Studies of Glycerol Transport Across the Rabbit Brush Border

GASTROENTEROLOGY 66: 378-383, 1974 Copyright © 1974 by The Williams & Wilkins Co.

Vol. 66, No.3

Printed in U.S.A.

STUDIES OF GLYCEROL TRANSPORT ACROSS THE RABBIT BRUSH BORDER ALLEN W. RUBIN, M.D., AND JULIUS J. DEREN, M.D.

Department of Medicine, University of Pennsylvania, and the Philadelphia Veterans Administrotion Hospital, Philadelphia, Pennsylvania

The unidirectional movement of glycerol across rabbit brush border was studied in vitro. The permeability coefficient for glycerol across the ileal brush border decreased from 3.41 x 10- 4 cm ·sec- 1 at 0.05 mM to 0.65 at 5 mM, and remained constant at this level from 5 to 50 mM. Glucose and phlorizin did not inhibit glycerol movement. N -ethylmaleimide inhibited glycerol by more than 55% from a. 0.05 mM solution, but did not affect glycerol uptake from a 50 mM solution. Glycerol movement across the ileal brush border measured from a 0.05 mM solution was 2 times greater than the movement across proximal intestine. By contrast, glycerol movement across proximal and distal intestine was similar when measured from a 50 mM solution. It is concluded that glycerol movement occurs both by diffusion via the aqueous regions of the membrane and by a saturable carrier-mediated mechanism. Studies during the past two decades have demonstrated that dietary triglyceride is hydrolyzed to glycerol, fatty acid, and monoglyceride. 1 The latter two components are quite lipid-soluble, although less so than the parent compound, and are passively absorbed across the brush border2 after solubilization into the aqueous phase by bile salts. 3 Little is known, however, of the site or mechanism of absorption ofthe water-soluble lipolytic product, glycerol. The studies to be described demonstrate that glycerol movement across rabbit brush border occurs by both a diffusive movement via the aqueous regions of the

membrane and by a saturable carriermediated mechanism.

Experimental Procedures

Received September 25, 1972. Accepted September 24, 1973. Address requests for reprints to: Dr. Julius J. Deren, 503 Johnson Pavilion, University of Pennsylvania. School of Medicine, Philadelphia, Pennsylvania 19104. Supported in part by National Institute of Health Research Grant AM-13089, Training Grant AM-5462, and a gift from J. Aron and Company. 378

Technique. The uptake of glycerol was measured using chambers similar to those described by Schultz et al.· Unfasted, white New Zealand rabbits were anesthetized with barbiturates, and the small bowel was excised, rinsed in cold isotonic saline, and opened along the mesenteric border. Sheets of either proximal (area immediately caudad to the ligament of Treitz) or distal (area cephalad to the sacculus rotund us) small intestine were impaled onto the lower section of a Plexiglas chamber with the serosal surface facing a piece of moistened filter paper. The mucosal surfaces were exposed to a compartment via an aperture of 1.13 cm 2 to which 1 to 2 ml of Krebs-Ringer bicarbonate bufferS was added. The entire chamber was placed in a constant temperature apparatus at 37 C and attached to a gas source which allowed for continous stirring of the mucosal solution by humidified 5% CO 2 -95% O 2 • After a 3D-min equilibration period, the bathing solution was replaced with 1.0 ml of fresh buffer containing uniformly labeled HC-glycerol, and tritiated

March 1974

GLYCEROL TRANSPORT ACROSS THE RABBIT BRUSH BORDER

methoxyinulin . At intervals ranging from 15 to 45 sec, the test solution was rapidly removed, and the tissue preparation was rinsed with several milliliters of ice-cold mannitol. The portion of the mucosal strip that had been exposed to the test solution was excised with a steel punch, rinsed briefly in isotonic mannitol, and extracted overnight in 0.1 N nitric acid . Portions of the incubation solutions and the tissue extracts were counted in a liquid scintillation counter at settings appropriate for simultaneous counting of 14C and 3H. The labeled inulin was used to measure the quantity of incubation fluid not removed during the rinsing procedure. Calculations. Results were expressed as permeability coefficients which have the dimensions of centimeters per second. 6 Basically, the permeability coefficient is a flux measurement (amount of movement per unit area per unit time) divided by the concentration of the transported substrate in the test solution. It is to be emphasized that, because of the villous and microvillous projections of the mucosal surface, the precise area across which absorption occurs cannot be readily calculated. The surface area employed in these calculations is merely the surface area of the aperture of the chamber facing the mucosal surface. For compounds which move passively and hence do not show saturation, the permeability coefficient is independent of the concentration and remains constant when the concentration of the permeant molecule is varied. By contrast, compounds which move via a saturable mechanism, the permeability coefficient decreases when the concentration of the permeant molecule approaches a value at which at any given moment most of the carrier sites are occupied. Materials. U-UC-glycerol was purchased from either Amersham/Searle, Arlington Heights, Illinois, or the New England Nuclear Corporation, Boston, Massachusetts. The glycerol obtained from Amersham was a 2: I mixture of glycerol-I-C" and glycerol-2-C 14 , while the glycerol obtained from the New England Nuclear Corporation was prepared from uniformly labeled D-fructose-14C. The radiochemical purity of the compounds was determined by the manufacturer and were stated to be greater than 97 % pure by several chromatographic methods. Both preparations yielded similar results. 3H-methoxyinulin was purchased from the New England Nuclear Corporation. Glycerol was purchased from the J. T. Baker Chemical Company , Phillipsburg, New Jersey, D-

379

glucose from the Mallinckrodt Chemical Works, New York, phlorizin from the Mann Research Laboratories, New York, and N-ethylmaleimide from the Sigma Chemical Company, St. Louis, Missouri. Statistics. Student's t-test for either paired or unpaired groups of unequal size was used when appropriate.

Results Uptake of glycerol across rabbit brush border with time. As shown in figure 1, the rate of uptake of glycerol across the brush border of rabbit intestine remained linear over a 45-sec time interval. Hence, the rate of entry of the L·C label represents the unidirectional flux of glycerol across the brush border and was unaffected by intracellular metabolism or back-diffusion.' In all subsequent experiments, a 30-sec incubation period was employed. Permeability coefficient of the brush border of distal ileum for glycerol at various concentrations (fig. 2). When the glycerol concentration on the bathing medium exposed to the mucosal surface was varied from 0.05 to 50 mM, the permeability coefficient decreased from 3.41 x 10- 4 cm· sec - L at 0.05 mM to 1.34 x 10- 4 at 0.5 mM, and then remained constant at about 0.65 x 10- 4 cm · sec - L from 5 to 50 mM. Effect of various compounds on glycerol transport (table 1). Glucose (10 mM), phlo8.0

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6.0

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5.0

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..."

;§-

time sec .

FIG. 1. Glycerol movement across the ileal brush border with time. Glycerol concentration in the incubation solution was 0.05 mM. Values given are the mean values obtained in three experiments.

380

RUBIN AND DEREN

rizin (0.5 mM), and ethylene glycol (10 mM) failed to alter glycerol uptake from a 0.05 mM solution. N-ethylamleimide (10 mM) inhibited glycerol uptake by more than 55% when glycerol uptake was measured from a 0.05 mM solution, but did not affect glycerol uptake from a 50 mM solution. Comparison of glycerol uptake by the proximal and distal rabbit intestine (table 2). Glycerol uptake by distal intestine measured from a 0.05 mM solution ex4 .0

I

111

I

1.0

(7)

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____

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ceeded by more than 2-fold the uptake observed with proximal intestine. In contrast, glycerol uptake by proximal and distal intestine did not differ when studied at a glycerol concentration of 50 mM. Discussion According to one of the current concepts of membrane transport, polar water-soluble compounds move across lipid-rich biological membranes by one of two mechanisms. 7 If the polar compound is small, movement can occur by simple passive diffusion through the aqueous chanTABLE 2. Comparison of the permeability of the proximal and distal rabbit brush border for glycerol

(55)

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~

10 2

10 '

Glycerol concentration (mM)

Small bowel segment

0.05 0.05

Distal Proximal Distal Proximal

Permeability coefficient

na

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II

10 4

p

em ·sec- l ·l0 - 4

(II) (01111)

____-L____-L____

glycerol concentration

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±

29

2.05 0.96

13 9

0.66 0.65

±

29

±

0.13' 0.14

< 0.001

0.04 0.14

NS'

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50.0 50.0

~M

2. Permeability coefficients of the ileal brush border for glycerol at various concentrations of glycerol in the incubation solution. Values given are mean values ± SEM. FIG .

TABLE

Vol. 66, No . 3

±

a Number of experiments. • Values given are mean values , NS, not significant.

±

SEM.

L Effect of various compounds on glycerol transport across the brush border of rabbit small bowela

Glycerol concen· tration (mM)

Inhibitor

n'

Permeability coefficient

P

em ·see - l·lO- ·

0.05} 0.05

Glucose (10 mM)

0.05 } 0.05

Phlorizin (0.5 mM)

0.05} 0.05

Ethylene glycol (10 mM)

0.05} 0.05

N-ethylmaleimide (10 mM)

50.0 } 50.0

N-ethylmaleimide (10 mM)

8

1.54 ± 0.23'} 1.67 ± 0.21

NS·

8

2.62 ± 0.45 } 2.46 ± 0.22

NS

8

2.90 ± 0.34 } 2.60 ± 0.23

NS

13

3.98 ± 0.37 } 1.72 ± 0.11

< 0.001

5

0.72 ± 0.07 } 0.72 ± 0.04

NS

a Ileal segments were used in this series of experiments. • Number of paired experiments. ,. Values giveh are mean values ± SEM. • NS, not significant.

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GLYCEROL TRANSPORT ACROSS THE RABBIT BRUSH BORDER

nels ("pores") which are thought to interrupt the lipoid membrane. 7 Larger watersoluble compounds are restricted from the pore and can move across the membranous barrier only by interreacting with specific components of the lipoid membrane that provide for specific pathways for the movement of these compounds (i.e., carriermediated).8 The data in this paper demonstrate that the movement of glycerol (mol wt 92) across the rabbit brush border cannot be explained solely on the basis of simple passive diffusion. Rather, this relatively small molecular weight substance appeared to enter the rabbit epithelial cell by two mechanisms. At low concentrations, a substantial fraction of glycerol entry can be best described to be via a carrier-mediated saturable system as evidenced by the decreasing permeability coefficient when the concentration of glycerol in the bathing medium was increased and the inhibition of uptake by 10 mM N-ethylmaleimide. The carrier-mediated mechanism has limited capacity and becomes saturated at concentrations of glycerol greater than 1 mM. Since movement through the pores is by simple passive diffusion, the rate of transfer by this route would not be expected to show saturation kinetics and hence would increase with incre~sing glycerol concentration. We observed a rate of uptake that was linearly related to the concentration in the incubation medium from 1 to 50 mM (constant permeability coefficient), and N-ethylmaleimide failed to inhibit glycerol movement across rabbit brush border when measured from incubating solutions containing 50 mM glycerol. Thus, at higher concentrations, greater than 1 mM, the major route of glycerol entry appeared to be simple passive diffusion with little contribution from the saturated carriermediated process. These studies do not distinguish between movement via the aqueous channel in the membrane itself or transfer between epithelial cells via the tight junctions. 9 Previous studies of glycerol transport were interpreted to indicate that glycerol movement was via simple passive diffusion. 10 However, such studies

381

were performed at glycerol concentrations greater than 5 mM, well above concentrations, at least in the case of the rabbit intestine, that saturate the carriermediated mechanism. In a previously published paper,11 the movement of a series of poly hydric alcohols across the rabbit brush border was studied. Transport of 2, 4, 5, and 6 carbon length polyhydric alcohols failed to show saturation or inhibition by N-ethylmaleimide. Accordingly; it was assumed that movement occurred via simple passive diffusion through the aqueous areas in the membrane. The movement of ethylene glycol was similar in both proximal and distal intestine, which indicated that the total available pore area was similar in both regions of the intestine. Greater restriction to the movement of 4, 5, and 6 carbon poly hydric alcohols across the distal bowel than the proximal intestine was observed, suggesting that the equivalent radius of the pores of the proximal bowel were larger than those of the distal bowel. These observations were in confirmation with conclusions drawn from human studies. 12 The permeability coefficient for glycerol observed in this study at concentrations greater than 1 mM approximated the values that would have been predicted from interpolating data from the previous study (fig. 3). Although movement of glycerol is restricted as compared with ethylene glycol in both proximal and distal intestine, the smaller pores in the distal intestine do not significantly restrict molecules the size of glycerol to any measureable extent more than the larger pores of the proximal intestine. Failure to observe a difference in glycerol movement across proximal and distal brush border in spite of the postulated difference in pore size can be explained by data obtained from artificial porous membranes. Renkin 13 studied diffusion across cellulose membranes and developed a function relating the restriction to diffusion to the radius of the pores and the radius of the permeant molecules. Examination of this function indicates that, given two membranes with different size pores, the difference in the restriction to diffusion

382

RUBIN AND DEREN

1.00

0

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3.5

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3.8

4.2

FIG. 3. Comparison of the permeability coefficient

Vol. 66, No.3

enzyme which is obligatory in the metabolism of glycerol is also present in greater abundance in the distal than in the proximal small bowel. 16 Finally, it is of interest to note that glycerol transport in other cell types also displays characteristics which would indicate that glycerol transport does not occur simply by passive diffusion. 17·20 In fact, one of the earliest examples of facilitated diffusion, a term subsequently coined by Danielli,21 was described by Jacobs et al. 22-24 for glycerol movement across the red blood cell.

(P) for glycerol across rabbit brush border with those

for ethylene glycol, erythritol, ribitol, and mannitol. The closed circles represent mean values obtained with proximal intestine, and the open squares those obtained with distal intestine. The values for ethylene glycol, erythritol, and mannitol were published previously." The estimation of molecular radii is based on viscometric analysis. 25

of smaller molecules would be less than that observed for larger molecules. The carrier mechanism for glycerol is distinct from the glucose-galactose carrier mechanism since neither 10 mM glucose, nor phlorizin (0.5 mM), altered glycerol movement. 14 Of interest is the failure of ethylene glycol to inhibit glycerol movement which stands in contrast to observations in red blood cells. 15 The uptake of glycerol measured at 0.05 mM was greater per cm 2 of distal bowel than proximal bowel. This suggests that, in all likelihood, there is a greater abundance of carrier in the distal small bowel, although the possibility that the carrier mechanism in the distal small bowel may have different kinetic characteristics cannot be excluded. During digestion, an appreciable concentration of glycerol would be achieved in the proximal bowel which would lead to a high rate of diffusive glycerol movement via the aqueous regions of the membrane. As the bolus travels down the intestinal tract, glycerol would be present at a much lower concentration and the higher affinity carrier-mediated mechanism would provide for the absorption of the residual glycerol. Glycerol kinase, an

REFERENCES 1. Isselbacher KJ: Biochemical aspects of fat absorption. Gastroenterology 50:78-82, 1966 2. Hofmann AF, Small DM: Detergent properties of bile salts: correlation with physiological function. Ann Rev Med 18:333-376, 1967 3. Johnston JM, Borgstrom B: The intestinal absorption and metabolism of micellar solutions of lipids. Biochem Biophys Acta 84:412-423, 1964 4. Schultz SG, Curran PF, Chez RA, et al: Alanine and sodium fluxes across mucosal border of rabbit ileum. J Gen Physiol 50:1241-1260, 1967 5. Krebs HA, Henseleit K: Untersuchungen uber die Harnstoffbildung im Tierkorper. Hoppe-Seyler's Z Physiol Chern 210:33-62, 1932 6. Davson H, Danielli JF: The permeability of natural membranes. Second edition. Cambridge, (England), University Press, 1952 7. Solomon AK: Characterization of biological membranes by equivalent pores. J Gen Physiol 51:335'- 364'" 1968 8. Wilbrandt W, Rosenberg T: The concept of carrier transport and its corollaries in pharmacology. Pharmacol Rev 13:109-183, 1961 9. Frizzell RA, Schultz SG: Ionic conductances of extracellular shunt pathway in rabbit ileum. Influence of shunt on transmural sodium transport and electrical potential differences. J Gen Physiol 59:318-346, 1972 10. Howard J, Jackson MJ, Smyth DH: Intracellular hydrolysis of short chain glycerides by rat small intestine in vitro and transfer of glycerol. J Physiol 208:461-471, 1970 11. Ross A, Rubin AW, Deren JJ: Differential permeability of the proximal and distal rabbit small bowel. J Clin Invest 51:2414-2419, 1972 12. Fordtran JS, Rector FC, Ewton MF, et al: Permeability characteristics of the human small intestine. J Clin Invest 44:1935-1944, 1965

March 1974

GLYCEROL TRANSPORT ACROSS THE RABBIT BRUSH BORDER

13. Renkin EM: Filtration, diffusion, and molecular sieving through porous cellulose membranes. J Gen Physiol 38:225-243, 1954 14. Crane RK: Intestinal absorption of sugar. Physiol Rev 40:789-825, 1960 15. Jacobs MH: A case of apparent physiological competition between ethylene glycol and glycerol. Bioi Bull 107:314-315, 1954 16. Haessler HA, Isselbacher KJ: The metabolism of glycerol by intestinal mucosa. Biochem Biophys Acta 73:427-436, 1963 17. Stein WD: Spontaneous and enzyme-induced dimer formation and its role in membrane permeability: II. The mechanism of movement of glycerol across the human erythrocyte membrane. Biochem Biophys Acta 59:47-65, 1962 18. Villegas R, Villegas GM: The endoneurium cells of the squid giant nerve and their permeability to ("C) glycerol. Biochem Biophys Acta 60:202-204, 1962

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19. LeFevre PG: Evidence of active transfer of certain non-electrolytes across the human red cell membrane. J Gen Physiol 31:505-527, 1948 20. Sanno Y, Wilson TH, Lin ECC: Control of permeation to glycerol in cells of Escherichia Coli. Biochem Biophys Res Commun 32:344-349, 1968 21. Danielli JF: Recent Developments in Cell Physiology. Edited by JA Kitching. New York, Academic Press, 1954, p 1-14 22. Jacobs MH: The permeability of the erythrocyte. Ergebn Bioi 7:1-55, 1931 23. Jacobs MH, Corson SA: The influence of minute traces of copper on certain hemolytic processes. Bioi Bull 67:325-326, 1934 24. Jacobs MH, Steward DR: Observations on an oligodynamic action of copper on human erythrocytes. Am J Med Sci 211:246, 1946 25. Schultz SG, Solomon AK: Determination of the effective hydrodynamic radii of small molecules by viscometry. J Gen Physiol 44:1189-1199, 1961