Mechanism of 3-0-methylglucose uptake by Hymenolepis diminuta

International Journalfor Parasitology Vol. 21, No. S,pp. 517-520. 1991 Printed in Great Britain 0

MECHANISM

OF 3-0-METHYLGLUCOSE HYMENOLEPIS DIMINUTA

002%7519/91 $3.00 + 0.00 Pergamon Press p/c Societyfir Pmzsiro/ogy

1991 Awrrolim

UPTAKE

BY

GARY L. UGLEM* and PETER W. PAPPAS? * School of Biological t Department

Sciences, University of Kentucky, Lexington, KY 40506, U.S.A. of Zoology, The Ohio State University, Columbus, OH 43210, U.S.A. (Received 14 November

1990; accepted 5 March 1991)

AbBtract-Ll~~~~ G. L. and PAPPASP. W. 1991. Mechanism of 3-0-methylglucose uptake by Hymenolepis diminuta. International Journal for Parasitology 21: 517-520. The mechanism by which Hymenolepis diminuta (Cestoda) absorbs 3-0-methylglucose (30MG) in vitro was analyzed. Influxes of 0.1 and 0.01 mu‘H-30MG during incubations ranging from 5 s to 60 min were not affected significantly when 10 mMunlabeled 30MGwas present as an inhibitor. After 60 min in 0.1 mt.+‘H-30MG, theconcentration of labeled substrate within tapeworms (0.04 pmol ml-’ worm water = 0.04 mM) was less than that of the bathing medium. Tapeworms incubated for 1 h with either 5 mr+glucose or 5 mr+~methylglucose (/3MG) gained 15-20% more water than did tapeworms in saline alone, but addition of 5 m&r-30MG to the saline had no significant effect on weight change. When the ‘H-30MG concentration was varied from 0.01 to 10 mM, influxes were a linear function of substrate concentrations. These analyses show that H. diminuta absorbs 30MG by simple diffusion alone. Thus, use of this monosaccharide to estimate the internal concentration of actively transported sugars (e.g. glucose or /3MG) in H. diminuta is invalid. INDEX KEY WORDS: glucose; pmethylglucose;

Hymenolepis diminutu; Cestoda; glucose content.

diffusion;

active transport;

3-0-methylglucose;

the same system. The only evidence provided by Comford (1990) to support this assumption was his statement that “5 mM-ghcOSe caused an 83% reduction in 0.01 mM-radiolabeled 3-0-methylglucose uptake” during a 5 s incubation. Unfortunately, no uptake rates, statistics, or supporting data were provided. Because Cornford’s (1990) report lacked empirical data to support his basic assumption, we reexamined 30MG uptake by H. diminuta, and the results are reported herein.

INTRODUCTION CONSIDERABLE evidence exists that uptake Of D-ghcOSe by the tapeworm Hymenolepis diminuta occurs via the

process of active transport. Glucose uptake is saturable, Na’-dependent, and phlorizin-sensitive, and tapeworms accumulate glucose against an apparent concentration difference (reviewed by Pappas, 1983). The glucose transport system is stereospecific, and analogues of glucose, such as p methylglucose (PMG), interact with the system and inhibit glucose uptake (Uglem, Love & Eubank, 1978; Rosen & Uglem, 1988). On the other hand, analogues such as 3-0-methylglucose (30MG) which do not inhibit uptake of glucose, or other monosaccharides known to interact with the glucose transport system, are assumed to be absorbed by simple diffusion alone (Rosen & Uglem, 1988). This concept of glucose transport in H. diminuta was contradicted recently when Cornford (1990) reported that the glucose concentration within tapeworms did not exceed that of the external medium-that is, glucose was not accumulated against a concentration difference and, therefore, not actively transported. In arriving at this conclusion, Cornford (1990) used the equilibrium distribution of 14C-30MG between the tapeworm and its environment to estimate ‘free glucose concentrations’ within the tapeworms. In support of using 30MG to estimate internal glucose, he assumed that 30MG and glucose are transported by

MATERIALS AND METHODS Radiolabeled 30MG (3-0-methyl-[1-‘H] glucose) was obtained from Amersham, and the purity was verified using descending paper chromatography (Gray & Fraenkel, 1953). Immediately before use, the stock isotope solutions were evaporated to dryness to remove contaminating ‘H,O. Unlabeled monosaccharides and phlorizin were obtained from Sigma Chemical Co. H. diminuta was maintained in male Sprague-Dawley rats and beetles (Tenebrio molitor). Rats were infected with 35 cysticercoids and adult tapeworms were recovered 12 days post-infection. Tapeworms were triplerinsed in Krebs-Ringer saline buffered at pH 7.4 with 25 mMTris-maleate (KRT of Read, Rothman & Simmons, 1963), randomized into groups of three tapeworms, and incubated in 15 ml of fresh KRT at 37°C for 15 min. Each group of tapeworms was transferred to 10 ml of pre-warmed KRT containing the substrate (‘H-30MG) and, when appropriate, inhibitor (unlabeled 30MG). After incubation at 37’C the tapeworms were removed, triple-rinsed in fresh KRT, blotted 517

G. L.

518

UGLEM

and P. W.

on damp filter paper, weighed and extracted overnight in 70% ethanol (3 ml per group). Uptake rates of ‘H-30MG were calculated based on the ethanol-extracted dry weight of tapeworms following drying at 95°C. The water content of tapeworms (i.e. worm water) was estimated assuming a wet weight to dry weight ratio of 1:0.25 (Read et al., 1963). Differences were analyzed using Student’s t test with a value of P
time was (Table 1) 10 mMwere not

I-EFFECTS

OF 10 II1M-UNLABELED

OF

0.01 mM-‘H-30MG

= With

10

mM

BY

Hymenolepis

diminuta

Incubation time (s)

Control 10 mM-30MG

5

20

60

5.8 f 0.62 6.5f0.29

3.0*0.37 3.5 f 0.70

2.1410.56 2.0f0.44

Each value represents the mean f s. D. of three replicates. Uptake values for controls and experimentals were not statistically different for any of the time periods.

inhibited at any time period indicating simple diffusion. Following a 60 min incubation of tapeworms in 0.1 mM-‘H-3OMG, the concentration of ‘H-30MG in the worms (z 0.04 pmol ml -’ worm water = 0.04 InM) was substantially less than that of the external medium (Fig. 1). Uptake of labeled 30MG was also examined as a function of substrate concentration in 60 s incubations (Fig. 2); rates were a linear function of 30MG concentration, again indicating simple diffusion. For comparative purposes, Fig. 2 contains data from previous publications demonstrating the active and 12 n

=

- = l

0

3-O-METHYLGLUCOSE

(~OMG)ONUPTAKE(NMOLGETHANOLEXTRACTEDDRYWT~’MIN~‘)

10

0.05

0.04

TABLE

PAPPAS

glucose PMG

= 30MG

a

30MG

6 0.03 = @MG (diffusion) 0.02

_..’

__,; __

0.01

I

0

0.00 0

10

20 Time

30

40

50

60

(minutes)

FIG. 1. Uptake of 0.1 mM-3H-3-0-methylglucose (30MG) by diminuta vs time in the absence (controls) and presence of 10 mrrr-unlabeled 30MG. Each value represents the mean of three replicates, and values for controls and experimentals were not statistically different for any of the time periods. Hymenolepis

2

4 [Substrate],

a

6

10

mM

FIG. 2. Uptake velocities (V) of glucose, Pmethylglucose (PMG) and 3-0-methylglucose (30MG) by Hymenolepis diminufa (solid lines, symbols as indicated in figure). Also shown are the passive or diffusion components for glucose and j3MG (dashed lines, symbols as indicated in figure). The data for 30MG represent the averages of three replicates, while the data for glucose and non-metabolized BMG are from Uglem & Prior (1980) and Uglem et al. (1978), respectively.

3-0-Methylglucose uptake by H. diminuta passive components for uptake of glucose and p methylglucose (/3MG) by H. dirninuta. Comparison of these data demonstrated clearly that H. diminuta absorbed glucose and /3MG via a saturable system, while uptake of 30MG was linear and not saturable. Moreover, the rate at which 30MG entered H. diminuta was comparable to the diffusion rates for glucose and /?MG. Worm wet weight increased 5% during a 1 h incubation in saline (Fig. 3). Addition of 5 mM-30MG to the saline had no significant effect on weight change at any time period. By contrast, worms rapidly gained 15-20% more water than did controls when either glucose or /3MG was added to the saline. This demonstrates the capacity of ‘actively transported’ sugars to osmotically ‘drive’ water uptake. Glucoseenhanced water uptake was blocked by 0.1 mMphlorizin, a potent inhibitor of the glucose transport system (Uglem & Love, 1977). Because H. diminuta is impermeable to phlorizin (Read, 1966) the active transporters, and thus the limiting step in osmoticallydriven water uptake, must be located in the external

0

=

Controls

.

=

With

30MG

.

=

With

glucose

v

=

With

PMG

q

=

With

glucose

phlorizin

0

15

30

45

60

519

membrane of the worm. This type of analysis also shows that there were no ‘internal compartments’ into which 30MG might be transported. Since the concentration of glucose within whole 6and 1&day-old tapeworms did not change during a 60 min incubation in KRT at 0°C it was assumed that the concentration of glucose within individual pieces of 1&day-old worms did not change during the time they were being collected for analysis (Fig. 4). Analysis of 10

6

FIG. 4. Internal glucose concentrations of whole 6-, 12- and 18-day-old Hymenolepis diminuta and individual pieces of 1% day-old tapeworms. Columns labeled ‘1’ through to ‘10’ represent the glucose concentrations of individual pieces of 18-day-old tapeworms cut into 10 pieces, with piece ‘1’ representing the anterior end (scolex) of the tapeworm (values are means f s. o. of six replicates). Column ‘6W(O)’ represents the glucose concentration of whole 6-day-old tapeworms maintained in KRT at 0°C for 60 min, while column ‘6W’ represents the glucoseconcentration ofwhole 6day-old worms immediately after removal from the rodent host (values are means fs. D of 10 replicates). Columns ‘18W(O)’ and ‘18w’ represent similar determinations for 18day-old tapeworms (mean f s. D. of 10 replicates). Column ‘12 (W)’ represents the glucose concentration of whole 12day-old tapeworms.

Time (minutes)

FIG.3. Increases in wet weight (%) of Hymenolepis diminuta during 60 min in the absence (controls) and presence of 5 mh+ 3-0-methylglucose (30MG), 5 mr+ghtcose, 5 mr+,+methylglucose @MC) or 5 mh+glucose plus 0.1 mhr-phlorizin (symbols as indicated in figure). Each point is the average of three replicates (three worms per group). After the 15 min pre-incubation, the worms were blotted on damp filter paper, weighed ( f 0.2 mg) and transferred to 15 ml of prewarmed (37°C) KRT without or with the monosaccharide. The worms were removed at 15 min intervals, blotted, weighed and returned to the saline.

these individual pieces demonstrated a steep gradient of internal glucose concentrations along the strobila of H. diminuta. The highest concentration of glucose (7.5 mM) was found in the anterior-most section of the tapeworm, and the glucose concentrations decreased progressively in the posterior sections. Thus, the glucose concentration in a whole tapeworm actually represents an average value for the tapeworm’s entire strobila, e.g. 4 mM in 12-day-old worms (Fig. 4).

G. L. UCLEM and

520 DISCUSSION

To determine whether a glucose gradient exists along the strobila of H. diminuta, Cornford (1990) incubated ICday-old tapeworms in vitro with 2 mMglucose plus ‘a trace concentration’ of radiolabeled 30MG. After 45 min the worms were rinsed and cut into four pieces (‘quartiles’) and the absorbed radioactivity was determined. The ‘free glucose level’, as indicated by the amount of ‘%-30MG absorbed, decreased progressively from 0.49 mM in the first quartile to 0.31 mM in the fourth. Because the ‘free glucose concentration’ (i.e. 30MG) was a ‘fraction’ (0.1-0.2) of the external medium (i.e. 2 mM glucose), Cornford concluded that H. riiminuta is unable to accumulate glucose against a concentration difference. Our data show that Cornford’s observations simple and satisfactory explanation-30MG,

have a

the indicator molecule used by Cornford, is absorbed by simple diffusion alone. The internal concentration of 30MG, or for that fact any molecule absorbed by diffusion alone, would never exceed that of the external medium. Thus, 30MG uptake is unrelated to glucose transport and utilization rates, as well as internal glucose concentration. The actual glucose concentration (see Fig. 4) would be underestimated. Cornford did observe a 30MG gradient of 0.49 rnM anteriorly to 0.31 rnr+.tposteriorly along the strobila after a single 45 min incubation in labeled 30MG. While he assumed that 30MG had reached equilibrium conditions (based on ‘H-OH), when a time course of 30MG uptake is determined (see Fig. 1), it is clear that worms require longer than 45 min to equilibrate, i.e. 30MG uptake was still increasing at 60 min. Thus, the small 30MG gradient observed by Comford would seem to show little more than the obvious differences in the surface area-to-volume ratios that exist between smaller pieces of worm (e.g. the scolex-neck, or first quartile) and larger ones (e.g.

P. W. PAPPAS mature proglottids, or the fourth quartile), and the effect of this ratio on uptake by diffusion. In other words, when calculated on a ‘per weight basis’, more 30MG would diffuse into small pieces of tapeworm than large ones. REFERENCES CORNFORDE. M. 1990. Glucose

utilization rates are linked to the internal free glucose gradient in the rat tapeworm.

Experimental Parasitology 70: 25-34. GRAY H. E. & FRAENKELG. 1953. Fructomaltose, a recently discovered trisaccharide from honeydew. Science 118:304305. LLOYD J. B. & WHELAN W. J. 1969. An improved method for enzymic determination of glucose in the presence of maltose. Analytical Biochemistry 30: 467-470. PAPPASP. W. 1983.Host-parasite interface. In: Biology of the Eucesioda (Edited by ARME C. L PAPPASP. W.), pp. 297334. Academic Press, New York. READ C. P., ROTHMAN A. H. & SIMMONSJ. W., JR 1963. Studies on membrane transport, with special reference to parasite-host integration. Annals of The New York Academy of Sciences 113: 154-205. READ C. P. 1966. Nutrition of intestinal helminths. In: Biology of Parasites (Edited by SOULSSYE.), pp. 101-126.

Academic Press, New York. & UGLEM G. L. 1988. Localization

ROSEN R.

of facilitated diffusion and active glucose transport in cysticercoids of Hymenolepis diminuta (Cestoda). International Journaifor Parasitology 18: 581-584. UGL~M G. L. & LOVE R. D. 1977. Hymenolepis diminuta: properties of phlorizin inhibition of glucose transport. Experimental Parasitology 43: 9499. UCLEM G. L., LOVE R. D. & EUBANK J. H. 1978. Hymenolepis diminuta: membrane transport of glucose and pmethylglucoside. Experimental Parasita~agy 45: 88-92. UGLEM G. L. & PRIOR D. J. 1980. Hymenolepis diminuta: chloride fluxes and membrane potentials associated with sodium-coupled glucose transport. Experimental Parasitology 50: 287-294.