition nonspecific binding was low, total binding was also concentrated in the terminal three positions. Both for total and specific binding, number of silver grains per position was higher in the villus tip and/or upper villus than in the crypts. Each villus position had more grains in HC than in NC mice. Nonspecific
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VILLUS
AND
CRYPT
high carbohydrate
TRANSPORTERS
no carbohydrate TOTAL
abc
BINDING -
rll+l bc
C
1’1
I
Fig. 4. Effect of dietary carbohydrate on binding of 0.005 ,uM [“Hlphlorizin to everted sleeves of mouse jejunum, studied by autoradiography. For purposes of counting grains, each villus was divided into 5 equally long segments (I, villus tip; 5, crypt). Each bar is mean t SE of 6 (NC mice) or 8 (HC mice) villi. Within a panel, bars sharing same letter do not differ significantly (P > 0.05). Note that specific binding in each segment is 2-4 times higher on HC diet than on NC diet.
bc
+l
C
r-h
villus
segment
binding was independent of position in NC-fed mice and varied with position in HC-fed mice. Experiment 3: Immunocytochemical
Detection
of Glucose Transporter
Immunofluorescent staining showed that the antibody against a peptide from the cloned glucose transporter recognized a protein in the brush border of enterocytes of the upper villus (Fig. 5A) but not of the crypts (Fig. 5B). This staining was apparent in both duodenum and jejunum. (Because of limited available quantities of antibody, we did not try to measure the lower quantities of transporter expected in ileum.) There was no staining of control tissues incubated in normal rabbit serum (Fig. 5C) nor of control tissues incubated in peptide-absorbed antiglucose transporter antibody. Control tissues incubated with goat antibody to rat sucrase exhibited staining of the brush border in these experiments, as we had also found in the experiments described in the first companion article of this series (16). DISCUSSION
Effect of Dietary
Carbohydrate
This article yields a set of unequivocal a set of more tentative conclusions.
conclusions, plus
Our unequivocal conclusions begin with our observation that the well-known two- or threefold stimulation of intestinal glucose transport by dietary carbohydrate is paralleled by a comparable increase in number of glucoseprotected phlorizin binding sites. This increase was measured by both methods that we employed: phlorizin binding to isolated cells fractionated by the Weiser method and phlorizin autoradiography of intact tissue sections. This conclusion in itself was not surprising, because we had previously found increased glucose-protectable phlorizin binding to intact intestinal sleeves (13). However, that previous study of intact sleeves could not localize the stimulatory effect along the crypt-villus axis. We now find, by both of the above-mentioned methods, that dietary stimulation of glucose-protectable phlorizin binding is unequivocally demonstrable along the whole terminal portion of the villus: the villus tip, upper villus, and midvillus (Table 1, Figs. 3 and 4). Our more tentative conclusions begin with our observation, also valid for intact sleeves (12, 13), that there appear to be not one but two types of glucose-protectable phlorizin binding sites, distinguished by their binding kinetics. The site with lower phlorizin affinity appears to account for less binding, to reside in the crypts, and also
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VILLUS
AND CRYPT
TRANSPORTERS
G1065
Fig. 5. Localization of glucose transporter in mouse jejunum by light microscopic (original magnification x400) immunofluorescence staining. A: villus tips incubated with anti-Na+-glucose transporter IgG, followed by fluorescein-conjugated anti-rabbit IgG. B: as in A but crypt region instead of villus tips. C: as in A except that normal rabbit serum was substituted as a control for anti-Na+-glucose transporter IgG. Note concentrated staining of brush border in villus tips (A, arrows) but not in crypts (B) nor in control tissue (C).
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G1066
VILLUS
AND CRYPT
to be upregulated by dietary carbohydrate as is the villus (higher-affinity) site. These conclusions are more tentative than are the conclusions of the preceding paragraph because of the larger SE values in our measurements of crypt cell binding. The difference in binding constant between crypt and villus cells did not achieve statistical significance in the present article but did in the larger samples reported in the following companion article (14). The effect of diet on site density either did achieve, or narrowly failed to achieve, significance, depending on the experimental method and sample size (this article and Ref. 14). Although, we thus conclude that dietary carbohydrate definitely increases phlorizin binding along the terminal villus and probably along the whole crypt-villus axis, this observation does not prove that the regulatory signal is perceived along the terminal villus or whole axis. In fact, the following (final) companion article of this series will present evidence that this signal is perceived only in the crypts (14). While our study is exclusively concerned with the brush-border glucose transporter, the site density and functional activity of the basolateral glucose transporter are also regulated by substrate levels, specifically, upregulated during hyperglycemia (8). Thus dietary regulation of intestinal glucose absorption involves regulation of transporters at both faces of the enterocyte. The remainder of this discussion section will consider in more detail the following issues: number of brushborder transporters per cell, phlorizin binding constants, the question of site heterogeneity, and the question of glucose transport in crypts. Number
of Transporters
Per Cell
From the number of cells per milligram
protein (-8 x binding sites per milligram protein (Table 1) for mouse intestine, we calculate that there are ~2.8 X lo6 or 1.4 X lo6 glucose transporters per cell in terminal villus segments of HC or NC mice, respectively. These values are similar to our previous estimate of lo6 to lo7 transporters per cell in HC 106: Ref. 16) and the density of phlorizin
Table 2. Kd of phlorizin
Mytilus
californius
Mytilus
edulis
gill
gill
TRANSPORTERS
mice, based on specific phlorizin binding to intact tissue (13). Similar transporter densities have been calculated for woodrat (15), hamster (10, 38), and chick (32) intestines while values up to lo-fold lower were obtained for rabbit (48) and iguana (15) intestines. Glucose transport rates per milligram of tissue vary in the same sequence, being similarly high in mouse, woodrat, hamster, and chicken intestines (15, 24) and up to an order of magnitude lower in rabbit (6) and iguana (24) intestines. Thus, as we discussed previously (15), species differences in glucose transporter density make an important contribution to species differences in intestinal glucose transport rates. Site Heterogeneity
and Distribution
Table 2 summarizes binding constants for phlorizin to glucose transporters of various tissues, studied in various types of preparations. There is a wide range of Kd values for the major binding site, from 0.0005 to 18 PM. Rabbit kidney and pig kidney as well as mouse intestine yield evidence of two binding sites differing in Kd. Values for the major site of mouse intestine are in the lower half of the range, with the value that we previously measured for intact tissue being somewhat below the value that we now find for isolated cells. Phlorizin binds not only to glucose transporters but also to a nucleoside transporter (28), as well as nonspecifically to membrane lipids. However, we infer that our measurements of what we term “specific phlorizin binding” constitute binding to glucose transporters, because we measure only the component of phlorizin binding blocked by glucose. [Measurement of Na+-dependent phlorizin binding yields the same value (12); the nucleoside transporter is not inhibited by glucose (28); and one would not expect binding to lipid to be Na+ dependent and glucose protectable.] Hence our observation of apparently differing glucose-protectable phlorizin binding kinetics in mouse intestinal crypts and villi raises two questions: whether there might also be two distinct intestinal glucose transporters and whether crypts transport some glucose.
to glucose transporters
Intact tissue
0.0005
BBMV
0.006
Isolated cells 0.05 LLC-PK1 pig kidney cells 0.07-0.2 Mouse intestine Intact tissue 0.2-0.5, l-4 Mouse intestine Isolated cells Rabbit kidney BBMV 0.2, 100 Pig kidney BBMV 0.4, 8 Chick intestine Isolated cells 0.9 BBMV 1 Rabbit kidney BBMV 3-5 Rat kidney BBMV 5 Rabbit intestine 6 Rabbit intestine cloned glucose transporter Injected oocytes 8,250O Rabbit kidney BBMV Rabbit intestine BBMV 9 BBMV 18 Guinea pig intestine Two values of & separated by a comma indicate that kinetic analysis revealed 2 binding sites with the failure to correct for nonspecific phlorizin binding led artifactually to much higher & values (e.g., 75 PM BBMV, brush-border membrane vesicles.
S. Wright and A. Pajor, personal communication S. Wright and A. Pajor, personal communication 2 12,13 14; this paper 44 27 32 3 20 42 23 8 23 4 stated & values. We omit studies in which for hamster intestine in the Ref. 10 study).
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VILLUS
AND
CRYPT
Studies of the possible heterogeneity of intestinal glucose transport have yielded conflicting results. On the one hand, evidence in favor of just a single intestinal glucose transporter includes the facts that only a single one has thus far been cloned (23), that a genetic defect in that cloned transporter was observed in a case of congenital glucose/galactose malabsorption in a human infant (43)) and that a recent careful kinetic analysis detected only one transporter in human intestinal brush-border membrane vesicles (BBMVs; 30). On the other hand, evidence pointing to the coexistence of multiple intestinal glucose transporters includes other kinetic analyses (e.g., 18, 19, 23, 25, 29, 41) plus studies of transporter differences in relative affinity for glucose and galactose (6, 37), susceptibility to upregulation by different dietary sugars (37), Na+ activation (19, 29), and temperature sensitivity (4). As deduced from differences in these properties, the multiple transporters differ from each other in their relative activities at different stages of ontogenetic development (6, 19), as well as in their relative activities in duodenum and jejunum and ileum (6, 19, 29, 37). Evidence regarding phlorizin binding and glucose transport in crypt cells is similarly conflicting. On the one hand, villus rather than crypt cells clearly account for most intestinal glucose transport. This was shown by the classic autoradiographic study of Stirling and Kinter (39) of [3H]galactose accumulation and was confirmed by many studies of glucose uptake by BBMVs from cell fractions separated by the Weiser method (e.g., 11, 18, 31). The cloned intestinal glucose transporter appears absent from crypts, as shown by immunocytochemistry (22; the present article) and by distribution of its mRNA (22). We correspondingly find that most glucose-protectable phlorizin binding is in the villi rather than in the crypts. On the other hand, phlorizin binding or glucose transport in crypts appears not to be negligible, as shown by the same BBMV studies just cited and by our phlorizin binding studies with cell fractions separated by the Weiser method. Perhaps the reconciliation of this apparent contradiction is simply that enterocytes along the whole crypt-villus axis do have some phlorizin-sensitive glucose transport sites, but that villus cells have more sites than do crypt cells. Even the classic study of Stirling and Kinter (39) detected some galactose accumulation in crypts. In addition, our phlorizin autoradiographic studies purposely employed very low phlorizin concentrations (0.005 pm) to distinguish specific phlorizin binding to glucose transporters from nonspecific binding. However, those low concentrations were incidentally much more suitable for detecting the high-phlorizin affinity site of villus cells than the putative low-phlorizin affinity site of crypt cells. Stirling (38) used much higher phlorizin concentrations (0.6-60 PM) and thereby detected phlorizin binding autoradiographically along the whole crypt-villus axis. Thus these questions about possible heterogeneity and crypt presence of glucose transport and phlorizin binding remain unresolved. Because glucose transport may drop from villus to crypt as a result of glucose absorption out of the poorly stirred intervillus space (40), it would not be surprising if the crypts transported glucose with high
Gl067
TRANSPORTERS
affinity but low capacity. The two phlorizin binding sites of rabbit kidney do differ in their affinity for glucose (44, 45). If crypts do carry out high-affinity glucose transport, it will remain to be determined whether there really are two unrelated glucose transporters and gene products differing in phlorizin affinity, one expressed in crypt cells and the other in mature villus cells or whether there is only one transporter, whose phlorizin binding and functional state change with microenvironment as enterocytes migrate from crypt to villus. We thank Dean Bok, Nathan Collie, Sharon Sampogna, Gail Strother, Ernest Wright, and Sasan Yasharpour for help. This study was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants GM-14772 and DK-17328 (University of California, Los Angeles, Center for Ulcer Research and Education). Present address of R. P. Ferraris: Dept. of Physiology, Univ. of Medicine and Dentistry of New Jersey, Newark, NJ 07103-2714. Address for reprint requests: J. Diamond, Dept. of Physiology, UCLA Medical Center, Los Angeles, CA 90024-1751. Received
7 October
1991;
accepted
in final
form
13 January
1992.
REFERENCES 1. Akaike, H. A new look at the statistical model identification. IEEE Trans. Auton. Control AC19: 716-723, 1974. 2. Amsler, K., and J. S. Cook. Linear relationship of phlorizinbinding capacity and hexose uptake during differentiation in a clone of LLC-PK, cells. J. CeZZ. Physiol. 122: 254-258, 1985. 3. Aronson, P. S. Energy-dependence of phlorizin binding to isolated renal microvillus membranes. J. Membrane Biol. 42: 81-98, 1978. 4. Brot-Laroche, E., M. A. Serrano, B. Delhomme, and F. Alvarado. Temperature sensitivity and substrate specificity of two distinct Na+-activated D-glucose transport systems in guinea pig jejunal brush border membrane vesicles. J. Biol. Chem. 261: 6168-6176, 1986. 5. Brot-Laroche, E., S. Supplisson, B. Delhomme, A. I. Alcalde, and F. Alvarado. Characterization of the D-glucose/ Na+ cotransport system in the intestinal brush-border membrane by using the specific substrate, methyl a-D-glucopyranoside. Biochim. Biophys. Acta 904: 781-780, 1987. 6. Buddington, R. K., and J. M. Diamond. Ontogenetic development of monosaccharide and amino acid transporters in rabbit intestine. Am. J. Physiol. 259 (Gastrointest. Liver Physiol. 22): G544-G555, 1990. 7. Cheeseman, C. I. Expression of amino acid and peptide transport systems in rat small intestine. Am. J. Physiol. 251 (Gastrointest. Liver Physiol. 14): G636-G641, 1986. 8. Cheeseman, C. I., and D. D. Maenz. Rapid regulation of D-glucose transport in basolateral membrane of rat jejunum. Am. J. Physiol. 256 (Gastrointest. Liver Physiol. 19): G878-G883, 1989. 9. Diamond, J. M., and W. H. Karasov. Effect of dietary carbohydrate on monosaccharide uptake by mouse small intestine in vitro. J. Physiol. Lord 349: 419-440, 1984. 10. Diedrich, D. F. Is phloretin the sugar transport inhibitor in intestine? Arch. Biochem. Biophys. 127: 803-812, 1968. 11. Dudeja, P. K., R. K. Wali, A. Klitzke, and T. A. Brasitus. Intestinal D-ghCOSe transport and membrane fluidity along cryptvillus axis of streptozocin-induced diabetic rats. Am. J. Physiol. 259 (Gastrointest. Liver Physiol. 22): G571-G577, 1990. 12. Ferraris, R. P., and J. M. Diamond. A method for measuring apical glucose transporter site density in intact intestinal mucosa by means of phlorizin binding. J. Membrane Biol. 94: 65-75, 1986. 13. Ferraris, R. P., and J. M. Diamond. Use of phlorizin binding to demonstrate induction of intestinal glucose transporters. J. Membrane Biol. 94: 77-82, 1986. R. P., and J. Diamond. Crypt-villus site of glucose 14. Ferraris, transporter induction by dietary carbohydrate in mouse intestine. Am. J. Physiol. 262 (Gastrointest. Liver Physiol. 25): Gl069G1073, 1992. 15. Ferraris, R. P., P. P. Lee, and J. M. Diamond. Origin of regional and species differences in intestinal glucose uptake. Am.
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J. Physiol. 257 (Gastrointest. Liver Physiol. 20): G689-G697, 1989. 16. Ferraris, R. P., S. A. Villenas, and J. M. Diamond. Regulation of brush-border enzyme activities and enterocyte migration rates in mouse small intestine. Am. J. Physiol. 262 (Gastrointest. Liver Physiol. 25): G1047-Gl059, 1992. 17. Ferraris, R. P., S. Yasharpour, K. C. K. Lloyd, R. Mirzayan, and J. M. Diamond. Luminal glucose concentrations in the gut under normal conditions. Am. J. Physiol. 259 (Gastrointest. Liver. Physiol. 22): G822-G837, 1990. 18. Freeman, H. J., G. Johnston, and G. A. Quamme. Sodiumdependent D-glucose transport in brush-border membrane vesicles from isolated rat small intestinal villus and crypt epithelial cells. Can. J. Physiol. Pharmacol. 65: 1213-1219, 1987. 19. Freeman, H. J., and G. A. Quamme. Age-related changes in sodium-dependent glucose transport in rat small intestine. Am. J. Physiol. 251 (Gastrointest. Liver Physiol. 14): G208-G217, 1986. 20. Gibbs, E. M., M. Hosang, B. F. X. Reber, G. Semenza, and D. F. Diedrich. 4-azidophlorizin, a high affinity probe and photoaffinity label for the glucose transporter in brush border membranes. Biochim. Biophys. Acta 688: 547-556, 1982. 21. Hirayama, B. A., H. C. Wong, C. D. Smith, B. A. Hagenbuch, M. A. Hediger, and E. M. Wright. Intestinal and renal Na+/glucose cotransporters share common structures. Am. J. Physiol. 261 (Cell Physiol. 30): C296-C304, 1991. 22. Hwang, E.-S., B. A. Hirayama, and E. M. Wright. Distribution of the SGLTl Na+/glucose co-transporter and mRNA along the crypt-villus axis of rabbit small intestine. Biochem. Biophys. Res. Commun. 181: 1208-1217, 1991. 23. Ikeda, T. S., E.-S. Hwang, M. J. Coady, B. A. Hirayama, M. A. Hediger, and E. M. Wright. Characterization of a Na+/ glucose cotransporter cloned from rabbit small intestine. J. Membrane BioZ. 110: 87-95, 1989. 24. Karasov, W. H., D. H. Solberg, and J. M. Diamond. What transport adaptations enable mammals to absorb sugars and amino acids faster than reptiles? Am. J. Physiol. 249 (Gastrointest. Liver Physiol. 12): G271-G283, 1985. 25. Kaunitz, J. D., and E. M. Wright. Kinetics of sodium D-glucose cotransport in bovine intestinal brush border vesicles. J. Membrane Biol. 79: 41-51, 1984. 26. Keljo, D. J., R. J. Macleod, M. H. Perdue, D. G. Butler, and J. R. Hamilton. D-Glucose transport in piglet jejunal brushborder membranes: insights from a disease model. Am. J. Physiol. 249 (Gastrointest. Liver Physiol. 12): G751-G760, 1985. 27. Koepsell, H., G. Fritzsch, K. Korn, and A. Madrala. Two substrate sites in the renal Na+-D-glucose cotransporter studied by model analysis of phlorizin binding and D-glucose transport measurements. J. Membrane Biol. 114: 113-132, 1990. 28. Lee, C. W., C. I. Cheeseman, and S. M. Jarvis. Transport characteristics of renal brush border Na+- and K+-dependent uridine carriers. Am. J. Physiol. 258 (Renal Fluid Electrolyte Physiol. 27): F1203-F1210, 1990. 29. Malo, C. Kinetic evidence for heterogeneity in Na+-D-glucose cotransport systems in the normal human fetal small intestine. Biochim. Biophys. Acta 938: 181-188, 1988. 30. Malo, C., and A. Berteloot. Analysis of kinetic data in transport studies: new insights from kinetic studies of Na+-D-glucose cotransport in human intestinal brush border membrane vesicles using a fast sampling, rapid filtration apparatus. J. Membrane BioZ. 122: 127-141, 1991.
TRANSPORTERS 31.
32.
33.
34. 35.
36. 37.
38. 39.
40.
41.
42.
43.
44.
45.
46.
47.
48.
Meddings, J. B., D. Desouza, M. Goel, and S. Thiesen. Glucose transport and microvillus membrane physical properties along the crypt-villus axis of the rabbit. J. CZin. Invest. 85: 10991107, 1990. Restrepo, D., and G. A. Kimmich. Phlorizin binding in isolated enterocytes: membrane potential and sodium dependence. J. Membrane Biol. 89: 269-280, 1986. Scharrer, E., S. Wolfram, W. Raab, B. Amann, and N. Agne. Adaptive changes of amino acid and sugar transport across the brush border of rabbit jejunum. In: Mechanisms of IntestinalAdaptation, edited by J. W. L. Robinson, R. H. Dowling, and E. 0. Riecken. Lancaster, UK: MTP, 1981, p. 123-137. Schwarz, G. Estimating the dimension of a model. Annu. Stat. 6: 461-464, 1978. Shirazi-Beechey, S. P., B. A. Hirayama, Y. Wang, D. Scott, M. W. Smith, and E. M. Wright. Ontogenetic development of lamb intestinal sodium-glucose co-transporter is regulated by diet. J. Physiol. Lond. 437: 699-708, 1991. Sokal, R. R., and F. J. Rohlf. Biometry. San Francisco, CA: Freeman, 1981. Solberg, D. H., and J. M. Diamond. Comparison of different dietary sugars as inducers of intestinal sugar transporters. Am. J. Physiol. 252 (Gastrointest. Liver Physiol. 15): G574-G584, 1987. Stirling, C. E. High-resolution radioautography of phlorizin-3H in rings of hamster intestine. J. CelZ BioZ. 35: 605-618, 1967. Stirling, C. E., and W. B. Kinter. High-resolution radioautography of galactose-“H in rings of hamster intestine. J. CelZ BioZ. 35: 585-618, 1967. Thomson, A. B. R., and J. M. Dietschy. Derivation of the equations that describe the effects of unstirred water layers on the kinetic parameters of active transport processes in the intestine. J. Theor. Biol. 64: 277-294, 1977. Thomson, A. B. R., M. L. G. Gardner, and G. L. Atkins. Alternate models for shared carriers or a single maturing carrier in hexose uptake into rabbit jejunum in vitro. Biochim. Biophys. Acta 903: 229-240, 1987. Toggenburger, G., M. Kessler, and G. Semenza. Phlorizin as a probe of the small intestinal Na,D-glucose cotransporter: a model. Biochim. Biophys. Acta 688: 557-571, 1982. Turk, E., B. Zabel, S. Mundlos, J. Dyer, and E. M. Wright. Glucose/galactose malabsorption caused by a defect in the Na+/ glucose cotransporter. Nature Lond. 350: 354-356, 1991. Turner, R. J., and A. Moran. Heterogeneity of sodium-dependent D-glucose transport sites along the proximal tubule: evidence from vesicle studies. Am. J. Physiol. 242 (Renal Fluid Electrolyte Physiol. 11): F406-F414, 1982. Turner, R. J., and A. Moran. Further studies of proximal tubular brush border membrane D-glucose transport heterogeneity. J. Membrane BioZ. 70: 37-45, 1982. Usukura, J., G. L. Fain, and D. Bok. 3H-ouabain localization of Na-K ATPase in the epithelium of rabbit ciliary body pars plicata. Invest. OphthaZmoZ. Visual Sci. 29: 100-108, 1988. Walters, J. R. F., and M. M. Weiser. Calcium transport by rat duodenal villus and crypt basolateral membranes. Am. J. Physiol. 252 (Gastrointest. Liver Physiol. 15): GYi’O-G177, 1987. Wright, E. M., and B. E. Peerce. Sodium-dependent conformational changes in the intestinal glucose carrier. Ann. NY Acad. of Sci. 0456: 108-117, 1985.
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diets stimulate brush-border glucose uptake in mouse, rat, and sheep intestine (9, 33, 35). This effect of dietary substrate is a specific one on the maximal velocity ( Vmax) of the glucose transporter, without change in mucosal mass or glucose passive permeability. We previously showed that this change in glucose transporter Vmax arises from proportionate changes in glucose transporter site density, as measured in intact intestinal sleeves by specific binding of phlorizin, a competitive inhibitor of the brush-border glucose transporter (13). In that study, we detected two types of binding sites, differing in their affinity constants for phlorizin (12). Density of the Na+-glucose cotransporter protein, detected by immunoreactivity, also changes in parallel with changes in the transporter Vmax measured in brush-border membrane vesicles (35). These observations beg the question how this substrate-dependent regulation of the glucose transporter relates to the well-known crypt-villus axis of intestinal mucosal organization. Crypt and villus enterocytes differ in structure and function, correlated with continual mitosis in the crypts, enterocyte migration along the villus, and eventual sloughing-off of mature enterocytes at the villus tips. Some of the resulting questions could be answered by studies of transporter distribution along the crypt-villus axis in the steady state. For example, where along the axis is each type of transport site expressed? Do both site types coexist in the same cell or does one type become transformed into the other as enterocytes migrate from crypt to villus? Where along HIGH-CARBOHYDRATE
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the axis does the diet-dependent difference in site density take effect? The present article, the second in a series (see Ref. 16 for the preceding article), examines these questions involving steady-state transporter distribution. We shall use three methods for localizing transporters along the crypt-villus axis in mice on a high-carbohydrate (HC) or no-carbohydrate (NC) diet: phlorizin binding to isolated enterocytes fractionated by the Weiser method, phlorizin binding detected autoradiographically in tissue sections, and immunocytochemistry. The following article (14) examines transporter distribution during transients after a sudden change in dietary carbohydrate level to deduce where the regulatory signal is perceived. MATERIALS Animals
AND METHODS
and Diets
Adult male white Swiss Webster mice were initially maintained on Wayne Lab-Blox chow and were then switched for 2 wk to either the HC or NC diet used in Refs. 14 and 16. We found previously that 2 wk is ample time for glucose transporter activity of mouse intestine to adapt to dietary carbohydrate level (9). During those 2 wk, we recorded initial body weight and then monitored food consumption and body weight 2-3 times per week. As we had found previously (13), food consumption differed slightly between the HC and NC diets (P < 0.025, 2tailed t test), but body weight gain (P > 0.2) and intestinal length (P > 0.1) did not. For HC and NC mice, respectively, daily food consumption was 5.4 t 0.3 (n = 18) and 4.2 t 0.2 g/mouse, final body weight was 43.7 t 1.0 (n = 18) and 41.6 t 1.6 (n = 17) g, and intestinal length was 54.8 & 0.5 (n = 18) and 52.5 t 1.2 (n = 17) cm. Experiment
1: [3H]phlorizin
binding
to isolated
enterocytes
We fractionated enterocytes along the crypt-villus axis by the Weiser procedure already described, with the following minor modifications. Jejunums were pooled from three mice, not two mice. We used 18 mice (6 experiments with 3 mice each) per diet. Following Walters and Weiser (47), we incorporated 5 mM fructose and 5 mM glutamine in the citrate solution, 0.025 mg/ml trypsin inhibitor in the incubation phosphate-buffered solution with EDTA (PBS), and we used 10 consecutive incubation steps lasting 18, 12, IO, 8, 4, 4, 7, IO, 12, and 20 min, respectively (a total of 105 min). The resulting enzyme profiles proved similar to those described previously (16). We pooled the resulting IO cell fractions into five pairs (fractions 1 and 2,3 and 4,5 and 6, 7 and 8, and 9 and IO), respectively, representing cells from the villus tip, upper villus, midvillus, lower villus, and crypt (see Fig. 1 of Ref. 16). Before use, cells were kept in PBS-P solution [PBS modified for phlorizin binding (in mM): 105 NaCl, 2.7 KCl, 7.6 Na,HPO,, 2.1 KH2P04, pH 7.01 plus 50 mM mannitol and 1 mg/ml bovine serum albumin (BSA). To measure phlorizin binding by those isolated enterocyte fractions, we modified the method of Restrepo and Kimmich (32). A lo-p1 cell aliquot containing 80-100 pg cell protein was preincubated for 1 min at 37°C in a 2.2-ml microcentrifuge tube.
1992 the
American
Physiological
Society
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We then added 110 ~1 of PBS-P solution at 37°C containing [“Hlphlorizin, 50 mM glucose or fructose (see below for rationale), and 1 mg/ml BSA. The mixture was vortexed for 5 s and rinsed after 2 s (i.e., total incubation time 7 s) with 2 ml ice-cold PBS-P solution plus 50 mM mannitol and 1 mg/ml BSA, spun down for IO s in a Beckman microfuge at 10,000 g, and the supernatant was discarded. The cell pellet was subjected again to the same rinse, dissolved in 200 ~1 of Protosol (New England Nuclear), and transferred to a scintillation vial for liquid scintillation counting of the [3H]phlorizin. We found that the first rinse of fructose-incubated cells removed 94 t 1% (n = 3) of the initial radioactivity, the second rinse 4.5 t 1.5%, and a third rinse 0.25). These time dependences of specific, nonspecific, and total binding in isolated enterocytes are very similar to those in intact intestinal sleeves, except that the hyperbolic phase of nonspecific and total binding and the initial rise of specific binding are slower in intact sleeves, due -presumably to their thicker unstirred layers (12). A priori, one might wonder whether calculating specific phlorizin binding as the difference between two measured larger numbers (total and nonspecific binding) would be sufficiently accurate. In practice, our values for all six time points were the same within experimental error, which was modest. The coefficient of variation was lowest at 7 s (16%), because shorter incubations were more difficult to time accurately, but longer incubations yielded larger nonspecific binding corrections. Hence, we chose an incubation time of 7 s to minimize nonspecific binding while ensuring completeness of specific binding. l
Delayed removal of cells does not affect specificphlorizin binding. The Weiser method removes villus tip cells first
and crypt cells last. We shall show (Table 1) that villus tip and crypt cells differ in their kinetics of specific phlorizin binding. Does that difference reflect a corresponding difference in native properties of villus tip and crypt cells or is it an artifact of the longer incubation of crypt cells l n 0
total binding nonspecific binding specific binding
Fig. 1. Binding of 0.015 FM phlorizin to isolated mid- and upp ler villus enterocytes, as a function of incubation time (means -c- SE, n = 4). Curves fitted through experimental points by least squares are a straight line for specific binding and a straight line plus a hyperbolic component for nonspecific and total binding.
TRANSPORTERS
during the Weiser cell fractionation procedure? We addressed this question by the same method that we used to address the corresponding question for enzyme activities (16): we decreased mechanical agitation from 90 to 30 Hz, thereby delaying release of villus cells from 30 to 75 min, nearly as long as the normal release time of crypt cells. Nonlinear ligand binding analysis with proportional weighting showed that this delay did not significantly change the dissociation constant (&; 0.260 t 0.049 PM for control cells vs. 0.276 t 0.075 PM for delayed cells, P > 0.8 by 2-tailed t test) nor the site density (23.2 t 3.4 vs. 17.0 t 3.4 pmol/mg protein, P > 0.1) of specific binding sites for phlorizin. Linear regression with simple weighting of Scatchard plot transforms of the data yielded the same conclusion: neither Kd [0.323 t 0.065 vs. 0.422 t 0.132 PM (P > 0.4)] nor site density [29.2 t 4.2 vs. 26.6 t 5.4 pmol/mg protein (P > 0.5)] differed between control and delayed cells. Hence a more than twofold increase in incubation time did not affect phlorizin binding kinetics, and the apparent difference in kinetics between villus tip cells and crypt cells is presumably real. AP activity. The steep decrease of AP activity (Fig. 2) from villus tip to crypt (P < 0.0001 by a 2-way ANOVA), the slightly (on average, 23%) higher AP activity on the NC than HC diet (P < 0.02), and the absolute values of activity were very similar to our previous results for AP activity in mouse jejunum (see Fig. 2, B and C, of Ref. 16). Effect binding.
of crypt-villus
position
on specific phlorizin
In four of our five cell fractions along the cryptvillus axis, specific phlorizin binding to the glucose transporter involved a single site (Table 1), as judged by linear Scatchard plots (Fig. 3) and by the criteria of Akaike (1) and Schwarz (34). The single sites in the three terminal positions (villus tip, upper and midvillus) shared similar Kd values, whereas the single site in the crypt was of much lower affinity (Kd 3-5 times higher) (Table 1). Data for the remaining cell fraction, the lower villus fraction that mixes some crypt and villus cells (16), were best fit by a two-site model, with Kd values of the high-affinity and low-affinity site similar to the Kd value of the single site in terminal positions and in the crypts, respectively. Because of large SEs on binding parameters of the lowaffinity site in the crypts, the difference between its Kd and that of the high-affinity site in terminal positions did not achieve statistical significance in the present study (P > 0.05 by 2-tailed t-test), but it did achieve significance in our subsequent study with larger sample sizes (P < 0.02, Ref. 11). Site density of the high-affinity site decreased twofold from villus tip to midvillus and fivefold from villus tip to lower villus in mice of both diet groups @-tailed t test, P < 0.05 in each case), but that site’s Kd did not change among those four terminal regions (P > 0.1). Effect of diet on specific phlorixin binding. Linear regression of Scatchard transforms and nonlinear regression both yielded the same conclusion: compared with the NC diet, the HC diet increased site density of specific phlorizin binding by 1.8- to 6.3-fold without affecting Kd
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VILLUS
Table 1. Effect of diet and crypt-villus
AND
position
Tip UPPer Mid Lower Site Site CrVpt
Kinetic constants are simple weighting (fitted the latter set is given in lower villus, whose data mice over the experimental two sites, the dissociation site 2 (note large SEs in
binding sites No-Carbohydrate
Diet Density, pmol/mg protein
Kci, PM
1 2
Gl063
TRANSPORTERS
on Kd and density of phlorizin
High-Carbohydrate Villus Segment
CRYPT
Diet
PM
Density, pmol/mg protein
19.223.0 12.5t3.7 10.5k2.0
&,
0.51t0.11 0.47t0.11 0.34&O. 10
37.9t6.3 33.8t5.8 18.4t4.2
0.36t0.07 0.36&O. 14 0.2620.07
0.38t0.18 0.70t0.18 1.4t0.9
4.7t2.6 22.4t3.8 27.0t14.2
0.23t0.19 0.3t39 1.2t0.7
4.1t3.8 68t99 4.3t2.4
given as estimates t SE of the estimate. Phlorizin binding data were analyzed by linear regression of Scatchard plots with lines of Fig. 3) or else by nonlinear regression with proportionate weighting. Both methods yielded very similar parameters; this table. By the criteria of Akaike (1) and Schwarz (34), a l-site model yielded the best fit for all villus segments except were best fitted by a 2-site model (site 1, high-affinity site; site 2, low-affinity site; site 2 nonsaturable in no-carbohydrate range of concentrations). For lower villus of no-carbohydrate mouse, our data sufficed to establish the coexistence of constant (&) and density values for site 1, and the density/& ratio for site 2, but not density and & separately for that case). m 0
1 t i p ---
2 ------___ VILLUS
no carbohydrate high carbohydrate
3 -----
4 _____
0 no carbohydrate 0 high carbohydrate
5 c ry
p t
SEGMENT
activity of alkaline phosphatase in jejunal cells fractionFig. 2. Specific ated along crypt-villus axis, from mice fed a HC or NC diet. Fraction 1, villus tip cells; fraction 5, crypt cells. Each bar gives mean t SE (n = 6). Within a diet, bars sharing same letter do not differ significantly (P > 0.05).
at any position (Table 1). The high-affinity site continued to characterize the villus tip, upper villus, and midvillus, with densities increased 1.9-, Z.7-, and I.&fold, respectively, over values in NC mice (P = 0.005, 0.004, and 0.052 by l-tailed t test). The low-affinity site continued to characterize the crypts, with 6.3-fold increased density (borderline significance, P = 0.06, due to large SEs). Experiment pH]phlorizin
2: Binding
Detected Autoradiographically
As summarized in Fig. 4, two-way ANOVA showed large effects both of diet and of villus position on total binding (P = 0.0002 and 0.003, respectively) and on specific binding (P c 0.0001 and P = 0.006, respectively). In total binding studies, HC mice greatly exceeded NC mice in number of silver grains per villus [251 t 28 (n = 8) vs. 100 t 17 (n = 6) respectively, P < O.OOl]. Because, however, nonspecific binding was low and independent of diet [16 t 2 (n = 8) vs. 21 t 3 (n = 6), P > 0.051, specific binding per villus was also three times higher in HC than in NC mice [234 t 28 (n = 8) vs. 80 t 14 (n = 6) respectively, P < O.OOl]. This factor of three for increased specific binding derived from autoradiography is in good agreement with the factor derived from liquid scintilla-
10
20
I 30
amount of phlorizin bound (pmoles/mg protein) Fig. 3. Effect of dietary carbohydrate on Scatchard plots of [3H]phlorizin binding by isolated enterocytes from villus tip (A) or crypt (B). Each point is mean rt SE (n = 6). Lines are least-squares linear fits of Scatchard-transformed data with simple weighting.
tion counting of everted sleeves of the same mice (HC mice 3.1 times higher than NC mice). Because we used a low phlorizin concentration, most specific binding was to the high-affinity sites located in the terminal three positions of the villus, regardless of diet. Because in addition nonspecific binding was low, total binding was also concentrated in the terminal three positions. Both for total and specific binding, number of silver grains per position was higher in the villus tip and/or upper villus than in the crypts. Each villus position had more grains in HC than in NC mice. Nonspecific
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AND
CRYPT
high carbohydrate
TRANSPORTERS
no carbohydrate TOTAL
abc
BINDING -
rll+l bc
C
1’1
I
Fig. 4. Effect of dietary carbohydrate on binding of 0.005 ,uM [“Hlphlorizin to everted sleeves of mouse jejunum, studied by autoradiography. For purposes of counting grains, each villus was divided into 5 equally long segments (I, villus tip; 5, crypt). Each bar is mean t SE of 6 (NC mice) or 8 (HC mice) villi. Within a panel, bars sharing same letter do not differ significantly (P > 0.05). Note that specific binding in each segment is 2-4 times higher on HC diet than on NC diet.
bc
+l
C
r-h
villus
segment
binding was independent of position in NC-fed mice and varied with position in HC-fed mice. Experiment 3: Immunocytochemical
Detection
of Glucose Transporter
Immunofluorescent staining showed that the antibody against a peptide from the cloned glucose transporter recognized a protein in the brush border of enterocytes of the upper villus (Fig. 5A) but not of the crypts (Fig. 5B). This staining was apparent in both duodenum and jejunum. (Because of limited available quantities of antibody, we did not try to measure the lower quantities of transporter expected in ileum.) There was no staining of control tissues incubated in normal rabbit serum (Fig. 5C) nor of control tissues incubated in peptide-absorbed antiglucose transporter antibody. Control tissues incubated with goat antibody to rat sucrase exhibited staining of the brush border in these experiments, as we had also found in the experiments described in the first companion article of this series (16). DISCUSSION
Effect of Dietary
Carbohydrate
This article yields a set of unequivocal a set of more tentative conclusions.
conclusions, plus
Our unequivocal conclusions begin with our observation that the well-known two- or threefold stimulation of intestinal glucose transport by dietary carbohydrate is paralleled by a comparable increase in number of glucoseprotected phlorizin binding sites. This increase was measured by both methods that we employed: phlorizin binding to isolated cells fractionated by the Weiser method and phlorizin autoradiography of intact tissue sections. This conclusion in itself was not surprising, because we had previously found increased glucose-protectable phlorizin binding to intact intestinal sleeves (13). However, that previous study of intact sleeves could not localize the stimulatory effect along the crypt-villus axis. We now find, by both of the above-mentioned methods, that dietary stimulation of glucose-protectable phlorizin binding is unequivocally demonstrable along the whole terminal portion of the villus: the villus tip, upper villus, and midvillus (Table 1, Figs. 3 and 4). Our more tentative conclusions begin with our observation, also valid for intact sleeves (12, 13), that there appear to be not one but two types of glucose-protectable phlorizin binding sites, distinguished by their binding kinetics. The site with lower phlorizin affinity appears to account for less binding, to reside in the crypts, and also
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VILLUS
AND CRYPT
TRANSPORTERS
G1065
Fig. 5. Localization of glucose transporter in mouse jejunum by light microscopic (original magnification x400) immunofluorescence staining. A: villus tips incubated with anti-Na+-glucose transporter IgG, followed by fluorescein-conjugated anti-rabbit IgG. B: as in A but crypt region instead of villus tips. C: as in A except that normal rabbit serum was substituted as a control for anti-Na+-glucose transporter IgG. Note concentrated staining of brush border in villus tips (A, arrows) but not in crypts (B) nor in control tissue (C).
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VILLUS
AND CRYPT
to be upregulated by dietary carbohydrate as is the villus (higher-affinity) site. These conclusions are more tentative than are the conclusions of the preceding paragraph because of the larger SE values in our measurements of crypt cell binding. The difference in binding constant between crypt and villus cells did not achieve statistical significance in the present article but did in the larger samples reported in the following companion article (14). The effect of diet on site density either did achieve, or narrowly failed to achieve, significance, depending on the experimental method and sample size (this article and Ref. 14). Although, we thus conclude that dietary carbohydrate definitely increases phlorizin binding along the terminal villus and probably along the whole crypt-villus axis, this observation does not prove that the regulatory signal is perceived along the terminal villus or whole axis. In fact, the following (final) companion article of this series will present evidence that this signal is perceived only in the crypts (14). While our study is exclusively concerned with the brush-border glucose transporter, the site density and functional activity of the basolateral glucose transporter are also regulated by substrate levels, specifically, upregulated during hyperglycemia (8). Thus dietary regulation of intestinal glucose absorption involves regulation of transporters at both faces of the enterocyte. The remainder of this discussion section will consider in more detail the following issues: number of brushborder transporters per cell, phlorizin binding constants, the question of site heterogeneity, and the question of glucose transport in crypts. Number
of Transporters
Per Cell
From the number of cells per milligram
protein (-8 x binding sites per milligram protein (Table 1) for mouse intestine, we calculate that there are ~2.8 X lo6 or 1.4 X lo6 glucose transporters per cell in terminal villus segments of HC or NC mice, respectively. These values are similar to our previous estimate of lo6 to lo7 transporters per cell in HC 106: Ref. 16) and the density of phlorizin
Table 2. Kd of phlorizin
Mytilus
californius
Mytilus
edulis
gill
gill
TRANSPORTERS
mice, based on specific phlorizin binding to intact tissue (13). Similar transporter densities have been calculated for woodrat (15), hamster (10, 38), and chick (32) intestines while values up to lo-fold lower were obtained for rabbit (48) and iguana (15) intestines. Glucose transport rates per milligram of tissue vary in the same sequence, being similarly high in mouse, woodrat, hamster, and chicken intestines (15, 24) and up to an order of magnitude lower in rabbit (6) and iguana (24) intestines. Thus, as we discussed previously (15), species differences in glucose transporter density make an important contribution to species differences in intestinal glucose transport rates. Site Heterogeneity
and Distribution
Table 2 summarizes binding constants for phlorizin to glucose transporters of various tissues, studied in various types of preparations. There is a wide range of Kd values for the major binding site, from 0.0005 to 18 PM. Rabbit kidney and pig kidney as well as mouse intestine yield evidence of two binding sites differing in Kd. Values for the major site of mouse intestine are in the lower half of the range, with the value that we previously measured for intact tissue being somewhat below the value that we now find for isolated cells. Phlorizin binds not only to glucose transporters but also to a nucleoside transporter (28), as well as nonspecifically to membrane lipids. However, we infer that our measurements of what we term “specific phlorizin binding” constitute binding to glucose transporters, because we measure only the component of phlorizin binding blocked by glucose. [Measurement of Na+-dependent phlorizin binding yields the same value (12); the nucleoside transporter is not inhibited by glucose (28); and one would not expect binding to lipid to be Na+ dependent and glucose protectable.] Hence our observation of apparently differing glucose-protectable phlorizin binding kinetics in mouse intestinal crypts and villi raises two questions: whether there might also be two distinct intestinal glucose transporters and whether crypts transport some glucose.
to glucose transporters
Intact tissue
0.0005
BBMV
0.006
Isolated cells 0.05 LLC-PK1 pig kidney cells 0.07-0.2 Mouse intestine Intact tissue 0.2-0.5, l-4 Mouse intestine Isolated cells Rabbit kidney BBMV 0.2, 100 Pig kidney BBMV 0.4, 8 Chick intestine Isolated cells 0.9 BBMV 1 Rabbit kidney BBMV 3-5 Rat kidney BBMV 5 Rabbit intestine 6 Rabbit intestine cloned glucose transporter Injected oocytes 8,250O Rabbit kidney BBMV Rabbit intestine BBMV 9 BBMV 18 Guinea pig intestine Two values of & separated by a comma indicate that kinetic analysis revealed 2 binding sites with the failure to correct for nonspecific phlorizin binding led artifactually to much higher & values (e.g., 75 PM BBMV, brush-border membrane vesicles.
S. Wright and A. Pajor, personal communication S. Wright and A. Pajor, personal communication 2 12,13 14; this paper 44 27 32 3 20 42 23 8 23 4 stated & values. We omit studies in which for hamster intestine in the Ref. 10 study).
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VILLUS
AND
CRYPT
Studies of the possible heterogeneity of intestinal glucose transport have yielded conflicting results. On the one hand, evidence in favor of just a single intestinal glucose transporter includes the facts that only a single one has thus far been cloned (23), that a genetic defect in that cloned transporter was observed in a case of congenital glucose/galactose malabsorption in a human infant (43)) and that a recent careful kinetic analysis detected only one transporter in human intestinal brush-border membrane vesicles (BBMVs; 30). On the other hand, evidence pointing to the coexistence of multiple intestinal glucose transporters includes other kinetic analyses (e.g., 18, 19, 23, 25, 29, 41) plus studies of transporter differences in relative affinity for glucose and galactose (6, 37), susceptibility to upregulation by different dietary sugars (37), Na+ activation (19, 29), and temperature sensitivity (4). As deduced from differences in these properties, the multiple transporters differ from each other in their relative activities at different stages of ontogenetic development (6, 19), as well as in their relative activities in duodenum and jejunum and ileum (6, 19, 29, 37). Evidence regarding phlorizin binding and glucose transport in crypt cells is similarly conflicting. On the one hand, villus rather than crypt cells clearly account for most intestinal glucose transport. This was shown by the classic autoradiographic study of Stirling and Kinter (39) of [3H]galactose accumulation and was confirmed by many studies of glucose uptake by BBMVs from cell fractions separated by the Weiser method (e.g., 11, 18, 31). The cloned intestinal glucose transporter appears absent from crypts, as shown by immunocytochemistry (22; the present article) and by distribution of its mRNA (22). We correspondingly find that most glucose-protectable phlorizin binding is in the villi rather than in the crypts. On the other hand, phlorizin binding or glucose transport in crypts appears not to be negligible, as shown by the same BBMV studies just cited and by our phlorizin binding studies with cell fractions separated by the Weiser method. Perhaps the reconciliation of this apparent contradiction is simply that enterocytes along the whole crypt-villus axis do have some phlorizin-sensitive glucose transport sites, but that villus cells have more sites than do crypt cells. Even the classic study of Stirling and Kinter (39) detected some galactose accumulation in crypts. In addition, our phlorizin autoradiographic studies purposely employed very low phlorizin concentrations (0.005 pm) to distinguish specific phlorizin binding to glucose transporters from nonspecific binding. However, those low concentrations were incidentally much more suitable for detecting the high-phlorizin affinity site of villus cells than the putative low-phlorizin affinity site of crypt cells. Stirling (38) used much higher phlorizin concentrations (0.6-60 PM) and thereby detected phlorizin binding autoradiographically along the whole crypt-villus axis. Thus these questions about possible heterogeneity and crypt presence of glucose transport and phlorizin binding remain unresolved. Because glucose transport may drop from villus to crypt as a result of glucose absorption out of the poorly stirred intervillus space (40), it would not be surprising if the crypts transported glucose with high
Gl067
TRANSPORTERS
affinity but low capacity. The two phlorizin binding sites of rabbit kidney do differ in their affinity for glucose (44, 45). If crypts do carry out high-affinity glucose transport, it will remain to be determined whether there really are two unrelated glucose transporters and gene products differing in phlorizin affinity, one expressed in crypt cells and the other in mature villus cells or whether there is only one transporter, whose phlorizin binding and functional state change with microenvironment as enterocytes migrate from crypt to villus. We thank Dean Bok, Nathan Collie, Sharon Sampogna, Gail Strother, Ernest Wright, and Sasan Yasharpour for help. This study was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants GM-14772 and DK-17328 (University of California, Los Angeles, Center for Ulcer Research and Education). Present address of R. P. Ferraris: Dept. of Physiology, Univ. of Medicine and Dentistry of New Jersey, Newark, NJ 07103-2714. Address for reprint requests: J. Diamond, Dept. of Physiology, UCLA Medical Center, Los Angeles, CA 90024-1751. Received
7 October
1991;
accepted
in final
form
13 January
1992.
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34. 35.
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48.
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