Kenneth B. Robbins
Sugar Absorption by C. stelleri
INTRODUCTION
The absorption of nutrients is one important phys¬
iological function of animal intestine. Absorption of
nutrients has been well-studied in vertebrates (for re¬
views, see Wilson, 1962; Crane, 1968; Curran, 1973),
revealing accumulation against an apprent concentration
gradient by means of the carrier-mediated process termed
active transport (for a review of membrane carrier trans¬
port, see Neame and Richards, 1972). Little work has
been done with invertebrates, however.
The study of monosaccharide active transport in in¬
vertebrate intestine has encompassed only two classes of
Echinodermata (see Lawrence and Mailman, 1967; Ferguson, 1964),
one class of Arthropoda (Randal and Derr, 1965), and two
classes of Mollusca (Lawrence and Lawrence, 1967; Wright,
1968). Very little is known about the exact site of mono¬
saccharide absorption in invertebrates, or the nature of
the mechanism by wheih that absorption occurs.
The first investigation demonstrating active transport
of monosaccharides by a molluscan intestine was done by
Lawrence and Lawrence (1967) using the amphineuran Crypto¬
chiton stelleri (Middendorff, 1846). D-glucose and 3-0-
methylglucose were found to be accumulated from the in¬
page 2
Kenneth B. Robbins
Sugar Absorption by C. stelleri
cubation medium into everted sacs of proximal anterior in¬
testine, while D-galactose was accumulated by everted sacs
of posterior intestine. Further studies have revealed a
socium coupled galactose transport system in the posterior
intestine of this organism (Lawrence and Mailman, 1967; Puddy,
1970; Lawrence et al., 1972).
Little or no work has been done on the kinetics or spec¬
ificity of the monosaccharide transport systems in the in¬
testinal tissue of Cryptochiton stelleri due to the exper¬
imental limitations of the everted sac technique. I
decided to approach the problem of sugar uptake and related
enzyme kinetics of this organism by measuring the accumulation
of sugar in intestinal tissue itself, as Crane and Mandelstam
(1960) did with hamster intestine. There are two basic
reasons for preferring this technique. First, it is
difficult to elucidate the actual transport mechanism using
everted sacs, as at least two separate processes are occurring
to yield net transport across the intestinal wall: initial
transport into the epithelial cells, followed apparently by
diffusion through the many underlying cell layers to the
serosal fluid. The connective tissue itself, which is 200 u
thick in the anterior intestine (Fretter, 1937), may act
as a significant barrier to free diffusion. With the use
of tissue slices, however, sugar need only enter the epithel¬
ial cells to be measured, and underlying tissue serves as a
page 3
Kenneth B. Robbins
page 4
Sugar Absorption by C. stelleri
repository similar to the serosal fluid of the everted sac.
A second advantage of the tissue accumulation technique is
that slices from different locations on the intestine and
from different animals can be pooled and distributed among
flasks so that the sugar absorbing potential of the tissues
in each flask is uniform. This is not possible using everted
sacs, as the transport rate of each sac varies. The use of
tissue samples with equivalent absorbing potential is essential
in the study of saturation kinetics, which is an important
means of characterizing any transport mechanism.
The properties of D-glucose, D-galactose, and, in
certain circumstances, 3-0-methylglucose transport were
thus studied using a tissue accumulation technique. The
two regions of intestine studied showed specific accumulation
of one or more of these sugars. Transport was notably slow
compared to sugar absorption by hamster intestinal tissue.
The D-galactose transport system of the proximal posterior
intestine of Cryptochiton stelleri, however, was found to
be more sensitive to low sugar concentrations than the
mammalian equivalent.
MATERIALS AND METHODS
Chitons (Cryptochiton stelleri) of both sexes weighing
between 600 and 1200 g were obtained subtidally near Mussel
Point, Pacific Grove, California. They were kept in aerated
page 5
Sugar Absorption by C. stelleri
Kenneth B. Robbins
seawater at 13°C at least three weeks before use.
The procedure for determining monosaccharide accumulation
in intestinal tissue was a modification of that of Crane and
Mandelstam (1960). The intestine and associated digestive
gland was removed from an experimental animal and placed in
chiton Ringer solution (Lawrence and Lawrence, 1967). The
digestive gland was carefully teased away from the intestine
and the intestine was elongated. The proximal half of the
anterior intestine and the proximal 30 cm of the posterior
intestine were the only portions studied in these experiments.
Fifteen centimeter portions of the experimental tissues
were everted over a glass rod, rinsed in chiton Ringer solu¬
tion, and cut with scissors to yield a number of 2-4 mm
wide tissue rings. These were distributed evenly among
incubation flasks containing 50 ml of chiton Ringer solution,
thus assuring a uniform sample in each flask.
Incubations flasks were given a 15-minute preincubation
period followed by the addition of specific unlabeled and
C-labeled monosaccharides to the medium. Both pre¬
incubation and incubation were carried out at 18°-20°C
on a rotary shaker (60rpm) under 100% oxygen. Concentrations
of sugar in the incubation medium ranged from 0.2-10 mM.
Incubation periods ranged from 0-8 hours.
Following incubation, two tissue samples containing
6-8 intestinal tissue slices were removed from an incubation
flask, blotted lightly, weighed, homogonized in 2.0 ml 2.0%
enneth B. Robbins
Sugar Absorption by C. stelleri
Znso 7H»0 followed by 2.0 ml 1.8% Ba(OH), for the purpose
of deproteinization, and centrifuged at low speed. The fil¬
trate was analyzed for the appropriate monosaccharides.
Original intestinal tissue, tissue dipped in the experimental
medium, original medium samples and final medium samples
were also analyzed for sugar content.
Determinations of D-glucose and D-galactose were made
by bothe chemical and radioactive techniques. Analysis of
3-0-methylglucose was done only by the radioactive method.
Chemical determinations were done colorimetrically using the
Glucostat and Galactostat reagents (Worthington Biochemical
Corporation). D-glucose-U- C, D-galactose-1-C, and 3-0-
methyl- C-D-glucose were obtained from New England Nuclear
Corporation. Tracer concentrations in the incubation media
were 0.040uc/ml ( 3x10M), added to the non-radioactive
analogues which were present at final concentration.
Radioacitivity was determined by liquid scintillation counting
in a solution of Omnifluor in Dioxane (8 g/1) using
proportional counting by a Nuclear Chicago Unilux II Scaler.
Quenching was consistent for known standards and all
experimental samples.
Data are presented as millimolar concentrations of
sugar in the tissue water. Tissue water was determined to
be approximately 80% of tissue wet weight for both anterior
and posterior intestinal samples, by comparing the wet weights
and dry weights (after 24 hours at 100°C) of pooled samples.
page 6
Sugar Absorption by C. stelleri
Kenneth B. Robbins
RESULT
D-galactose at gn original concentration of 4.5 mM in
the medium was found to be concentrated to 7.0 mM within
posterior intestinal tissue after a three hour incubation.
Results of the accumulation of 3.0 mM D-galactose over an
8-hour period by posterior intestinal tissue are presented
in fig. 1. D-galactose at a medium concentration of 4.5
mM gave an anterior intestinal tissue concentration of 4.4
mM after 8 hours, apparently indicating the lack of a D¬
galactose transport system in this region of the intestine.
Original tissue D-galactose concentrations were less than
0.4 mM, and thus were not a problem in analysis.
The results of an experiment demonstrating saturation
kinetics over a three-hour incubation period by the D¬
galact se transport system of the posterior intestine are
presented in fig. 2. D-galactose accumulation has been divided
into a hypothetical diffusion component and saturable, carrier¬
mediated component, the latter being the difference between
total transfer and the diffusion component. On this basis
the maximum velocity of the transport system (Vmay) is
0.8 millimoles D-galactose/l tissue fluid/hr, and K, the
concentration of substrate necessary to give half the maxi¬
mum velocity, is less than O.1 mM.
D-glucose at an original medium concentration of 5.5 mM
was found to be concentrated to approximately 8.0 mM in both
Page 7
Kenneth B. Robbins
Sugar absorption by C. stelleri
anterior and posterior intestinal tissue over a three-hour
period (fig. 3). Three-O-methylglucose, a non-metabolizable
glucose analogue in other transport systems (Curran, 1973,
p. 188), was not concentrated by posterior intestinal tissue.
The accumulation of D-glucose, but not of 3-0-methylglucose,
was substantiated by an experiment in which 3-0-methylglucose
tracer exhibited only passive diffusion in both a D-glucose
and 3-0-methylglucose medium (fig. 4). This demonstrates
that the difference in transport of the two sugars is due
to carrier specificity rather than the need for a metabolic
substrate in the medium.
The analysis of D-glucose transport was complicated
by the presence of an average of 3 mM D-glucose in the
original intestinal tissue of chitons starved for three
weeks. Chemical analysis thus gave higher D-glucose
concentrations than tracer analysis for the first few hours
of tissue incubation in a D-glucose medium. By the fourth
hour, however, radioactive and chemical analysis gave
approximately the same results. This was assumed to indicate
the replacement of original cellular D-glucose with D-glucose
from the medium by the process of exchange diffusion.
D-galactose transport in the posterior intestine was
not greatly inhibited by the presence of a relatively high
concentration of D-glucose, thus ruling out competition of
these two sugars for the same carrier. A concentration of
page 8
Kenneth B. Robbins
Sugar absorption by C. stelleri
0.2 mM D-galactose in the presence of 3.0 mM D-glucose gave
a tissue/medium D-galactose concentration ratio (T/M) av¬
eraging 14.5, while a 0.2 mM control gave a T/M of 17.0
over a three-hour period,
DISCUSSION
A number of assumptions were made in the calculationan
and interpretation of data. Tissue water was approximated
at 80% of tissue wet weight, and sugar was assumed to be
spread uniformly throughout that fluid. Tissue water,
however, is actually a composite of adherent medium,
extracellular fluid and intracellular fluid. There are
furthermore a variety of cell types composing the tissue,
not all of which may be participating in absorption. The
sugar concentrations presented are thus averages of all the
uncontrollable factors related to tissue structure and do not
represent true intracellular concentrations.
It is assumed that transport is a one-way process over
the tie interval studied, and the possible effects of dif¬
fusion and outward transport were not delat with quantitatively
in simple uptake experiments. These effects over a three¬
hour period may be considerable, however, since an everted
sac is presumably filled with sugar by the action of dif¬
fusion over that interval.
Other assumptions made were that all transported sugars
page 9
Kenneth B. Robbins
Sugar absorption by C. stelleri
were fee in solution in the cells, that tissue slicing dam¬
age was minimal, and that the contents of all cells and
organelles were released upon homogenization. The effects
of metabolism were ignored, since chemical and radioactive
tracer analysis gave approximately the same results in all
experiments when original tissue concentrations were
accounted for.
In consideration of all these factors, the concentrations
of sugar determined in these experiments are biased, if at
all, towards a lower tissue/medium concentration ratio.
Thus,
an accumulation of sugar above medium concentration by a
tissue definitely indicates active transport, while no
accumulation (T/M 1) does not necessarily negate that
possibility.
It is also important to realize that the rate of sugar
absorption by intestinal tissue varies from one animal
source to another, thus making the comparison of rates in
separate uptake experiments impossible.
Experimental results thus indicate active transport
of D-galactose by the proximal posterior intestine and of
D-glucose by both the anterior and posterior intestine.
D-glucose transport by the proximal anterior intestine, and
D-galactose transport by the posterior intestine were both
observed in the everted sac studies of Lawrence and Lawrence
(1967). The fact that D-galactose was not accumulated by
anterior intestinal tissue which accumulated D-glucose, and
the fact that D-galactose transport by posterior intestinal
page 10
Kenneth B. Robbins
page 11
Sugar absorption by C. stelleri
tissue was not substantially inhibited by high concentrations
of D-glucose support the belief of Lawrence and Lawrence
(1967) that at least two monosaccharide transport systems
are operating in the intestine of this organism.
The velocity of D-galactose accumulation in the
experiment demonstratin saturation of galactose transport
(fig. 2) is represented by the final tissue concentration
after a three-hour incubation period. The experimental
values thus do not directly represent initial rates, as is
desired. However, transport is slow enough that it is
assumed the final concentrations are proportional to initial
rates. Transport analyzed in this way yields a curve which
can be divided hypothetically into a saturable and un¬
saturable component, the linear unsaturable component being
attributed to net diffusion. The value for Vpgy determined
for the saturated D-galactose system, 0.8 mM/hr, may actually
be somewhat lower than the true value due to the effects
of outward transport which were not considered in this
experiment. Nevertheless, even with a two-fold increase
in rate, transport remains extremely slow when compared to
a corresponding mammalian system. The D-galactose transport
system of hamster intestine, for example, has aI
of
max
120 mM/hr (Crane, 1960). The difference in rates is not
unexpected, however, as there are great differences in the
metabolic rates and energy requirements of these two
Kenneth B. Robbins
Sugar absorption in C. stelleri
organisms. The low temperature environment (11°-14°C)
of Cryptochiton stelleri may, in particular, depress
the rate of any carrier-mediated transport process
(Lawrence and Lawrence, 1967a).
Another unique feature od D-galactose transport in
Cryptochiton stelleri is the extremely low K of the system.
The value cannot be accurately determined from the graph
of carrier transport in fig. 2, but it is definitely in
the range of 0.1 mM or less. The corresponding Ky for
hamster intestinal D-galactose transport is 2.2 mM (Crane,
1960). The D-galactose transport system of Cryptochiton
stelleri thus responds most efficiently at low concentrations,
perhaps compensating somewhat for the low transport rate.
The D-glucose transport system in the proximal posterior
intestine seems to be extremely specific, as 3-0-methylglucose
is not apparently concentrated. Lawrence and Lawrence (1967)
did not detect an accumulation of D-glucose in everted
sacs of posterior intestine. Nevertheless, a decrease
in D-glucose concentration and no decrease in 3-0-methyl¬
glucose concentration was noted in the serosal compartment.
The decrease in D-glucose was attributed to metabolism.
My results suggest, however, that D-glucose was not
metabolized, but merely accumulated in the tissue, while
3-0-methylglucose was not accumulated. The absorption of
D-glucose within the posterior intestinal tissue without
Page 12
Sugar absorption in C. stelleri
Kenneth B. Robbins
unidirectional transport across the intestinal wall seems
reasonable, as Gabe and Prenant (1949) have reported
significant glycogen stores in that tissue for three species
of chitons. Presumably, D-glucose was actively transported
into epithelial cells, but was not converted to glycogen
during the course of the experiments.
Although this study demonstrates the accumulation of
sugar by intestinal tissue, a process which most certainly
is important in the trans-intestinal transport process, no
investigation of Cryptochiton stelleri has yet determined
the entire mechanism by which sugars and other nutrients
are transported across the intestinal wall and into the
body fluids. Histological studies of chiton intestine have
been done (Fretter, 1937; Gabe and Prenant, 1949), but
no cells show the characteristic microvilli of mammalian
epithelia. In mammals, transport is localized in the
microvilli region of the epithelial cells (Wilson, 1962),
thus explaining the unidirectional flux of nutrients across
the gut wall. Exactly how unidirectional flux is achieved
by the intestinal epithelia of Cryptochiton stelleri
remains the greatest enigma of this invertebrate transport
system.
SUMMARY
1. The uptake of D-glucose and D-galactose by the intestine
of Cryptochiton stelleri (Middendorff, 1846) was studied
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Kenneth B. Robbins
Sugar absorption by g. stelleri
in vitro by the accumulation of sugar within intestinal
tissue.
2. Proximal anterior intestinal tissue actively absorbs
D-glucose.
3. Proximal posterior intestinal tissue actively absorbs
D-glucose and D-galactose, via separate transport systems.
4. The posterior intestinal D-glucose trasnport system is
apparently incapable of transportin 3-0-methylglucose, a
non-metaboizable glucose analogue in other transport systems.
5. D-galactose uptake by the posterior intestine is very
slow in mammalian terms (Va
= 0.81 mM/hr), although the
D-galactose transport system of Cryptochiton stelleri
shows a greater sensitivity (K, - 0.1 mM) to D-galactose
than does the equivalent system in hamster intestine.
This indicates a slow but efficient accumulation of D¬
galactose at low concentrations by this invertebrate.
ACKNOWLEDGMEN
The author wishes to thank Dr. Frederick A. Fuhrman and
the rest of the faculty and staff of Hopkins Marine Station
for encouragement in this endeavor.
page 14
KEnneth B. Robbins
page 15
Sugar absorption of C. stelleri
LITERATURE CITED
Crane, Robert K.
1960. Studies on the mechnism of the intestinal absorption
of sugars. Biochem. biophys. Acta 45: 477-482; 4 figs.
(23 April 1960)
Crane, Robert K.
1968. Absorption of sugars. Pages 1323-1351 in Charles
F. Code, ed. Alimentary canal. Vol. III: Intestinal
absorption. Section 6 of Handbook of physiology.
Washington, D.C. (American Physiological Society)
Crane, Pobert K., & Paul Mandelstam
1960. The active transport of sugars by various prep¬
arations of hamster intestine. Biochim. biophys.
Acta. 45: 460-476.
Curran, Peter F.
1973. Active transport of amino acids and sugars.
Functional and Structural Nature of Biomembranes: II.
New York (MSS Information Corporation) pp. 185-202.
Ferguson, J. C.
1964. Nutrient transport in starfish II: Uptake of
nutrients by isolated organs. Biol. Bull. 126: 391-406.
Fretter, Vera.
1937. The structure and function of the alimentary canal
of some species of Polyplacophora (Mollusca). Trans.
Roy. Soc. Edin. 70 (4): 119-164.
(enneth B. Robbins
Sugar absorption by C. stelleri
Gabe, M., & M. Prenant.
1949. Contribution a l'etude cytologique et histochimique
du tube digestif des Polyplacaphores. Arch. Biologie
60: 39-77.
Lawrence, A. L., & D.C. Lawrence
1967. Sugar absorption in the intestine of the chiton,
Crytochiton stelleri. Comp. Biochem. and Physiol.
22: 341-357.
Lawrence, D. C., & A. L. Lawrence
1967a. Effects of temperature upon mechanisms for active
transport of monosaccharides in the intestine of a chiton,
Cryptochiton stelleri. Amer. Zool. 7: 194.
Lawrence, A. L. & D. S. Mailman
1967. Electrical potentials and ion concentrations across
the gut of Cryptochiton stelleri. Journ. Physiol. 193:
535-545.
Lawrence, A. L., D. S. Mailman, & Robert Earl Puddy
1972. The effect of carbohydrates on the intestinal
potentials of Cryptochiton stelleri. Journ. Physiol.
225 (3): 515-527.
Neame, K. D., & T. G. Richards
1972. Elementary Kinetics of Membrane Carrier Transport.
i - xii + 120 pp; illus. New York (John Wiley & Sons)
Puddy, Robert Earl
1970. Ion and carbohydrate absorption in the posterior
intestine of Cryptochiton stelleri. Ph.D. thesis, Univ.
of Houston.
page 16
Sugar absorption by C. stelleri
page 17
Kenneth B. Robbins
Randal, D. D., & R. F. Derr
1965. Trehalose: occurence and relation to egg, diapause,
and active transport in the differential grasshopper,
Melanopus differentialis. Journ. of Insect Physiol.
II: 329-335.
Wilson, T. Hastings
1962. Intestinal Absorption. i-ix + 263 pp.; illus.
Philadelphia (W. B. Saunders Company)
Wright, R. O.
1968. Sugar transport in the gut of a limpet, Megathura
crenulata. Masters Thesis. Univ. of Houston.
Kenneth B. Robbins
Sugar absorption by C. stelleri
FIGURE CAPTIONS
fig. 1. Uptake of D-galactose by posterior intestinal
tissue over an 8-hr incubation period. Initial medium con¬
centration was 3.0 mM. Samples were taken in duplicate at O.
1, 2, 3, 4, 6, and 8 hrs, and analyzed chemically and
radioactively for D-galactose. Both methods yielded similar
results; radioactive analysis is presented here.
fig. 2. Relationship of the rate of D-galactose uptake to
the concentration of D-galactose in the incubation medium.
Rate of uptake is plotted as the concentration of D-galactose
in posterior intestinal tissue after a 3-hr incubation
period. Radioactive and chemical analysis gave equivalent
results; radioactively-determined data are presented here.
Total transport is divided into a hypothetical insaturable
component and saturable component, attributed to net diffusion
and carrier transport, respectively. On this basis, Vmay
equals 0.81 mM/hr, and K 0.1 mM. Vpay
may actually be
somewhat higher, due to the possible effects of outward
transport which were not considered in analysis.
fig. 3. The uptake of D-glucose by intestinal tissue.
Proximal anterior and proximal posterior intestinal tissue
were incubated for 3 hrs in a 5.5 mM D-glucose medium.
Hourly samples were recovered in duplicate and analyzed by
page 18
enneth B. Robbins
Sugar absorption by C. stelleri
radioacitve and chemical techniques. Chemical analysis
gave slightly higher values for the first two hours, due to
the presence of 1.8 mM D-glucose and 3.1 mM D-glucose in
original anterior and posterior tissue samples. Complete
exchange diffusion seemed to occur by the third hour, however,
as radioactive and chemical analysis gave equivalent results,
Radioactively-determined data are presented here, as these
data definitely indicate accumulation of D-glucose from
the medium and are less biased to high tissue/medium D¬
glucose ratios.
fig. 4. Specificity of the posterior intestinal D-glucose
transport system. Incubations of posterior intestinal
tissue were carried out in the monosaccharide media described
beolow. Samples were taken in duplicate at 0, 2, and 4 hrs,
analyzed chemically for D-glucose and radioactively to
determine the behavior of tracers in each environment. The
contribution of added tracers to the total sugar concentration
was negligible ( 3x10 M).
A. D-glucose at 3.0 mM plus 0.04 uc/ml D-glucose-u-c.
Both D-glucose and labeled D-glucose were accumulated by the
tissue. The initial difference between chemical and radio¬
active analysis was due to the presence of 3.0 mM glucose,
in the tissue before addition of sugar to the medium. This
was apparently completely exchanged for D-glucose from the
medium by the fourth hour of incubation.
page 19
Kenneth B. Robbins
Sugar absorption by C. stelleri
B. Three-O-methylglucose at 3.0 mM plus 0.04 uc/ml 3-0-
methyl-+C-D-glucose. Labeled 3-0-methylglucose was not
apparently concentrated by the tissue. Presumably, original
tissue glucose dropped due to passive diffusion.
C. D-glucose at 3.0 mM plus 0.04 uc/ml 3-0-methyl-c-
D-glucose. D-glucose conentration, determined chemically,
rose within the tissue as in A. Labeled 3-0-methylglucose,
however, behaved similarly to the labeled 3-0-methylglucose
in B, and apparently was not concentrated.
page 20
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