J. Physiol. (1975), 252, pp. 681-700 With 2 text-ftgure8 Printed in Great Britain

681

EFFECTS OF FASTING AND SEMISTARVATION ON THE KINETICS OF ACTIVE AND PASSIVE SUGAR ABSORPTION ACROSS THE SMALL INTESTINE IN VIVO

BY E. S. DEBNAM* AND R. J. LEVIN From the Department of Physiology, University of Sheffield, Sheffield S10 2TN

(Received 25 March 1975) SUMMARY

1. The effects of dietary restriction on the kinetics of absorption in vivo of glucose, galactose and a-methyl glucoside were assessed by electrical and chemical methods in the rat jejunum. 2. The 'apparent Ki', maximum absorption or Vmax (jc-mole/ 1O cm. 15 min) and maximum potential difference (p.d.max) were obtained for the jejunal electrogenic active transfer mechanism from the transfer p.d.s and the chemical absorption data corrected for diffusion using various graphical kinetic plots. 3. Fasting for 3 days greatly decreased the 'apparent K.s', obtained from electrical or chemical data, for all the sugars but had no effect on those for L-valine or L-methionine. Semistarvation caused a less pronounced reduction of the 'apparent Kms' for the sugars. The dietaryinduced change in 'apparent K.' for glucose was also observed in the fasted hamster. One interpretation of these changes is that the affinity of the carriers for sugars increases during dietary restriction; the greater the level of restriction the greater the increase. 4. Fasting and semistarvation caused large reductions in the Vmax. These reductions were correlated with a reduced enterocyte population estimated by changes in enterocyte column size. 5. The reduction in the VmaX for galactose was mainly accounted for by the decrease in enterocyte population. In the case of glucose, other factors such as reduced enterocyte metabolism or changes in the carriers must be involved to explain the discrepancy between the large decrease in Vmax and the enterocyte column size. 6. Fasting and semi-starvation had complex, differential actions on the * Present address: Department of Physiology, Royal Free Hospital School of Medicine, Hunter Street, London, WC1N IBP.

E. S. DEBNAM AND R. J. LEVIN p.d.max for glucose, galactose and x-methyl glucoside. These changes did not correlate with those observed in the Vmax measured chemically. 7. A standard diet obtained from two commercial sources was found to differ greatly in its effect on the electrogenic transfer system for cx-methyl glucoside but had no effect on those for galactose and glucose. 682

INTRODUCTION

There is much confusion concerning the effects of dietary restriction on the intestinal absorption of sugars measured in the rat in vivo. Starvation has been reported to increase (Levinson & Englert, 1972) or decrease (Larralde, 1947; Levin, Newey & Smyth, 1965; Crouzoulon-Bourcart, Crouzoulon & P6res, 1968; Levin, 1970) sugar absorption, while semistarvation has been claimed to increase the absorption of glucose (Kershaw, Neame & Wiseman, 1960) but not that of 3-0-methyl glucose or of high concentrations of glucose (Esposito, 1967). Some of the differences can be attributed to the type of dietary restriction and some to the different techniques and parameters used to assess absorption (Levin, 1969, 1970, 1974). The major criticism of the previous work, however, is the absence of characterization of the active absorption mechanism using a wide range of substrate concentrations, a critical fault in a process known to be concentration dependent (see Dietschy, 1970, for discussion). The assessment of absorption in dietary restriction at specific sugar concentrations results in interpretation of the data correct for these specific concentrations but invalid for others. Moreover, measurements at idiosyncratic concentrations of the solute do not allow any inferences to be made about the mechanisms undertaking the transfer. The present study attempts to rectify some of these inadequacies by applying recently developed chemical and electrical techniques of estimating the kinetics of the active and the passive components of hexose absorption measured in vivo (Debnam & Levin, 1975). The active absorption component for sugars (glucose, galactose and x-methyl glucoside) has been characterized by estimating the operational kinetic parameters of 'apparent Km' and the maximum transfer capacity (Vmax) in fed, fasted and semistarved rats. In addition, some histological and morphological characteristics were studied to correlate the functional measures with the structural changes induced by dietary restriction. Preliminary accounts of this work have been published (Debnam. & Levin, 1971, 1973, 1974; Levin, 1974).

DIETARY RESTRICTION AND SUGAR ABSORPTION 683 METHODS Animals. Albino male rats of the Sheffield strain weighing approximately 250 g were used. A group of male Golden Hamsters (approximate weight 85 g), obtained from Stewarts, Tarvin, Chester, were used for species comparison. Diets. Some experiments were performed on groups of rats fed ad lib. with Diet 86 manufactured by Burnhill, Cleckheaton, while other experiments used groups fed with Diet 86 obtained from Oxoid Ltd, London. Marked differences in some results were obtained using animals fed these different diets. Because of this, in any series of experiments where the absorption of sugar was being tested and compared, the diet was standardized throughout. Animals that were fasted had their food removed for 1, 2 or 3 days before the commencement of the experiment, but were allowed ad lib. access to water throughout the duration of the fast. The semi-starved rats were each given approximately 5 g food per day for 9 days, about one quarter of their normal food intake. Their weighed food was given at 10 a.m. (± 30 min) each day. The rats had unlimited access to water. All animals in the dietary restricted groups were caged individually in suspended wire bottom cages to minimize coprophagy. A final group of rats were deprived of solid food for 3 days but were allowed ad lib. access to isotonic (5-4 %) glucose dissolved in their drinking water. In vivo preparation and measurement of transmural potential difference (p.d.). The method used for circulating the fluids through rat mid-jejunum and recording the electrical activity generated was identical to that described previously (Debnam & Levin, 1975). Two amino-acids, L-methionine and L-valine were also used over the concentration range 2, 4, 8, 16, 32 mm. When the hamster was employed, slight modifications in the technique were necessary. Because the hamster has a shorter intestine, a smaller segment of midjejunum was cannulated, the length being 6-5 cm compared to the 20 cm in the rat. The serial concentrations of glucose circulated were 2, 4, 8, 16, 32 mM. Chemical and radioactive estimations of sugar absorption. These were carried out as described previously (Debnam & Levin, 1975). Intestinal weight, histology and morphology. The small intestine (from the ligament of Treitz to the ileo-caecal junction) was removed from an anaesthetized rat, washed through with 0 9 % NaCl at 370 C and then everted over a glass rod. The length was measured by laying the intestine on damp blotting paper so that it was just extended without applying any tension. The middle 10 cm was cut out, blotted to remove excess fluid on damp filter paper (Whatman no. 50) and weighed. The remainder was weighed after similar blotting. The mucosal-submucosal layer of the 10 cm segment was removed by scraping with the edge of a glass microscope slide exerting as constant a pressure as possible until the serosal muscle layer was reached. The process was carried out under a Nikon dissecting microscope. The separated mucosalsubmucosal scrape (mucosa) and the remainder of the intestinal segment (serosa) were weighed individually. After drying these and the rest of the intestine to a constant weight at 1000 C, the weighing was repeated to obtain the dry weight. Light microscope investigation of rat jejunum. Rats were anaesthetized with i.P. Nembutal and 2 cm sections of non-everted jejunum were isolated, washed through with 0 9 % NaCl at 370 C, and placed in 10% formol saline. The sections were later blocked in paraffin wax, sectioned longitudinally (5-7 jam) and stained with haemotoxylin and eosin. Using a microscope (Zeiss model no. 67577) with a screen projection attachment, villus heights, villus counts and enterocyte column size (counts of the number of enterocytes along one side of the villus) were determined in sections of intestine from fed, fasted and semi-starved rats.

684

E. S. DEBNAM AND R. J. LEVIN

Estimation of kinetic parameters from electrical and chemical absorption data. This was carried out as described previously (Debnam & Levin, 1975). The plot of Lineweaver & Burk (1934) was used to obtain estimates of the kinetic parameters of 'apparent Kn' (mM), Vmax (maximum absorptive capacity expressed as ,s-mole/ 10 cm mid-jejunum 15 min) and p.d.max (the maximum p.d. generated during absorption expressed as mV). The values obtained were compared with those calculated by using four different graphical methods (Woolf, 1932; Hofstee, 1959; Dixon, 1972; Eisenthal & Cornish-Bowden, 1974) and an iterative method for a Wang 700 desk computer (Wang programme no. 3504). Chemical. Glucose was obtained from May & Baker Ltd, a-methyl glucoside, L-valine and L-methionine from B.D.H. Ltd and galactose from Mann Research Labs. [14C]a-methyl glucoside was purchased from The Radiochemical Centre and phlorrhidzin from Phase Separation Ltd, Flintshire.

RESULTS

Kinetic parameters of sugar absorption in vivo obtained from electrical measurements in fed, fasted and semistarved animals. Apparent Km. The 'apparent Kms' measured in vivo for glucose, galactose and az-methyl glucoside are listed in Table 1. It is clear that in the rat, starvation dramatically lowers the 'apparent Kin' for the three sugars. The 25 and 26% decrease for glucose and galactose respectively after 9 days of semi-starvation, although significant, was more modest. The reduction in 'apparent Kin' for glucose absorption in hamster small intestine following a 3-day fast indicates that the decreases observed in the rat were not species specific. Fasting did not induce a significant change in the 'apparent Kin' in vivo in the rat for either L-valine or L-methionine, two actively transferred, essential amino-acids. P.d.m.. The values obtained for the maximum transfer p.d. generated in vivo for glucose, galactose and a-methyl glucoside absorption are shown in Table 2. Fasting for 3 days led to significant decreases in the p.d.max for glucose and a-methyl glucoside in the rat, but not for galactose. No significant changes were recorded for either L-valine or L-methionine absorption in the rat or for glucose absorption across hamster small intestine. The effect of semistarvation on the p.d.max in the rat was complex. It produced an insignificant increase in p.d.max for glucose but a highly significant.increase in that for galactose absorption (P < 0-01). Kinetic parameters of sugar absorption in vivo obtained from chemical measurements in fed, fasted and semistarved rats Apparent Km. The method of correcting chemically measured absorption data in vivo for the 'diffusive' (phlorrhidzin-insensitive, non-electrogenic) component of absorption (Debnam & Levin, 1975) was used to assess the active absorption component of sugar absorption in fed, fasted and semistarved rat jejunum. In the presence of 5 x 10-4 M phlorrhidzin, fasting had

DIETARY RESTRICTION AND SUGAR ABSORPTION 685 no significant effect on the 'passive' absorption of glucose, galactose and c-methyl glucoside compared to the fed condition (Fig. 1). Chemical absorption curves corrected for the 'diffusion' component exhibited saturation kinetics in fed, fasted and semi-starved conditions. The 'apparent Kms' calculated from these absorption curves for glucose, galactose and TABLE 1. The 'apparent Kin' for the jejunal electrogenic active transfer mechanism estimated by Lineweaver-Burk analysis of the transfer p.d.s generated during the absorption in vivo of glucose, galactose and a-methyl glucoside. The animal groups used were control rats fed Oxoid Diet 86 (fed), rats starved for 3 days (fasted 3 days), rats fed 5 g Oxoid Diet 86 for 9 days (semi-starved), fed hamsters and hamsters starved for 3 days. The results are given as the mean + S.E. of the mean. The number in brackets represents the number of animals used Apparent Km (mMr)

Fasted (3 day) 7*9 ± 0 5 (16)

Semistarved 13-2 + 0 4 (7)

Species Rat

Fed 17-9 ± 04 (20)

Rat

P < 0.001 P < 0*001 ± 23-0+2-0 (8) 31-1 1'2 (15) 14-7±0-8 (13)

a-methyl glucoside

Rat

P < 0-001 P < 0-001 37-7 ±28 (7) 14X0 + 1X6 (7)

L-valine

Rat

P < 0*001 5 4+0 3 (7) 6-2+0-3 (6)

L-methionine Rat

P > 0*05 < 01 4-2±+ 0 5 (5) 41 +0-2 (5)

Glucose Galactose

Glucose

Hamster

P > 0-8 < 0*9 6-8 ± 0 3 (5) 10-9 ± 06 (5)

P < 0.001

c-methyl glucoside are shown in Table 3. Starvation caused large decreases in 'apparent Ki' for all three sugars, but semistarvation caused less profound reductions. The value for glucose in semistarved intestine was significantly different from both fed intestine (P < 0.005) and fasted intestine (P < 002). The reduced, 'apparent Ki' for galactose in the semistarved rat was not significantly different from the value found in fed intestine (P > 0-2 < 0-3), but it was significantly higher than that of fasted intestine (P < 0.005). Good agreement was observed between ' apparent Kms' obtained from electrical measurements (Table 1), and the chemical values (after correction) listed in Table 3.

E. S. DEBNAM AND R. J. LEVIN Vman. The corrected maximum absorptive capacity of rat jejunum was reduced significantly by 39 % for galactose and 51% for glucose following a 3 day fast, and by 36% for galactose and 46% for glucose after 9 days of semistarvation (Table 3). In both fed and fasted conditions the corrected Vmax values ranked in the order galactose > glucose > a-methyl glucoside. 686

TABLE 2. The 'p.d...' for the jejunal electrogenic active transfer mechanism estimated by Lineweaver-Burk analysis of the transfer p.d.s generated during the absorption in vivo of glucose, galactose and a-methyl glucoside. The animal groups used were control rats fed Oxoid Diet 86 (fed), rats starved for 3 days (fasted 3 days), rats fed 5 g Oxoid Diet 86 for 9 days (semistarved), fed hamsters and hamsters starved for 3 days. The results are given as the mean + s.E. of the mean. The number in brackets represents the number of animals used

PAd. (mV) Species Glucose

Galactose

Rat Rat

Fed

10*3 ±04 (20)

Fasted (3 day) 8-6 + 0 5 (16)

P > 0.01 < 0-02 P = 0*02 9.5 ± 07 (15) 9-4 + 0*4 (13) 12*5 ± 07 (8) P = 0*9 P < 0.001 11*9 +07 (7) 7 0 +06 (7)

a-methyl glucoside

Rat

L-valine

Rat

P < 0*001 5-1 +03 (6) 4-8+0*4 (7)

L-methionine Rat

P= 0*6 3*1 0*2 (5) 30 +0*2 (5)

Glucose

P= 09 7 0+ 0 4 (5) 7-3+ 0*5 (5)

Hamster

Semistarved

10*5 + 0*3 (7)

-

P = 0*9

Comparison of estimates of kinetic parameters obtained from various graphical transformations. The 'apparent Ki' and Vmax derived by five graphical methods and an iterative computer method are shown in Tables 4 and 5. It is clear that over the range of luminal concentrations of sugar employed, all the methods used to estimate chemically corrected and electrical 'apparent Kms', corrected Vmax and p.d.max produced qualitatively similar patterns for fed, fasted and semistarved intestine. While there was excellent agreement between electrical and chemical values of

DIETARY RESTRICTION AND SUGAR ABSORPTION 687 ' apparent K,, ' for the absorption of glucose and galactose in fed and semistarved conditions, in the case of the fasted 'apparent Kms', those obtained by the electrical technique were lower than those obtained from chemical measurements. This feature was not previously observed when only Lineweaver-Burk analysis was used (Tables 1 and 3). 100

l(8) I(6)

C

E us

17VE 0

0I:k 50 E

0

(5) 11

16

32 Sugar concn. (mM)

64

Fig. 1. The absolute absorption (#s-mole/10 cm. 15 min) of glucose, galactose and ac-methyl glucoside from the mid-jejunum at various luminal concentrations in the presence of 5 x 10-4 M phlorrhidzin in fed and 3 dayfasted rats. The results are given as the mean + S.E. of the mean where appropriate. The numbers in brackets represents the number of rats used. The symbols represent, for glucose, absorption in fed (@) and fasted (0) rats; for galactose, absorption in fed (A) and fasted (A) rats and for amethyl glucoside, absorption in fed (a) and fasted (El) rats. To avoid confusing overlapping of the data the results have been slightly separated horizontally at each concentration of sugar used.

The time dependence of the decrease in 'apparent Kmi' caused by fasting for 3 days. The 'apparent Km' for the electrogenic absorption of glucose, galactose and a-methyl glucoside after fasting for 1, 2 or 3 days are shown in Table 6. In the case of glucose, the decrease occurred 24-48 hr after removal of food from the animals and no further reduction was observed after 72 hr. In the case of galactose and a-methy] glucoside the decreases

E. S. DEBNAM AND R. J. LEVIN

688

TABLE: 3. The 'apparent Kin' and '4... of the jejunal electrogenic transfer mechanism for glucose, galactose and cz-methyl glucoside estimated by Lineweaver-Burk analysis from the chemical sugar absorption after correction for diffusion (see Methods section). The animal groups used were control rats fed Oxoid Diet 86 (fed), rats starved for 3 days (fasted 3 days) and rats fed 5 g Oxoid Diet 86 for 9 days (semi-starved). The results are given as the mean + s.E. of the mean. The number in brackets represents the number of animals used

Apparent Km. (mm) Fasted Glucose Galactose

a-methyl glucoside

(3 days) 10-2 ± 0-4 (12) 16-8 ± 1-1 (10) 11-5 ±0-8 (6)

Fed 22-6+± 1-3 (10) 32-4+ 2-3 (7) 31-2 + 1-8 (7)

Semistarved 14-6+ 1-5 (6)

28-0+±2-7 (7)

..(/s-mole/10 cm. 15 min) Glucose Galactose

az-methyl glucoside

85-8+ 4-1 (10) 113-0+ 11-0 (7) 48-8 ± 3-9 (7)

42-3± 3-5 (12) 68-8 ±3-4 (10) 27-0 ± 3-0 (6)

46-3 ±6-7 (6) 71-8 ±5-7 (7)

TABLE 4. The mean 'apparent Kin', p~.,,, and Vnax of the jejunal electrogenic active transfer mechanism for glucose estimated from transfer p.d.s (upper columns) or from the sugar absorption (corrected for diffusion) measured chemically (lower columns) using various techniques of obtaining the kinetic parameters. The parameters were obtained by using the mean values of either the transfer p.d.s or the corrected chemical absorption data calculated from the various results in fed, fasted and semi-starved rats. The group sizes from which the means were obtained are the same as those given in Tables 1, 2 and 3. The asterisked parameters calculated from the Eisenthal & Cornish-Bowden (1974) parameter-space plot are median values and not means like the rest of the Table

Fasted

Fed

Km Lineweaver & Burk

18-2

(1934) Woolf (1932) Hofstee (1959) Wang Dixon (1972)

17-0 15-3 19-4 16-0

PRd.max

Semistarved

Pd.da

P.d *max 10-0

Km 8-2

8-3

13-3

10-7

10-0 9-5

7-6 7-5

7-5

12-2 11-6 12-2 7-6 11.0*

10-2

10-3 10-8

8-4 8-2 8-0 7-6

8.9*

4-0

6.6*

7.5*

Km

10-0 10-2 9-5

9.7*

Eisenthal & CornishBowden (1974)

14.2*

Km

TM1

Km

Lineweaver & Burk

20-0

77-0

11-8

42-1

15-9

47-0

17-2 18-2 23-0 14-6

72-0 71-0 93-5 69-0

10-4 10-5 7-2

40-0 43-4 42-0 47-0

13-6 14-3 12-8 13-0

41-8 41-0 39-9 46-0

12.0*

40.5*

14.0*

36.6*

(1934) Woolf (1932) Hofstee (1959)

Wang Dixon (1972) Eisenthal & CornishBowden (1974)

17.8*

73.0*

11-1

IVnAz

Km

VMnaX

DIETARY RESTRICTION AND SUGAR ABSORPTION 689 TABLE 5. The mean 'apparent Kin', p.d.m.. and Vmaz of the jejunal electrogenic active transfer mechanism for galactose estimated from transfer p.d.s (upper columns) or from the sugar absorption (corrected for diffusion) measured chemically (lower columns) using various techniques of obtaining the kinetic parameters. The parameters were obtained by using the mean values of either the transfer p.d.s or the corrected chemical absorption data calculated from the various results in fed, fasted and semistarved rats. The group sizes from which the means were obtained are the same as those given in Tables 1, 2 and 3. The asterisked parameters calculated from the Eisenthal & Cornish-Bowden (1974) parameter-space plot are median values and not means like the rest of the Table Semistarved Fasted Fed

Km Lineweaver & Burk (1934) Woolf (1932) Hofstee (1959) Wang Dixon (1972) Eisenthal & CornishBowden (1974)

Km

Pd.max

Km

P.d.max

29-5

93

13-4

9-1

23-5

13-3

30-0 30-5 35-2 28-0

9-7 9-8 10-2 12-0

13-5 12-2 12-2 12-0

8-7 8-5 8-6 8-7 7.9*

22-8 20-2 22-5 22-0 21.6*

13-4 12-8 13-1 13-0 12.3*

26.4*

8.6*

Km

VMa

Km

max

105

19-0

77-0

28-6

62-0

29-0 30-0 33-5

103-5 108-0 114-4 100-0

21-0 15-8 16-9 12-8

77-2 65-4 65-1 64-0

24-0 30-0 27-6 18-8

14.2*

61.0*

22.4*

62-4 72-0 69-0 72-0 55.5*

32-0 25.0*

Im.

10.6*

33-0

Km Lineweaver & Burk (1934) Woolf (1932) Hofstee (1959) Wang Dixon (1972) Eisenthal & CornishBowden (1974)

P.d max

97.0*

TABLE 6. The 'apparent Kin', obtained from electrical methods, for the jejunal electrogenic transfer of glucose, galactose and a-methyl glucoside after 1, 2 and 3 days of starvation. The results are given as the mean+S.E. of the mean with number of animals used in brackets. All animals were maintained from weaning on Burnhill Diet 86 'Apparent Kn' (mM) Fed

1 day fasted 15-0 ± 1-9 (10)

2 day fasted 8-2 + 0-4 (6)

3 day fasted 7-1 +0-4 (7)

Glucose

17-3 + 0-5 (11)

Galactose

P < 0-001 P < 0-1 P > 0-2 15-1 + 1-5 (6) 13-5 1-1 (5) 13-9 31-2 + 1-9 (7)

a-methyl

glucoside

1-4 (7)

P > 0-4 P > 0-8 < 0-9 P < 0-001 8-4 ± 0-4 (6) 6-9 + 0-7 (6) 17-8 ± 0-9 (8) 10-0 ± 0-8 (6) k P 005 001). The progressive reduction shown by x-methyl glucoside was not observed with glucose or galactose.

Effect of dietary restriction on unstirred layer thickness To determine the importance of the thickness of unstirred layers in influencing the evaluation of kinetic parameters in the various dietary states, we utilized the techniques described by Dainty & House (1966) and Diamond (1966). We measured the half-time (t1) for generation of an osmotically induced p.d. brought about by 64 mM mannitol in the different dietary conditions. The values for ti in fed, fasted and semistarved intestine were 1-20 + 0 03 (31), 1'19 + 0 04 (30) and 1P23 + 0037 (30) min respectively. These correspond to unstirred layer thickness of 416, 414 and 421 jam for the fed, fasted and semi-starved intestine, respectively.

Effect of the type of the commercial feeding Diet 86 on dietary-induced changes of sugar absorption kinetics measured in vivo electrically A comparison of 'apparent K.' and p.d.mmax values obtained from electrical measurements when rats had been maintained from weaning on Diet 86 manufactured by either Burnhill Ltd or Oxoid Ltd is shown in Table 7. It can be seen clearly that the two diets produced no difference in the 'apparent Kms' and p.d.maxs for either glucose or galactose in the three dietary conditions used (fed ad lib., 3 day-fasted, and 3 day-fasted allowed ad lib. isotonic glucose throughout the fast). While there was a reduction of the 'apparent Kms' during the 3 day fast, the 'apparent Ki' for glucose was maintained at the fed level if alimentation with isotonic glucose was allowed (24.4 g drunk per rat over the 3 days). The 'apparent Ki' for galactose absorption was, however, not maintained by glucose feeding. In the case of ac-methyl glucoside absorption, the 'apparent Kin' obtained in rats fed on Burnhill Diet 86 was much lower than that measured in rats fed the Oxoid Diet 86. This difference occurred in both fed and fasted conditions. Surprisingly, when animals were fed isotonic glucose during the three-day fast, the 'apparent K.' for a-methyl glucoside absorption was maintained at the fed level in the Oxoid-fed rats, but remained at the fasting level in rats fed the Burnhill Diet. Significant differences were also obtained for the p.d.max under the same dietary conditions. The p.d.maxs for glucose and galactose absorption were identical for each dietary state when the rats were fed either Diet 86, but those for a-methyl glucoside were reduced in rats fed the Burnhill Diet.

DIETARY RESTRICTION AND SUGAR ABSORPTION 691 TABLE 7. The 'apparent Ki' and p~d.m.. of the jejunal electrogenic active transfer mechanism for glucose, galactose and a-methyl glucoside estimated by LineweaverBurk analysis from the transfer p.d.s generated during the absorption of these sugars in vivo. The animals used were controls fed either Oxoid Diet 86 or Burnhill Diet 86 from weaning, similar rats fasted for 3 days and those fasted for 3 days but allowed ad lib. access to isotonic glucose (5-4 %) in their drinking water bottles throughout the fast. The results are given as the mean + s.E. of the mean with the number of animals used in brackets Apparent Km (mM) Fed Oxoid Diet 86 Burnhill Diet 86 Fasted Oxoid Diet 86 Burnhill Diet 86 Fasted + 5-4 % glucose Oxoid Diet 86 Burnhill Diet 86

Fed Oxoid Diet 86 Burnhill Diet 86 Fasted Oxoid Diet 86 Burnhill Diet 86 Fasted + 5-4 % glucose Oxoid Diet 86 Burnhill Diet 86

Glucose 17-9 ± 0-4 (20) 17-3±0-5 (11)

Galactose 31-1 + 1-2 (15) 31-2 + 1-9 (7)

a-methyl glucoside 37.7 ± 2-8 (7) 17-8 ± 0-9 (8)

7-9 ± 0-5 (16) 7-1 ± 0-4 (7)

14-7 ± 0-8 (13) 13-9 ± 1-4 (7)

14-0± 1-6 (7) 6-9± 0-7 (6)

18-9±1-1 (12) 16-1 ± 0-9 (6)

18-0± 1-2 (14) 16-5 + 1-5 (8)

28-3 ± 2-0 (7) 7-3 ± 0-6 (6)

PA.Max 10-3 ± 0-4 (20) 10-5±0-5 (11)

9-5 + 0-7 (15) 8-5 ± 1-2 (7)

11-9 ± 0-7 (7) 7-6 ± 0-2 (8)

8-6 ± 0-5 (16) 7-8 ± 0-5 (10)

9-4 ± 0-4 (13) 9-2 ± 0-6 (6)

7-0 ± 0-6 (7) 5-6±0-3 (6)

9-2±0-3 (12) 8-9 ± 0-5 (7)

10-8±0-7 (14) 9-8 + 0-6 (8)

10-0 ± 0-8 (7) 7-8 ± 0-4 (6)

Drinking isotonic glucose during the fast maintained the p.d.max for the glucoside at the level of the fed rats in both Burnhill and Oxoid-fed animals. Weight of normal and dietary-restricted rat whole intestine. The body weight, small intestinal (combined jejunum and ileum) wet and dry weights in fed, fasted and semistarved conditions are shown in Table 8. In comparison to fed animals, fasting for 3 days resulted in a 15 % decrease in body weight (P < 0-001) compared to the 18% (P < 0-001) decrease following 9 days of semi-starvation. After fasting for 3 days, the whole intestinal wet and dry weights fell by 32% (P < 0-001) and by 34 % (P < 0-05) respectively, while semistarvation caused reductions of 36 % (P < 0-001) and 40 % (P < 0-01) respectively. Total small intestinal length was reduced significantly by 11-6 % after fasting (P < 0-01) and by 9-8 % following semistarvation (P < 0-005).

692

E. S. DEBNAM AND B. J. LEVIN

TABLE 8. Effect of fasting and semistarvation on the rat body weight, combined jejunum and ileum wet and dry weights and combined jejunum and ileum length. The results are the mean + S.E. of the mean with the number of animals used shown in brackets Fed Fasted Semistarved Initial animal 247-4 4 248-5 ± 5-3 (7) 249-6±5-1 (7) ± +9 (13) body weight (g) Final animal 251-9 + 3-5 (13) 211-4±2-1 (7) 205-0±61 (7) body weight (g) 4-04 + 0-08 (7) Jejunum-ileum 4-31 + 0-19 (7) 6-34+0-21 (13) wet weight (g) Jejunum-ileum 1-38 + 0-19 (13) 0-91 ± 0-05 (7) 0-82 + 0-02 (7) dry weight (g) Jejunum-ileum 68-3 ± 1-0 (13) 60-4 ± 2-5 (7) 61-6 ± 1-7 (7) length (cm) TABLE 9. Various parameters of intestinal morphology for 10 cm of the mid-jejunum from fed (Oxoid Diet 86), 3-day fasted (fasted) and 9-day semistarved (semistarved) rats. Intestinal wet weight (unscraped) and dry weight (unscraped) refers to the whole intact 10 cm of jejunum. The wet and dry weight of mucosa refers to the weight of the mucosa and submucosa scraped from the intestine, leaving the wet and dry serosa behind. The villus height and enterocyte column size were obtained from histological preparations of the jejunum. The results are given as the mean+ s.E. of the mean. The number of animals used is given in round brackets except in the case of the villus height and enterocyte column size. In these cases the number in brackets represents the number of villi measured from three rats from each group. The % reduction of each measurement from the fed control group is given in the square brackets. All the decreases were statistically significant except those marked with an asterisk Fed Fasted Semistarved Intestinal 750-5 + 33-9 (11) 550-0+ 15-4 (7) 479-9 ± 22-7 (7) wet weight (mg) [-27%] [-36%] (unscraped) 120-7 ± 4-3 (7) Intestinal 178-6+ 11-4 (11) 102-9 ± 6-3 (7) dry weight (mg) [-33 %] [-42%] (unscraped) 444-4 + 40-3 (11) 294-1 ± 9-2 (7) Wet weight of 254-1 ± 8-1 (7) mucosa (mg) [-34%] [-43%] 131-7+ 10-0 (11) 67-5 ± 3-5 (7) Dry weight of 79-4+ 1-3 (7) mucosa (mg) [-40%] [-49%] 177-7 + 16-8 (7) 207-5+ 14-9 (11) Wet weight of 154-3±11-1 (7) serosa (mg) *[ -14 %/] [-25 %] 47-3+3-6 (11) Dry weight of 41-1±4-1 (7) 35-4+3-1 (7) serosa (mg) [-25%] *[ -13 %/] Villus height 560+5-5 (118) 448±3-1 (138) 438 ± 3-2 (129)

(#m) Enterocyte column size

95-5 + 1-7 (48)

[-20%]

[-27 %]

72-0+ 1-0 (48)

64-0± 1-0 (48)

[-25%]

[-33 %]

DIETARY RESTRICTION AND SUGAR ABSORPTION 693 Weights of normal and dietary-restricted rat mid-jejunum. The weight results in Table 9 refer to those obtained from the middle 10 em of the combined jejunum and ileum. The percentage reductions in jejunal wet and dry weights (unscraped intestine) following dietary restriction were similar to those of the combined jejunum and ileum listed in Table 8. Thus in fasted rats, jejunal wet and dry weights were decreased by 27 and 33 % respectively while after semi-starvation the jejunal wet and dry weight loss was 36 and 42 % respectively. Following 3-day fasting and 9-day semistarvation, serosal weights showed a much lower decrease than unscraped intestine (compared to the fed values), the reduction after fasting and semistarvation being 14 and 25 % respectively. An interesting feature of the results for serosal dry weights is the finding that there is a much greater and significant decrease in the value for semistarved intestine (P < 0-02 > 0 01) than that of fasted intestine (P < 0 3 > 0Q2) when both are compared to the fed value. Effect of dietary restriction on intestinal mucosal structure. The estimates of villus height and enterocyte column size in fed, 3-day fasted and semistarved rat jejuna are listed in Table 9. Compared to fed intestine, fasting for 3 days resulted in a 20 % decrease in villus height (P < 0 001) and a 25 % decrease in enterocyte column size (P < 0-001). Semistarvation caused a 22 % reduction in villus height (P < 0 001), and a 33 % decrease in enterocyte column size (P < 0 001). DISCUSSION

Influence of dietary restriction on kinetic parameters of active absorption 'Apparent Km8. ' The severity of the dietary restriction influences the changes observed in the 'apparent Km.s' for glucose, galactose and c-methyl glucoside active absorption. Fasting caused large decreases for all the sugars while semistarvation caused less profound decreases for glucose and galactose. The changes are not artifactually caused by any particular graphical technique of handling the absorption data nor by dietary induced non-specific alterations of the intestinal milieu (e.g. blood flow, microflora, bile and pancreatic secretions, etc.) as the Kms for two amino acids remained unchanged and the behaviour of the Kms for the sugars over the 3-day fast was different (Table 4). Alterations in the unstirred layers that could create artifacts in 'apparent Kms' (Winne, 1973; Wilson & Dietschy, 1974) can also be excluded as there was no significant difference in their thickness in fasting or semistarvation. Thus, as far as interpretation of operational kinetic parameters allows inferences to be made about transfer mechanisms, the decreases in 'apparent Kms' in dietary restriction probably represent an enhanced

E. S. DEBNAM AND R. J. LEVIN affinity of the 'carrier' part of the active transfer mechanism for sugars (Schultz & Zalusky, 1964; Crane, 1968). The fact that decreases in the 'apparent Kin' also occur for jejunal glucose transfer in fasted hamster indicates that the dietary-induced adaptations are not species specific. Correlation of changes in Van with changes in morphology. Dietary restriction induces dramatic decreases in the Vmax for sugar absorption. As the Vmax was measured as It-mole of sugar absorbed per 10 cm of midjejunum per 15 min the decreases could be due to (1) a reduced number of enterocytes per unit length of jejunum and/or (2) a reduced ability of the enterocytes to transfer sugars either because the cellular metabolism linked to the transfer mechanisms is altered or there is a reduced number of 'carriers'. We have tried -to assess the importance of such factors by using three indices of enterocyte number in fed, 3-day fasted and semistarved rats and compared the percentage reductions of these indices with the percentage decreases in the Vmaxs ,using the fed intestine as the basis for comparison. Fasting caused a 40 °% decrease in mucosal dry weight but only a 20 % decrease in villus height and a 25 % decrease in enterocyte column size. Semistarvation caused a 49 % fall in mucosal dry weight concomitant with a 22 % decrease in villus height and a 33 % decrease in enterocyte column size. The much larger decrease in mucosal dry weight compared to enterocyte column size and villus height is probably because the mucosal dry weight includes transferring and non-transferring enterocytes and other tissues present in the mucosa and submucosa. Many of these are also affected by the decreased nutrition, e.g. lymphoid tissue is very sensitive to starvation (Shields, 1968). Mucosal dry weight is thus an inaccurate index of the decrease of enterocytes during dietary restriction. Villus height and enterocyte column size appear more specific indices yet even these can be criticized as they are one-dimensional measures of the complex, three-dimensional shape of the rat villus (Clarke, 1973, 1974). A number of studies have found good correlations, however, between villus height or column size and the absorptive capacity of the intestine in various experimental conditions (Menge, Bloch, Schauml6ffel & Riecken, 1970; Roche, Bognel, Bognel & Bernier, 1970; Riecken & Martini, 1973; Riecken, Menge, Bloch, Lorenz-Meyer, Wram & Ihloff, 1974). We therefore chose the enterocyte column size as our primary index of changes in the enterocyte population during fasting and semistarvation despite the fact that it assumes that all enterocytes possess identical transfer activity. This fault, however, is common to all indices. The relationship between the decreases in Vmax and in enterocyte column size are shown in Fig. 2. It is clear that the percentage decreases in enterocyte column size do not match exactly the percentage reductions in Vmax. The discrepancy was much greater for glucose than for galactose. In either dietary-restricted state the 694

DIETARY RESTRICTION AND SUGAR ABSORPTION 695 decreases in enterocyte column size account for approximately half of the decrease in the Vmax for glucose. In the case of galactose, the decrease in column size accounts for nearly all the reduction in Vmax in semi-starvation but not in fasting. Bearing in mind the assumptions and experimental errors involved, the observed reductions in enterocyte column size could 50 r

1+

+ + +

+

+

+

40F

+.

+ ++

I

+.

301-

4_

N

+ ++

E

1 40

+ + 4 + ++ + + +

A, F+

+x

+ ++ ++ ++ .4 + +44

++

+

Sc

+++ 4.44

+ + + ++

+++4+ + 4 + +4 4

13

W

LI

+ ++ +4+4. + + 4

U

+ ++

0

2

+ +

0u 410

20 F

E

41 +4+

120

C c C

._

(U 0

la

10

-

1

0"o

4.4.4 + +1+

U

b

****i

.&

.1

-

L

Fasted Semi-starved in Fig. 2. The mean percentage decreases the V.. for glucose (cross-filled bars) and for galactose absorption (dotted-filled bars) from 3-day fasted and semi-starved jejuna compared to the values of fed controls. The open bars represent the mean % decreases of the enterocyte column size in these fasted and semistarved intestines. The vertical arrows represent the % decrease in the V. for glucose (continuous line) and for galactose (interrupted line) not accounted for by the percentage reduction in the number of enterocytes.

account for the reductions in the galactose Vmax. With glucose, however, the much larger reductions in Vmax compared to those of the column size suggest that fasting and semi-starvation also have a direct action on the transferring capacity of enterocytes. This may be due to alterations in the number of carriers or of the metabolic energy supply to the transfer mechanisms. The metabolism of glucose by the jejunum is reduced by fasting (Levin et al. 1965) and glycolysis and the enzymes metabolizing

E. S. DEBNAM AND B. J. LEVIN glucose are decreased (Srivastava & Hubscher, 1966; Srivastava, Shakespeare & Hubscher, 1968). Studies in our laboratory have indicated that enterocytes possess multiple sugar carriers (Debnam & Levin, 1971, 1974; Levin & Syme, 1971) a concept that has been independently confirmed (Honegger & Semenza, 1973; Honegger & Gershon, 1974). It is possible that the greater depression of glucose absorption may be due to selective changes in the carrier sites for glucose. 696

Influence of dietary restriction on 'apparent K.' and p.d.max obtained from electrical data Different methods of calculating the 'apparent Kin' from electrical and chemical data showed similar values and qualitative patterns for fed and semi-starved rats (Tables 4, 5). However, the 'apparent K.s' obtained from electrical data in fasting rats were regularly lower than those obtained from chemical data. At the present we are unable to conclude whether this bias indicates a real difference between the 'apparent Kms' after complete restriction of food intake. Fasting and semistarvation have different, complex effects on the p.d.max for the three sugars (Table 2) which do not appear to be simply related to the changes in Vmax measured from the chemical data. This lack of correlation of the p.d.max with the Vmax in vivo has been discussed previously (Debnam & Levin, 1975). As the relationship between the active transfer of hexoses and the sodium gradient is the centre of much controversy and contradictory results (Crane, 1968; Forster & Hoos, 1972; Kimmich, 1973; Fisher & Gardner, 1974; Schultz, Frizzell & Nellans, 1974; Bieberdorf, Morawski & Fordtran, 1975) definitive explanations of the correlation, or lack of, between the p.d.max and Vmax must remain sub judice. Despite such a caveat, the p.d.max may still be a useful parameter in human malabsorption (Read, Holdsworth & Levin, 1974). The differential changes in the 'apparent Kms' for glucose, galactose and a-methyl glucoside during the 1-, 2- or 3-day fasts (Table 6) appear incompatible with the accepted model of a common hexose transfer mechanism (Wilson, 1962). Fasting 'uncovers' separate carriers or electrogenic transfer mechanisms. No further discussion will be made on this topic as it is the subject of separate studies (Debnam & Levin, 1971, 1974) which are to be published later elsewhere (Debnam & Levin, 1975). The effects of different commercial diets 86 on the kinetics of absorption of ac-methyl glucoside. The kinetic parameters obtained electrically for the absorption of ac-methyl glucoside were different in animals fed Diet 86 obtained from two separate commercial sources but those for glucose and galactose

DIETARY RESTRICTION AND SUGAR ABSORPTION 697 remained unaffected (Table 7). Other studies have observed that the two nominally similar Diets 86 can significantly alter the performance of the electrogenic transfer system of rat small intestine (Levin & Syme, 1973, and work submitted for publication). It is clear that there are unidentified dietary constituents) that selectively influence the jejunal mechanism for the active transfer of ac-methyl glucoside. Different commercial diets are known to alter the sensitivity of intestine to hormonal actions (Ramsey & Bern, 1972) and influence the levels of various enzymes involved in digestion and transport (Heitanen, 1975). Relation of results to previous studies The reduction of jejunal sugar absorption at concentrations above their Kms or near those of their Vmax (Larralde, 1947; Levin et al. 1965; Levin, 1970; Esposito, 1967; Crouzoulon-Bourcart et al. 1968) and the increased absorption at concentrations below their Km (Esposito, 1967) in dietary restricted rats is to be expected by a transfer mechanism with an enhanced affinity but a reduced Vmax. A similar effect is observed for serosal glucose transfer in vitro across fasted jejunum (Newey, Sanford & Smyth, 1970). The increased absorption in vivo of glucose in semistarved rats at a concentration just above the Km reported by Kershaw et al. (1960) raises an important experimental point. They injected a small volume of 23 mM glucose into the tied-off intestine which was left unstirred for up to 30 min. The concentration of glucose in the immediate vicinity of the brush border and adjacent unstirred layers will be depleted rapidly by the hexose active transfer mechanism. The filling of the carrier in the unstirred conditions becomes rate limited by glucose diffusion from the luminal bulk phase. Carriers in the fasted enterocytes will operate with a distinct advantage over those in fed enterocytes as the former have a greater affinity for the low concentrations of hexose present in the unstirred layers and transfer glucose from the low concentrations more effectively. The method of measuring absorption in vivo can thus influence the results obtained. One of the authors (E.S.D.) is indebted to the Medical Research Council for a Scholarship during the tenure of which this work was carried out. REFERENCES

BIEBERDORF, F. A., MORAWSKI, S. & FORDTRAN, J. S. (1975). Effect of sodium, mannitol, and magnesium on glucose, galactose, 3-0-methylgucose and fructose absorption in the human ileum. Gastroenterology 68, 58-66. CLARKE, R. (1973). Progress in measuring epithelial turnover in the villus of the small intestine. Digestion 8, 161-175.

~E. S. DERNAM AND B. J. LEVIN 698 698 CLARKyE, R. (1974). Morphological description of intestinal adaptation: measurements and their meaning. In Inte~tinal Adaptation, ed. DOWLINGr, R. H. & RIECKEN, E. 0., pp. 11-17. Stuttgart: F. K. Schattauer Verlag. CRAN, R. K. (1968). Absorption of sugars. In Handbook of Phy8iology, section 6: Alimentary Canal, vol. 3, ed. CODE, C. F. & HEIDEL, W., pp. 1323-1351. Washing. ton: American Physiological Society. CROUJZOULON-BouIRcART, C., CRouzouroN, G. & P#,Rts, G. (1968). Reoherches sur l'absorption intestinales des hexoses. I. Effets du j eeine sur l'absorption du glucose et le metabolism de la muqueuse intestinale. C. r. Se~anc. Soc. Riol. 162, 21312136.

DMn-rY, J. & HousE, C. R. (1966). 'Unstirred layers' in frog skin. J. Phy~iot. 182, 66-78.

DEIBNAM, E. S. & LEVIN, R. J. (1971). Evidence for separate hexose carriers in the small intestine uncovered by fasting and glucose alimentation. J. Phy~iol. 218, 38-39P.

DEBNAM, E. S. & LEVIN, R. J. (1973). Assessment of the effects of starvation and of semi-starvation on the operational kinetic parameters of the active transfer of hexoses measured in vivo. J. Phyaiol. 231, 21-2 3P. DEBNAM, E. S. & LEVIN, R. J. (1974). Dietary sugars and the maximum absorptive capacity of the small intestine; further evidence for multiple hexose transfer mechanisms. J. Phy~iol. 238, 81-82 P. DEBNAM, E. S. & LEVIN, R. J. (1975). An experimental method of identifying and quantifying the active transfer electrogenic component from the diffusive component during sugar absorption measured in vivo. J. Physiol. 246, 181-196. DIAMOND, J. M. (1966). A rapid method for determining voltage concentration relations across membranes. J. Phyeiol. 183, 83-100. DIETSCHY, J. M. (1970). Difficulties in determining valid rate constants for transport and metabolic processes. Ga8troenterology 58, 863-874. DIXON, M. (1972). The graphical determination of Km and Kj. Biochem J. 129, 197-202. EiSENTHAI, R. & CORNISH-BOWDEN, A. (1974). The direct linear plot. Biochem. J. 139, 715-720. ESPOSITO, G. (1967). Intestinal absorption of sugars in semi-starved rats.-Proc. Soc. exp. Riot. Med. 125, 452-455. FIsHLER, R. B. & GARDNER, M. L. G. (1974). Dependence of intestinal glucose absorption on sodium, studied with a new arterial infusion technique. J. Phy-8iot. 241, 235-260. FORSTER, H. & Hoos, I. (1972). The excretion of sodium during the active absorption of glucose from the perfused small intestine of rats. Hoppe-Seyler'8 Z. phy8iol. Chem. 353, 88-94. HEITANEN, E. (1975). The effect of different diets on the alkaline phosphatase (ATPase) and disacch'aridase levels of the small intestine- of the rat. Comp. Biochemi. Phy~iol. 50, 41-46. HOFSTEE, B. H. J. (1959). Non-inverted versus inverted plots in enzyme kinetics. Nature, Lond. 184, 1296-1298. HONEGGER, P. & GERSHON, E. (1974). Further evidence for the multiplicity of carriers for free glucalogues in hamster small intestine. Biochim. biophy8. Acta 352, 127-134. HONEGGER, P. & SEMENZA, G. (1973). Multiplicity of carriers for free glucalogues in hamster small intestine. Biochim. biophy8. A cta 318, 3 90-41 0. KERSHAW, T. G., NEAME, K. D. & WISEMAN, G. (1960). The effect of semi-starvation on absorption by the rat small intestine in vitro and in vivo. J. Phy8iol.- 152, 182-190.

DIETARY RESTRICTION AND SUGAR ABSORPTION 699 KIMMICH, G. A. (1973). Coupling between Na+ and sugar transport in small intestine. Biochim. biophy8. Acta 300, 31-78. LARRALDE, J. (1947). Influencia del ayuno sobre la absorci6n intestinal de glucosa. Revta esp. Fieiol. 3, 31-38. LEvrN, R. J. (1969). The effects of hormones on the absorptive, metabolic and digestive functions of the small intestine. J. Endocr. 45, 315-348. LEvIN, R. J. (1970). The intestinal absorption of some essential and non-essential amino-acids in fed and fasting rats. Life Sci. Oxford 9, 61-68. LEvIN, R. J. (1974). A unified theory for the action of partial or complete reduction of food intake on jejunal hexose absorption in vivo. In Intestinail Adaptation, ed. DOWLING, R. H. & RIECKEN, E. O., pp. 125-133. Stuttgart: F. K. Schattauer Verlag. LEvIN, R. J., NEWEY, H. & SMYTH, D. H. (1965). The effects of adrenalectomy and fasting on intestinal function in the rat. J. Phyaiol. 177, 58-73. LEVIN, R. J. & SYME, GABRIELLr (1971). Differential changes in the 'apparent K.' and maximum potential difference of the hexose and amino acid electrogenic transfer mechanisms of the small intestine induced by fasting and hypothyroidism. J. Physiol. 213, 46-48P. LEvIN, R. J. & SYME, GABRIELLE (1973). Changes in the performance of the electro. genic transfer mechanism of the small intestine related to different sources of a commercial feeding diet for rats. J. Phyeiol. 231, 109-1 lOP. LEVINSON, R. A. & ENGLERT, E. (1972). Small intestinal absorption of D-galactose and water in fasted and alloxan diabetic rats. Experientia 28, 1039-1040. LINEWEAVER, H. & BURK, D. (1934). The determination of enzyme dissociation constants. J. Am. chem. Soc. 56, 658-666. MENGE, H., BLOCH, R., SCHAUMLOFFEL, E. & RIEcKEN, E. 0. (1970). Transportstudien, morphologische, morphometrische und histochemische Untersuchungen zum Verhalten der Dunndarmschleimhaut im operativ ausgeschalteten jejunal abschnitt der Ratte. Z. ge8. exp. Med. 153, 74-90. NEWEY, H., SANFORD, P. A. & SMYnI, D. H. (1970). Effects of fasting on intestinal transfer of sugars and amino acids in vitro. J. Phyeiol. 208, 705-724. RAMSEY, D. H. & BERN, H. A. (1972). Stimulation by ovine prolactin of fluid transfer in everted sacs of rat small intestine. J. Endocr. 53, 453-459. READ, N. W., HOLDSWORTH, C. D. & LEVIN, R. J. (1974). Electrical measurement of intestinal absorption of glucose in man. Lancet, ii 624-627. RIECKEN, E. 0. & MARTINI, G. A. (1973). The classification of abnormal small intestinal mucosal appearances. Germ. Med. 3, 120-127. (Translated from Dtech. med. Wechr. (1973), 98, 998.) RIECKEN, E. O., MENGE, H., BLOCH, R., LORENZ-MEYER, H., WRAM, K. & IHLOFF, M. (1974). Effect of pair feeding on intestinal adaptation after hypophysectomy. In Intestinal Adaptation, ed. DOWLING, R. H. & RIECKEN, E. O., pp. 239-247. Stuttgart: F. K. Schattauer Verlag. ROCHE, A. C., BOGNEL, J. CL., BOGNEL, C. & BERNIER, J. J. (1970). Correlation between the histological changes and glucose intestinal absorption following a single dose of 5 fluorouracil. Digestion 3, 195-212. SCHULTZ, S. G., FRIZZELL, R. A. & NELLANS, H. N. (1974). Ion transport by mammalian small intestine. A. Rev. Phyeiol. 36, 51-91. SCHULTZ, S. G. & ZALUSKY, R. (1964). Ion transport in isolated rabbit ileum. II. The interaction between active sodium and active sugar transport. J. gen. Phyaiol. 47, 1043-1059. SHIELDS, J. W. (1968). Intestinal lymphoid tissue activity during absorption. Am. J. Gaatroenterol. 50, 30-36.

700

E. S. DEBNAM AND R. J. LEVIN

SRIVASTAVA, L. M. & HUBSCHER, G. (1966). Glucose metabolism in the mucosa of the small intestine. Biochem. J. 100, 458-466. SRIVASTAVA, L. M., SHAKESPEARE, P. & HUBSCHER, G. (1968). Glucose metabolism in the mucosa of the small intestine. Biochem. J. 109, 35-42. WILSON, F. A. & DIETSCHY, J. M. (1974). The intestinal unstirred layer: its surface area and effect on active transport kinetics. Biochim. biophy8. Acta 363, 112-126. WILSON, T. H. (1962). In Inte8tinal Ab8orption, chap. 4, pp. 69-109. Philadelphia: Saunders. WINNE, D. (1973). Unstirred layer, source of biased Michaelis constant in membrane transport. Biochim. biophy8. Acta 298, 27-31. WOOLF, B. (1932). In Allegemeine Chemie der Enzyme, ed. HALDANE, S. & STERN, K. G. Dresden: Steinkopff Verlag.