Sugar absorption by the mouse small intestine following infection with Schistosoma mansoni

55 TRANSACTIONS

OF THE ROYAL SOCIETY OF TROPICAL

Sugar absorption

MEDICINE

AND HYGIENE,

VOL. 73, No.

by the mouse small intestine Schistosoma

Department

1, 1979

following

infection

with

mansoni

P. B. VENGESA* AND H. J. LEESE** of Biology, University of York, York YOl SOD, England

Summary The effect of acute schistosomiasis on the structure and function of the mouse small intestine has been examined. Whole 100~s of small intestine from normal mice, and mice seven weeks post-infection were incubated in an in vitro perfusion apparatus. The transport of glucose, 3-0-methylglucose, sorbitol and fluid were all markedly impaired in loops from the infected animals. Kinetic analysis of the data for glucose transport indicated that schistosome infection brought about a reduction in the total number, but not the affinity, of the absorptive sites for glucose. Under the scanning electron microscope, villi from normal mice were tall and erect, with intact surfaces devoid of mucus, whereas the villi from the infected animals were partially covered with strands of mucus and appeared swollen and eroded, with lesions around their apices. Introduction It has been estimated that Schistosoma spp. infect up to 200 million people in Africa, the Americas, the Caribbean and the Far East (WRIGHT, 1968). Although cases of constant and intermittent watery diarrhoea have been reported for more than 50 9; of people with chronic schistosomiasis in Brazil (MACARIO, 1959) and of watery diarrhoea with traces of blood in all patients with acute bilharziasis in Puerto Rico (DIAZ-RIVERA et al., 1956), there have been very few studies on intestinal absorption either in patients or experimental animals with schistosomiasis (FIKRY et al., 1962; FIKRY, 1964; DE WITT, 1957; DOMINGO & WARREN, 1969). We now report the results of in vitro studies on the effects of acute S. mansoni infection on the absorptive function of the small intestine of white mice. This species has been used extensively in studies on other aspects of schistosomiasis, ~~~MADGE (1962, 1975) has demonstrated imnaired intestinal absorption following bacterial mfection in mice. A preliminary account of our findings has been published (VENGESA & LEESE, 1977). Materials and methods Albino male mice, Mus musculus (of the Laca strain), body-weight 20 to 25 g, were infected with 250 to 300 cercariae of S. mansoni (of Puerto Rican origin) as described by SMITHERS & TERRY (1965). The control and infected mice were caged separately but housed in the same room and allowed free access to food and water. The experiments were carried

out in a gas-lift perfusion apparatus similar to that described bv FISHER & PARSONS (19491. 30 ml of a modified Krebs-Ringer bicarbonate medium (BRONK & PARSONS, 1965) was recirculated through the lumen of the isolated whole gut by a stream of 95’s’, oxygen and 5y0 CO* (V/V). The serosal surface of the gut was bathed in a similar medium. The preparation and setting up of the small intestine and the measurements of fluid transport were carried out as described by PARSONS & WINGATE (1961). All incubations were at 37°C and were of one hour duration. D-glucose was added to the mucosal and serosal fluids-to give an initial concentration of 11.1 mM. Serial samnles of the fluids were assaved for glucose by an auiomated fluorimetric method (L~ESE & BRONK, 1972). At the end of the experiments, the gut length and wet weight were measured and the gut dried to constant weight at 11O’C. The results are expressed as pmol sugar disappearing from the mucosal fluid (values presented as negative) or appearing in the serosal fluid (values presented as positive) per mg dry weight of intestine. In some experiments, the transfer of 3-0-methylglucose and sorbitol were measured using trace amounts of [ U-i”C] 3-0-methylglucose and D-[ 1-3H] sorbitol, obtained from The Radiochemical Centre, Amersham, UK. These samples were counted in a Nuclear Chicago liquid scintillation counter. Procedure for electron microscopy Small pieces (60-80 mg wet wt) of intestinal tissue from normal or schistosomeiinfected mice were fixed in 49; elutaraldehvde in 100 mM phosphate buffer at” a ;H of 7.2-for two hours at 2 to 4°C. The tissue was washed several times in 100 mM phosphate buffer solution for 15 to-20 minutes and split open to remove residual debris from the intestinal lumen. The specimen was dehydrated in water/acetone mixtures graded at 20?:, intervals for 45 minutes in each mixture. The material was then dried bv a critical point method using a Polaron apparatus. Dry acetone was reolaced first with liauid CO., which in turn was evaporated from the-tissue by warming the polaron * Present address : Department of Anatomy, School of Medicine, Case Western Reserve University, Cleveland. Ohio 44106. U.S.A. **Address for correspondence: Dr. H. J. Leese, Department of Biology, University of York, York YOl 5DD, England.

56

SUGAR

ABSORPTION

Table I-Effect of schistosome infection length and dry and wet weights Mouse k)

wt

IN

MURINE

on mouse

Food intake (g/day)

Wet wt intestine (g)

SCHISTOSOMIASIS

body-weight,

food

Dry wt intestine (g>

intake

and small

Dry wt Wet wt

intestinal Length of intestine (cm)

Normal

36.4

+ 0.05

8.2 f 0.9 (5)

1.64 + 0*04

0.35

:k 0.012

0.218

i 0.002

52.2 + 1.3

Infected

23.5

* 0.05

4.2 i 0.5 (5)

2.33 + 0.26

0.46

i 0.02

0.188

f 0.002

46.5 + 2.1

Infected

%

65

51

142

132

85

89

Normal Values are mean & s.e. of mean of at least 20 determinations, mice were used after 48-50 days of infection.

to 35 to 36”C, a temperature above the critical temperature (31°C) of liquid CO,. The dried tissues were mounted on aluminium stubs and coated with a layer of gold-palladium in a s.e.m. coating unit E 5000. The specimens were subsequently examined under a scanning electron microscope (Cambridge Stereoscan 600). Electron micrographs were taken on a 125 ASA film and developed in microdol.

Fig. 1. The transport of glucose (11’ 1 mM) by segments of small intestine from normal (.) and schistosome-infected (0) mice. Values are mean 5 S.E. of eight determinations. In this and all the following Figures, the schistosome-infected mice were used after 48-50 days of infection.

Results Effect of infection on body weight, food intake and small intestinal weights The effect of seven weeks of schistosome inection on body-weight, food intake and some

except where stated. The schistosome-infected

physical parameters of the mouse small intestine are summarized in Table I. The infected mice gained weight at a similar rate to the controls up to six weeks after infection, but during the following week lost weight dramatically so that by the end of seven weeks they weighed only 65 U0 of the controls. The infected mice then continued to lose weight and had usually died by the tenth week after infection. Food intakes by normal and schistosome-infected mice were similar up to six weeks after infection, but by the end of week seven the food intake by the infected animals had dropped to one half that of the controls, and continued to drop in subsequent weeks. The fresh weight of the mouse whole small intestine seven weeks after infection was greater than that of the controls and, of the extra weight, more was fluid than dry matter, so that the dry weight/wet weight ratio was slightly diminished after infection. All the infected mice used in this study exhibited frank watery diarrhoea, sometimes with traces of blood. Sugar transfer experiments Fig. 1 shows the results for the transfer of glucose present at an initial concentration of 11.1 mM in both the mucosal and serosal media. Glucose disappeared steadily from the mucosal fluid bathing the guts from normal mice, and about 30”” of the glucose disappearing could be accounted for by an increase in the glucose content of the serosal medium. The final serosal to mucosal concentration ratio for glucose was 8.99 5 1.58 (6), indicating that the uphill or active transport of glucose had taken place. Sugar transport by segments taken from the infected animals was markedly impaired, and the results were significantly different (p <0.05) from those for normal mice, whether they were expressed in terms of gut length, dry or wet weight. from Only about 4 ” L of the glucose disappearing the mucosal fluid could be accounted for by the increase in the glucose content of the serosal medium, and the final serosal to mucosal concentration ratio for glucose was reduced to 2.41 -1 0.15 (6).

P. B. VENGESA

Phlorizin at a concentration of 2 X lo-*M completely inhibited the mucosal disappearance of glucose, in preparations from both normal and infected animals. The kinetics of glucose absorption In order to determine whether or not schistosome infection altered the kinetic parameters for glucose transfer by the mouse small intestine, a study was carried out following the principles outlined by NEAME & RICHARDS (1972). The kinetic equation

v max. s v = ^----- -S

-I-

Kt

was used where Kt = substrate concentration at which v = ; V,,, (analogous to the MichaelisMenten constant Km), v = rate of uptake of substrate, and s = substrate concentration. Since the analysis only applies if the initial rates of uptake are considered, i.e. while the uptake is essentially unidirectional, measurements of the mucosal disappearance of glucose were made within the first ten minutes of incubation. The glucose concentration was varied between 5.55 and 28 mM. The apparent Kt values in the normal and infected guts were 5.65 & 0.19 mM and 5.45 $ 0.17 mM, while the values for V,,, were 0.67 & 0.02 pmol. mg-’ 10-r min-l and 0.124 & 0.009 pmol. mg-l . 10-l mini respectively. These data strongly suggest that schistosome infection brings about a reduction in the total number, but not the affinity, of the functionally active absorptive sites for glucose in the small intestine.

AND

57

H. J. LEESE

measurements of fluid transfer were made in intestinal segments taken from normal and infected mice. The glucose concentration in these experiments was varied between 0 and 28 mM and the results are shown in Fig. 2. The transfer of fluid by segments from normal animals increased with increasing glucose concentration up to 16.65 mM and then levelled off. On the other hand, fluid transfer by segments from the infected mice was drastically impaired, and showed only a small statistically insignificant (p> O-05) response to increasing glucose concentration. PO-methyl glucose and sorbitol transfer In order to determine whether the impaired glucose transport bore some relationship to glucose catabolism in the diseased gut (VENGESA & LEESE, 1976) and/or whether it reflected some alteration in the passive permeability of the gut, experiments were carried out with the actively transported, non-metabolizable analogue, 3-O-methyl D-glucose and with sorbitol, which is considered to be transported passively. In these experiments, each sugar was added at 5.55 mM to the mucosal and serosal compartments. In the case of the 3-0-methylglucose experiments, a trace of the radio-active sugar was added to the mucosal compartment only, and the appearance of radio-activity in the serosal compart-

Fluid transfer Since water movements across the gut wall are closely linked to solute fluxes (PARSONS, 1966), 30-

$

20 -

$ 9

T/

T o-0-0 A

-r J.

-r I

: /

0

20

40 incubation

0 FO

IO Glucose

20 concentration

(mM)

Fig. 2. Effect of glucose concentration on the net transport of fluid by segments of small intestine from normal (e) and schistosomeinfected (0) mice. Values are mean Y& S.E. of eight determinations.

Fig. 3. mucosal normal * S.E.

60

time (mid

The transport of 3-0-methylglucose to the serosal medium, by segments (0) and schistosome-infected (0) of five determinations.

(5.55 mM) from the of small intestine from mice. Values are mean

ment monitored. In the case of the sorbitol transfer experiments, a trace of the radio-active sugar was

58

SUGAR

ABSORPTION

IN

added to each compartment in turn in order to follow its appearance in the other compartment. The data in Figs. 3 and 4 indicated that the serosal transfers of 3-0-methylglucose and sorbitol were reduced by about 50% in the preparations taken from the infected mice. A similar result was found for the serosal to mucosal transfer of sorbitol. Since the data suggested that schistosome infection had brought about some fundamental impairment in the capacity of the mouse small intestine to transfer sugars, the surface of the small intestine of control and infected mice was examined under the scanning electron microscope. Villi from normal mice (Fig. 5) were tall and erect, with intact surfaces indented with shallow sulci. They were devoid of mucus. In marked contrast, the villi from schistosome-infected mice were partially covered with thick strands of mucus (Fig. 6). The villi were swollen, eroded, indented with deep sulci and exhibited lesions or disruptions around their apices (Fig. 7).

MURINE

SCHISTOSOMIASIS

Fig. 5. Scanning electron micrograph intestinal villi from a normal mouse.

(S.E.M.)

of a group

of small

o.o1a-

Fig. 6. S.E.M. Note pronounced

I’/ 0

1

I

I

I 40

20 lncubotion

time

I

of a group of villi from a schistosome-infected strands of mucus.

mouse.

I 60

hin)

Fig. 4. The transport of sorbitol (5.55 mM) from the mucosal to the serosal medium, by segments of small intestine from normal (l ) and schismsome-infected (0) mice. Values are mean -J= S.E. of five determinations.

Discussion

Adult schistosomeworms establishthemselvesin the portal and mesentericvesselsof their host four weeksafter infection. The female worms then each start to lay 200 to 300 ova per day, the oviposition reaching a maximum 45 to 55 days after infection,

Fig. 7. S.E.M. of the apex infected mouse.

of a single

villus

from

a schistosome-

I’. B. VENGESA

coinciding with the period chosen for the present study. The schistosome egg is considered the main pathogenic agent in schistosomiasis, and the present results indicate that the small intestine is severely affected, structurally and functionally by the onset of oviposition. Since anorexia and diarrhoea occurred simultaneously, it is difficult to assess the relative importance of each to the loss in body-weight exhibited by the schistosome-infected mice. The absorption of glucose, 3-0-methylglucose and sorbitol were all reduced in the intestinal segments taken from the infected mice. We have evidence for a comparable reduction (about 50%) in glucose absorption by segments from the infected mice incubated in viva (VENGESA, B. I?. & LEESE, H. J., unpublished), and it is interesting to note that this figure corresponds closely with the reduction in glucose accumulation obtained with isolated rings of small intestine also taken from schistosomeinfected mice (VENGESA & LEESE, 1976). MADGE (1972), using an everted sac technique and a glucose concentration of 8 mM, showed that Salmonella infection reduced the final serosal to mucosal concentration ratio from 11.1 for control animals to 1.2 for inoculated mice. Bearing in mind the different species, preparations and glucose concentrations, these figures are of the same order as those obtained in the present study (8.99 and 2.41 respectively). The fact that the bi-directional transfer of sorbitol was reduced after infection (Fig. 4) makes it unlikely that the diminished capacity of the infected guts to transfer glucose was due to the development of pores or holes in the mucosa, since the movements of a passively transported sugar would be expected to increase if this were the case. In order to correlate the data on the kinetics of glucose transport with the surface appearance of the intestinal mucosa after infection, one has to suppose that the damaged villi contain fewer sugar absorptive sites. Since the apparent affinity of the sites for glucose was unaltered, this suggests that the mucus strands seen on the surface of the infected mucosa were not acting as a barrier or unstirred layer to prevent or restrict the access of sugar molecules to their sites of uptake. MADGE (1974), also using the scanning electron microscope, compared the surface appearance of the mucosal surface of mice infected with Salmonella enteritidis with that from normal mice. The normal mucosa appeared similar to that observed in the present work (Fig. 5), and disruption of the villus tip after infection was also apparent. However, the pattern of disruption differed from that seen in the present work (Fig. 7) and the pronounced strands of mucus seen in Fig. 6 were absent. Extrapolation of the data on murine schistosomiasis to man involves a number of uncertain assumptions (CHEEVERS, 1969) partly arising out of the lack of adequate human data. However, in order to mimic the watery diarrhoea sometimes with traces of blood seen in patients with bilharzia (DIAZ-RIVERA et al., 1956; MACARIO, 1959) we deliberately infected the mice used in the present study with a heavy dose of cercariae, and the

AND H. J. LEESE

59

derangement in fluid transport seen in Fig. 2 may go some way to explaining these symptoms. Our approach contrasts with that of DOMINGO & WARREN (1969) who used mice mildly infected with chronic 20-week-old schistosomiasis mansoni and found no impairment in the intestinal absorption of glucose, methionine and sodium propionate. In view of this it would be of interest to carry out absorption studies on mice at stages of infection intermediate between those of DOMINGO & WARREN and our own. In addition to the impairment in sugar and fluid transport reported in the present paper, we have shown that the oxygen uptake, lactate production and carbohydrate-splitting enzyme activities of the small intestine are-also reduced after schistosome infection. (VENGESA. B. I?. & LEESE. H. T.. umublished). This raises- the question of the” extent to which the abnormalities in the structure and function of the small intestine which follow infection are primary events in the development of acute schistosomiasis, or secondary consequences of some other primary interaction between the parasite ova and the host. Acknowledgements We thank Dr. Peter Ingham and Dr. Alan Wilson for many valuable discussions. P.B.V. thanks the Commonwealth Scholarship Commission for a research studentship. References Bronk, J. R. & Parsons, D. S. (1965). The polarographic determination of the respiration of the small intestine of the rat. Biochimica et Biophysics. Acta, 107, 397-404. Cheever, A. W. (1969). Quantitative comparison of the intensity of Schistosoma mansoni infections in man and experimental animals. Transactions C$ the Royal Society of Tropical Medicine and Hygiene, 63, 781-795. De Witt, W. B. (1957). Effects of S. mansoni infections on the ability of mice to digest and absorb dietary fats and proteins. Journal of Parasitology, 43, 32. Diaz-Rivera, R. S., Ramos-Morales, F., Koppisch, E.. Garcia-Palmieri, M. R., Cintron-Rivera. A. A., Marchand, E< J., Gonzales, 0. & Torregrosa, M. V. (1956). Acute Manson’s schistosomiasis. American Journal of Medicine, 21, 918-943. Domingo, E. 0. & Warren, K. S. (1969). Pathology and pathophysiology of the small intestine in murine schistosomiasis mansoni including a review of the literature. Gastroenterology, 56, 231-240. Fikry, E. M. (1964). Disturbance of digestion and absorution in bilharzial hepatic fibrosis : partial and selective correction after portalcaval -shunt oueration. Yournal of TroPical Medicine and hygiene, 67; 204-208.. Fikry, E. M., Aboul, Wafa, M. H. & Loutfy, K. D. (1962). Intestinal absorption in bilharzial hepatic fibrosis.Journal of Tropical Medicine and Hygiene, 65, 318-321. Fisher, R. B. & Parsons, D. S. (1949). A preparation of surviving rat small intestine for the study of absorption. Journal of Physiology, 110,36-46.

60

SUGAR

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IN

Leese, H. J. & Bronk, J. R. (1972). Automated fluorimetric analysis of micromolar quantities of ATP, glucose and lactic acid. Analytical Biochemistry, 45, 211-221. Macario, G. M. D. (1959). Ritmo intestinal na esquistossome mansoni. Estudo clinic0 de 100 cases. Arquivos Brasileiros de Medicina, 49, 57-62. Madge, D. S. (1972). Intestinal absorption in experimental Salmonella enterocolitis in mice. Comparative Biochemistry and Physiology, 42A, 445-463. Madge, D. S. (1974). Scanning electron microscopy of normal and diseased mouse small intestinal mucosa. Journal de Microscopic, 20, 45-50. Madge, D. S. (1975). Effects of Staphylococcus aureus on in vitro intestinal absorption in mice. Comparative Biochemistry and Physiology, 52A, 395-401. Neame, K. D. & Richards, T. G. (1972). Elementary kinetics of membrane carrier transport. Oxford: Blackwell Scientific Publications. Parsons, D. S. (1966). Sodium chloride absorption by the small intestine and the relationships between salt transport and the absorption of

MURINE

SCHISTOSOMIASIS

water and some organic molecules. Proceedings the Nutrition Society, 26, 46-55. Parsons, D. S. & Wingate, D. L. (1961). The effect of osmotic gradients on fluid transfer across rat intestine in vitro. Biochimica et BioDhysica Acta, - 46, 170-183. Smithers, S. R. & Terry, R. J. (1965). The infection of laboratory hosts with cercariae of S. mansoni and the recovery of the adult worms. Parasitology, 55, 695-700. Vengesa, B. I?. & Leese, H. J. (1976). Glucose accumulation bv rings of small intestine from normal and schistoiome-infected mice. Biochemical Society Transactions, 4, 274-277. Vengesa, P . B. & Leese, H. J. (1977). Structure and function of the mouse small intestine after infection with Schistosoma mansoni. Gut, 18, A965-A966. Wright, W. H. (1968). Schistosomiasis as a world problem. Bulletin of the New York Academy of Medicine, 44, 301-312. of

Accepted for publication

9th June, 1978.

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