BIOCHIMICA ET BIOPHYSICAACTA
717
BBA 75059
T H E E F F E C T OF ESTROGEN ON SUGAR TRANSPORT IN T H E RAT
UTERUS
ROBERT ROSKOSKI, JR." AND DONALD F. STEINER
Department of Biochemistry, University of Chicago, Chicago, Ill. ( U.S.A .)
(Received April 3rd, 1967)
(Revised manuscript received May 26th, 1967)
SUMMARY
I. The characteristics of sugar transport in the isolated rat uterus were studied
using the non-metabolized glucose analogue 3-0-methyl-D-glucose. The effect of
estrogen injection into ovariectomized rats on the transport rate was also determined.
2. Estrogen injection increased the initial rate of 3-0-methylglucose transport
into uterine cells. The effect was evident at 2 h, and maximal at 4 h. After 12 h the
control value was attained again.
3. Sugar transport obeyed Michaelis-Menten kinetics. Estrogen increased the
Vmax 2-fold, but did not change the Kin.
4. Sugar transport exhibited substrate specificity, competition between sugar
pairs, and countertransport.
5. Estrogen increased the rate of sugar effiux from the uterus.
6. Estradiol, insulin, N-ethylmaleimide, NaCN, diisopropylphosphorofluoridate, p-chloromercuribenzoate and dinitrophenol did not alter the transport rate
when added in vitro.
7. It is concluded that estrogen increases the rate of sugar transport in the
rat uterus and that sugar transport takes place by means of a specific mobile carrier
transport system.
INTRODUCTION
The stimulation of uterine anabolic activity by estrogenic hormones is accompartied by significant early changes in the carbohydrate metabolism of this tissue.
These include: an increase in glucose uptake in isolated rat uteri 4 h after estrogen
injection 1, an increase in glycogen content2, s which is detectable after 2 h of estrogen
treatment 4, and an increase in the incorporation of uniformly labeled [14C]glucose
* Presently Captain, USAF, MC, Wilford Hall USAF Hospital, Aerospace Medical Division
(AFSC), Lackland Air Force Base, Texas, U.S.A.
Biochim. Biophys. Acta, x35 (z967) 7z7-726
718
R. ROSKOSKI, Jr., D. F. STEINER
into uterine lipid, RNA, protein and CO, within 1-2 h after estrogen treatment 5.
SZEGO AND ROBERTS1 and NICOLLETTE AND GORSKI5 have suggested that glucose
transport may be stimulated by estrogen administration. However, HALKERSTONet al. ~
found no significant changes in the distribution of D-xylose in uteri from ovariectomized rats 1.5 and 6.0 h after estrogen injection, and concluded that estrogen does
not alter sugar permeability in uterus. The available literature on effects of estrogen
on sugar and amino acid transport in the uterus has been reviewed recently 7-9.
In the present study the rate of transport of the non-metabolized glucose
analogue 3-O-methylglucose was measured. This compound is transported into
erythrocytes at a rate that very closely approximates that of glucose 1°. These
studies differ from those of HALKERSTON et al. in that short incubations in vitro,
and not administration of glucose analogues in vivo, were employed to measure the
transport rate. The present study demonstrates the existence in the uterus of a
sugar transport system with many characteristics similar to transport systems of
other tissues, but also responsive to estrogen.
METHODS AND MATERIALS
Chemicals
The 3-O-methyl-D-glucopyranose, D-mannose, D-galactose, D-xylose, L-arabinose, and D-sorbitol were obtained from Calbiochem Corp. The D-lyxose and 2deoxy-D-glucose were obtained from Nutritional Biochemicals Corp. The D-glucose
was purchased from Pfanstiehl Laboratories, Inc. The 3-0-[14C!methyl-D-glucose
(2.72 mC/mmole) and D-EI-3H]sorbitol (168 mC/mmole) were obtained from New
England Nuclear Corp. Crystalline I7fl-estradiol was purchased from the Sigma
Chemical Co.
Incubation medium
Incubations were carried out at 37 ° in Krebs-Ringer bicarbonate previously
equilibrated with O,-CO, (95:5, v/v) (pH 7.4) 11. The incubation medium contained
o.io/zC of 3-O-[14C'methyl-D-glucose and 0.50 ~uC of D-[I-3H:sorbitol per ml. Carrier
3-O-methyl-D-glucose and D-sorbitol were added to a final concentration of I.O mM
except where otherwise specified. All additions to the buffer were made by adding
5 or IO/~1 of concentrated solution per ml of buffer or by dissolving the substance
directly in fleshly prepared buffer to give the desired final concentration. Each
uterine horn was incubated in 0.5 ml of the medium for the time specified.
Animals
Sprague-Dawley rats weighing about 11o-13o g were ovariectomized by the
lateral approach using ether anesthesia. Animals operated on the same day were
used for a given experiment 7-30 days postoperatively. The animals were allowed
access to Rickland rat/mouse diet and water at all times.
Hormone administration
A stock solution of 2.0 mg/ml I7fl-estradiol in 95% ethanol was diluted to
20/zg/ml in 20% ethanol before each experiment. I/~g (0.05 ml) was injected subcutaneously at the time indicated.
Biochim. Biophys. Acta, 135 (I967) 717-726
719
SUGAR TRANSPORT IN UTERUS
Preparation and incubation of the uterus
The animals were sacrificed b y decapitation. The abdomen was opened and the
uterine horns were excised and placed in o.15 M NaCI at ambient temperature. The
uterine horns were transferred to a filter paper moistened with normal saline and the
excess connective tissue was removed. Each uterine segment was blotted on dry
filter paper and transferred to o.5 ml incubation medium in a round-bottom plastic
tube in a Dubnoff metabolic shaking incubator under an atmosphere of O2-CO 2
(95:5, v/v). After the incubation period, the segments were immersed briefly in
physiologic saline at room temperature to remove adhering drops of medium, incised
longitudinally, blotted on dry filter paper, and weighted on a Roller Smith torsion
balance. Each uterine horn was transferred to a I2-ml graduated conical centrifuge
tube containing about o.5 ml of water. To prepared controls unincubated uterine
segments were added to conical centrifuge tubes with a small aliquot of the incubation medium and standard amounts of 3-O-[14C]methyl-D-glucose and D-[I-ZH]sorbitol in o.5 ml of water. All of the tubes were placed in a boiling-water bath for
15 min, and then allowed to cool to room temperature. The contents of each tube
were transferred to a small, conical, glass tissue grinder (Duall tissue grinder, Kontes
Glass Co.), homogenized for about 15 sec and returned to the graduated tube. The
homogenizer was rinsed twice with o.5-ml portions of distilled water which were also
transferred to the conical tube. The homogenates were deproteinized by adding
o.Io ml o.15 M Ba(OH)2, followed after 1o min by o.Io ml 5 % ZnSO4, and centrifuged at 75o × g for 15 min. The solution volumes were recorded and I.o-ml aliquots were added to 15 ml of BRAY'S solution 1~ in a plastic vial for 14C and 3H determination in a Packard Tri-Carb liquid-scintillation counter. The counts due to each
isotope were calculated b y the method of OKITA et al. 18.
Definitions
3-O-Methyl-D-glucose space. This is defined as the volume of tissue water necessary to contain the 3-O-methyl-D-glucose of the tissue at the concentration of the
incubation medium.
3-O-Methyl-D-glucose space (/,l/g)
counts/rain 3-O-[14C]methyl-D-glucose per g tissue
counts/rain 3-O-[14C]methyl-D-glucose per/,1 incubation m e d i u m
D-Sorbitol space. This is defined and calculated in a manner analogous to that
for the 3-O-methyl-D-glucose space.
Intracellular 3-O-methyl-D-glucose space. This is the difference between the 3O-methyl-D-glucose space and the D-sorbitol space:
Intracellular 3-O-methyl-D-glucose space = 3-O-methyl-D-glucose space -- sorbitol space
RESULTS
Sugar uptake in ~terine horns from untreated and 4-h estrogen-treated ovariectomized
rats
The upper two curves (Fig. I) show the rate of 3-O-methyl-D-glucose uptake
in the estrogen-treated and untreated cases, the middle curve shows the rate of
Biockim. Biophys. Acta, I35 (1967) 717-726
720
R. ROSKOSKI, Jr., D. F. STEINER
sorbitol u p t a k e in b o t h cases, a n d the lower two curves show t h e r a t e of i n t r a cellular 3-O-methyl-D-glucose u p t a k e . The l a t t e r curves indicate t h a t t h e initial r a t e
of 3-O-methyl-D-glucose a p p e a r a n c e in t h e i n t r a c e l l u l a r space was increased a b o u t
lOO% w i t h estrogen t r e a t m e n t . The r a t e of sorbitol u p t a k e was t h e s a m e in b o t h t h e
control a n d e s t r o g e n - t r e a t e d tissues. This indicates t h a t the r a t e of a p p e a r a n c e of
i n c u b a t i o n m e d i u m in t h e e x t r a c e l l u l a r space was t h e s a m e in each case. Therefore,
t h e increased r a t e of t r a n s p o r t of 3-0-methyl-D-glucose c a n n o t be ascribed to a n y
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Fig. I. Sugar uptake in uterine horns from estrogen-treated (4 h) and untreated ovariectomized
rats. The sugar spaces were determined as described in METHODSAND MATERIALS.Open symbols
represent data from estrogen-treated animals, and solid symbols represent data from untreated
animals. Each point represents the mean of eight determinations. The standard error of t h e
mean of each point is 4- 4.0 #1 or less. O - - O , 3-O-methyl-D-glucose space; A - - A , D-sorbitol
space; F]--[3, intracellular 3-O-methyl-D-glucose space.
Fig. 2. Determination of Km and Vmax of 3-0-methyl-D-glucose transport in uterine horns from
estrogen-treated (4 h) and untreated ovariectomized animals. Initial rates were determined
during a 5-rain incubation for the estrogen-treated group and a Io-min incubation in the control
group. Each point represents the mean of eight determinations. O - - O , treated; O--11, untreated.
difference in t h e r a t e of transfer of 3-O-methyl-D-glucose from the m e d i u m to the
site of t r a n s p o r t .
The w a t e r c o n t e n t of t h e u t e r u s was 7 9 % in t h e c o n t r o l u t e r u s (79 ° / , l / g ) a n d
83 % in t h e u t e r u s from t h e 4-h e s t r o g e n - t r e a t e d a n i m a l (830/,l/g). The 3 - 0 - m e t h y l D-glucose space c o r r e s p o n d e d to 9 6 % of t h e t o t a l cell w a t e r in t h e control (76o#1
p e r g: 79 ° #1 p e r g) a n d to 95 % of t h e t o t a l cell w a t e r in t h e t r e a t e d case (775/xl
p e r g: 830/zl p e r g). Thus, t h e u t e r u s d i d n o t c o n c e n t r a t e 3-O-methyl-D-glucose
relative to t h e m e d i u m . A l t h o u g h these results could be e x p l a i n e d b y t h e simple
diffusion of 3-O-methyl-D-glucose from t h e m e d i u m into t h e tissue cells, evidence
to be p r e s e n t e d indicates the existence of a specific sugar t r a n s p o r t system.
Biochim. Biophys. Acta, 135 (i967) 717-726
721
SUGAR TRANSPORT IN UTERUS
Determination of Kn, and Vr~x of transport in uteri from untreated and estrogen-treated
animals
The Km and Vmax for 3-O-methyl-D-glucose transport were determined b y
means of double reciprocal plots (I/V vs. I/[S] as shown in Fig. 2. Initial velocity of
transport was approximated b y using short incubation periods (i.e., influx >> efflux).
The uteri from untreated animals were incubated for IO min, and those from 4-h
estrogen-treated animals were incubated for 5 min in order to utilize the more
linear portion of the uptakes curves (Fig. I). As a result of this restriction 3-0methyl-D-glucose and sorbitol did not equilibrate completely with the extracellular
tissue fluid and therefore the cells of the uterus were exposed to a lower average concentration of the 3-O-methyl-D-glucose than that in the medium. This undoubtedly
produced a systematic error such that the observed Km of approx. 22 mM in both
cases represents a m a x i m u m estimate only. Correction for this error was not attempted
because it would require several assumptions concerning the diffusion of the substrate into the tissue, and it would not alter the interpretation with regard to either
the mechanism of transport on the effect of estrogenic hormone. The calculated Vmax
in the estrogen-treated group was about twice that in the untreated group
(168 m#moles/g •min vs. 92 mffmoles/g •min). These results can be interpreted as indicating saturation of a finite number of sites of diffusable membrane carriers; they
cannot be reconciled with the kinetics of simple diffusion which would not exhibit
saturation ~.
Countertransport and competition between 3-O-methyl-o-glucose and glucose
The upper and lower curves (Fig. 3) were determined using uteri from untreated
ovariectomized animals incubated without glucose or with 20 mM glucose, respectively. Glucose inhibited 3-O-methyl-D-glucose transport into the uterine cells. Presumably this is due to mutual competition for transport sites, and is consistent with
a carrier-mediated transport mechanism.
To further distinguish a mobile carrier transport system from simple diffusion
or other possible types of facilitated diffusion, countertransport experiments were
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Fig. 3. C o u n t e r t r a n s p o r t o f 3-O-methyl-D-glucose in u t e r i n e h o r n s f r o m u n t r e a t e d (left) and
estrogen-treated (4 h, right) o v a r i e c t o m i z e d rats. T h e e x p e r i m e n t is described u n d e r R~SULTS.
E a c h p o i n t r e p r e s e n t s t h e a v e r a g e of six d e t e r m i n a t i o n s . T h e vertical lines r e p r e s e n t 2 standard
errors o f t h e m e a n .
Biochim. Biophys. Aaa, i 3 5 (i967) 7 1 7 - 7 z 6
722
R. ROSKOSKI, J r . , D. F. STEINER
carried out in vitroZ°,14,15. Countertransport is defined as a shift in the distribution
of a sugar between the intracellular and extracellular compartments induced by a
difference in concentration across the cell membrane of another sugar that competes
for the same carrier site.
Equilibration of 3-0-methyl-D-glucose was allowed to occur b y incubating
uterine horns in glucose-free medium for 30 min. They were then transferred to a
second medium containing 20 mM glucose. The intracellular 3-0-methyl-D-glucose
space decreased when glucose was added, as shown in Fig. 3. This reflects a decrease
in the intracellular concentration of 3-0-methyl-D-glucose which, under these conditions, was transported from the region of lower concentration (intracellular) to the
region of higher concentration (extracellular) against a concentration difference.
These results are best explained by a mobile carrier transport system44°, ~5.
Substrate specificity
Various sugars were examined for their ability to produce countertransport of
3-O-methyl-D-glucose, as a means of determining the substrate specificity of the
glucose transport system. Addition of a substrate that competes with 3-0-methyl-Dglucose for its carrier will produce a net decrease in the 3-0-methyl-D-glucose space.
Other substrates should not influence the system. Uterine horns were incubated for
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Fig. 4- S u b s t r a t e specificity of s u g a r t r a n s p o r t in u t e r i n e h o r n s f r o m u n t r e a t e d (left) a n d e s t r o g e n t r e a t e d (4 h, right) o v a r i e c t o m i z e d rats. E a c h u t e r i n e h o r n w a s i n c u b a t e d in m e d i u m c o n t a i n i n g
I m M 3-O-methyl-D-glucose for 3o min. I t w a s t h e n t r a n s f e r r e d to m e d i u m t h a t c o n t a i n e d t h e
t e s t s u g a r a t a c o n c e n t r a t i o n of 2o m M a n d i n c u b a t e d 15 rain. T h e b a r r e p r e s e n t s t h e m e a n of
s i x d e t e r m i n a t i o n s , a n d t h e vertical line t h r o u g h t h e b a r r e p r e s e n t s 2 s t a n d a r d errors of t h e m e a n .
30 min in a medium containing I mM labeled 3-O-methyl-D-glucose. The intracellular 3-O-methyl-D-giucose space was determined in control uteri, and others
were transferred to the same medium containing 20 mM of the test sugar and were
incubated for 15 min longer. The results obtained with uterine horns from untreated and 4-h estrogen-treated ovariectomized rats were similar qualitatively
(Fig. 4)- D-Glucose, D-mannose, 2-deoxy-n-glucose, and D-xylose decreased the intracellular 3-O-methyl-D-glucose space, indicating competition with 3-0-methyl-Dglucose for its carrier. On the other hand, D-galactose and its C5 analogue L-arabinose
Biochim. Biophys. Acta, 135 (1967) 717-726
SUGAR TRANSPORT IN UTERUS
723
d i d n o t c o m p e t e w i t h 3-0-methyl-D-glucose t r a n s p o r t . The reason for t h e difference
in b e h a v i o r of D-mannose a n d its C 5 analogue, D-lyxose, is n o t known. BATTAGLIA
AND RAI~DLE le h a v e r e p o r t e d t h a t D-galactose a n d L-arabinose, are t r a n s p o r t e d ind e p e n d e n t l y in t h e r a t d i a p h r a g m .
Sugar e : u x from uterine horns from untreated and estrogen-treated animals
B o t h u t e r i n e horns from e s t r o g e n - t r e a t e d (4 h) a n d u n t r e a t e d a n i m a l s were
i n c u b a t e d in i mM 3-O-methyl-D-ghicose for 45 min. The 3-O-methyl-D-glucose
space was d e t e r m i n e d in one horn from each animal. T h e o t h e r horn was t h e n t r a n s ferred to successive o.6-ml aliquots of fresh sugar-free K r e b s - R i n g e r b i c a r b o n a t e
solution a t o, 5, io, a n d 15 min. The 3-0-methyl-D-glucose space of this horn was
d e t e r m i n e d after i n c u b a t i o n for a t o t a l p e r i o d of 30 min in the sugar-free m e d i u m ,
3-0-methyl-D-glucose efflux (3-O-methyl-D-ghicose space - - sorbitol space) was calc u l a t e d after 5, IO, 15, a n d 30 min of i n c u b a t i o n in t h e sugar-free m e d i u m . As shown
in Fig. 5 t h e initial efflux from t h e uteri of the e s t r o g e n - t r e a t e d animals was a b o u t
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Fig. 5. 3-O-Methyl-D-glucose efflux from 4-h estrogen-treated and untreated ovariectomized rats.
The experimental procedure is described under RESULTS. Each point represents the mean of six
determinations, and the vertical line represents 2 standard errors of the mean. O - - O, untreated;
O 1 0 , 4-h estrogen-treated ovariectomized rats.
Fig. 6. Time course of the estrogen-induced sugar transport increase. Experimental animals received i.o/~g of i7~-estradiol in 20% ethanol at zero time. They were sacrificed at the stated
time, and the rate of uterine 3-O-methyl-D-glucose transport was determined during a io-min
incubation period. Controls received vehicle only for 4 h. Each point represents the mean of five
determinations. The vertical lines represent 2 standard errors of the mean.
twice as g r e a t as t h a t from t h e uteri of t h e u n t r e a t e d animals (235/z#moles/g. m i n
vs. 8 8 / , # m o l e s / g - m i n ) . Thus, estrogen p r o d u c e s b o t h a n increase in 3-O-methyl-Dglucose influx as well as efllux, as w o u l d be e x p e c t e d from t h e mobile carrier model.
Time course of the transport response to estrogen
3-O-Methyl-D-glucose t r a n s p o r t was d e t e r m i n e d in o v a r i e c t o m i z e d r a t s sacrificed after v a r i o u s periods o f estrogen t r e a t m e n t . I n c u b a t i o n periods of i o m i n were
e m p l o y e d in o r d e r to d e t e r m i n e t h e initial t r a n s p o r t velocities. A f t e r I h of estrogen
Biochim. Biophys. Acta, x35 (x967) 7x7-726
724
R. ROSKOSKI, J r . , D. F. STEINER
treatment, the transport rate was increased a shght, but not significant, amount
(P < 0.2) (Fig. 6). A 3-fold increase occurred by 2 h (P < o.oi). The maximal response occulred at 4 h and thereafter the rate decreased to the initial value by 12 h.
Effect of x7#-estradiol and insulin in vitro on sugar transport
Uterine horns from untreated ovariectomized rats were preincubated in
various concentrations of ITfl-estradiol in Krebs-Ringer bicarbonate for 15 mill at
37 °. 3-O-Methyl-D-glucose transport was measured during a subsequent I5-min incubation period in a medium containing the same amount of I7/5-estradio]. Contralateral horns were treated in a similar manner in estrogen-free media. The results
plotted in Fig. 8 show that I #M, 3.8 #M and IO #M I7/5-estradiol does not affect
transport under these conditions in vitro. Insuhn, which increases glucose transport
in vitro by diaphragml~, is and heart 1~, did not significantly increase 3-O-methyl-Dglucose transport in the isolated uterus (Fig. 7).
Effect of various metabolic inhibitors on sugar transport
Uterine horns from untreated ovariectomized animals were preincubated for
15 mill in Krebs-Ringer bicarbonate containing the specified compound. Transport
was measured during a subsequent Is-min incubation period in the presence of
the inhibitor. Control horns were treated similarly in the absence of inhibitor.
2,4-Dinitrophenol, N-ethylmaleimide, iodoacetate, p-chloromercuribenzoate, diisopropylphosphorofluoridate and NaCN, at the concentrations indicated, did not
affect the rate of 3-O-methyl-D-glucose transport significantly (Fig. 8). Under similar
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Fig. 7- Effect of IT/~-estradiol and insulin in vitro on s u g a r t r a n s p o r t . The e x p e r i m e n t is described u n d e r RBSULTS. E a c h b a r represents the m e a n of five d e t e r m i n a t i o n s and the vertical ]ines
represents 2 s t a n d a r d errors of the mean.
Fig. 8. Effect of various metabolic inhibitors on s u g a r t r a n s p o r t . The e x p e r i m e n t is described
u n d e r RESULTS. E a c h b a r represents the m e a n of five d e t e r m i n a t i o n s a n d the vertical lines rep r e s e n t 2 s t a n d a r d errors o£ the mean. Abbreviations: NEM, 2V-ethylmaleimide; PCMB, pchloromercuribenzoate; D F P , diisopropylphosphorofluoridate and D N P , 2,4-dinitrophenol.
Biochim. Biophys. Acre, i35 (1967) 717-726
SUGAR TRANSPORT IN UTERUS
725
conditions 2,4-dinitrophenol and NaCN have been reported to increase sugar transport in the rat diaphragrnlL 18 and the peffused isolated rat heart 19. BATTAGLIA
AND RANDLEle have reported that N-ethylmaleimide and p-chloromercuribenzoate
inhibit D-xylose accumulation in the rat diaphragm, but that diisopropylphosphorofluoridate is without effect.
DISCUSSION
The following observations support the theory that a specific diffusable
carrier transport system for sugars exists in rat uterus: (I)3-0-Methyl-D-glucose
transport exhibits saturation kinetics. This behavior can be interpreted most readily
in terms of saturation of a finite number of combining sites necessary for transport.
(2) The system exhibits substrate specificity as evidenced by the competitive inhibition of 3-0-methyl-D-glucose influx by D-mannose, D-glucose, 2-deoxy-Dglucose, and D-xylose, whereas D-galactose, L-arabinose, and D-lyxose did not compete. (3) The demonstration of counteitransport establishes the existence of a specific
mobile carrier type of transport system. It rules out simple diffusion and irreversible
transport mechanisms. (4) The sugar is not concentrated intracellularly.
HALKERSTON et aL e have reported that D-xylose distributes to almost all of the
cell water (92%) in the castrate uterus, and that estrogen treatment produces no
change. In their experiments D-[14Clxylose was injected into the rats 1.5 h before
sacrifice. The 92% figure for distribution indicates that equilibration had nearly
occurred and, therefore, their experiments gave no measure of the rate of entry of
the sugar into the uterine cell water. We have found also that uteri from castrate and
estrogen-treated animals transport sugars to approximately the same volume of
uterine cell water after incubation for I h. However, the rate of transport in the
latter case clearly is increased when measured after short inbation periods.
In this study the sorbitol space was defined as the extraceUular space. It was
found to be about 55 °/,l/g or 55%. HALKERSTON and co-workers e-8 found similar
values for the uterus using inulin, sucrose, and Na + distribution, but found values
of approx. 20% for other tissues (adrenal, thymus, liver diaphragm and gastrocnemius). Because of the discrepancy between the uterus and other tissues, these
authors concluded that inulin and sucrose penetrate uterine cells. This assumption
is unnecessary since the castrate uterus obviously does contain an exceptionally
large extracellular space. This is borne out by the high collagen content of the
tissue. The uterus from an ovariectomized rat contains about 60 mg of collagen per
g of wet tissue ~1, whereas rabbit striate muscle has only 4 mg of collagen per g wet
weight ~°, corresponding to its extraceUular space of about 200/0.
The administration of physiologic doses of I7fl-estradiol causes a 2-fold increase or more in the initial velocity of sugar transport (both influx and efflux)
(Figs. I, 2, 6) without any measurable change in the apparent K m of transport
(Fig. 2). The response appeared between i and 2 h. Incubation in the presence of
ITfl-estradiol was without effect in vitro. The fact that more than I h after administration of estrogen is required to produce an effect suggests that the increase is not due
to a primary physicochemical interaction of the hormone with the transport system
in the cell membrane. The studies of JENSEN AND JACOBSON~ with immature rats
indicate that I7fl-estradiol is already present in the uterus within 20 min after
Biochim. Biophys. Acta, 135 (x967) 717-726
726
R. ROSKOSKI, Jr., D. Y. STEINER
s u b c u t a n e o u s a d m i n i s t r a t i o n . Therefore, it seems likely t h a t the estr0gen-induced
increase in t r a n s p o r t depends u p o n a n t e c e d a n t metabolic changes.
E n h a n c e d t r a n s p o r t occurs as early as the increased glucose utilization in vitro
d e m o n s t r a t e d b y SZEGO AND ROBERTS1. I t occurs w i t h i n the same time period as the
increase in the i n c o r p o r a t i o n of glucose into COy RNA, lipid, a n d protein which
was d e m o n s t r a t e d b y NICOLLETTE AND GORSKI5. I t is n o t possible to determine
whether e n h a n c e d t r a n s p o r t c o n t r i b u t e s to the increase t h e y observed or whether
the changes simply occur simultaneously. Differences due to the a n i m a l age a n d
strain, as well as to the dosage of estrogen also m u s t be considered.
Increased t r a n s p o r t precedes the estrogen-induced a c c u m u l a t i o n of glycogen
in the u t e r u s reported b y BITMAN et al. 4. T h e y found only a small increase in glycogen
at 2 h a n d a steady increase u n t i l 12-14 h. Our results show t h a t b y 2 h after adm i n i s t r a t i o n of estrogen the rate of t r a n s p o r t already is increased 2-4-fold. The
a c c u m u l a t i o n of glycogen m a y result indirectly from increased t r a n s p o r t of sugar,
b u t a direct effect of estrogen u p o n reactions involved in glycogen synthesis or deg r a d a t i o n also m u s t be considered*a, 24.
ACKNOWLEDGEMENTS
The a u t h o r s wish to t h a n k Miss J. KING a n d Miss J. MALACHOWSKI for able
technical assistance.
This research was supported b y g r a n t s from the N a t i o n a l I n s t i t u t e s of H e a l t h
(AM-o4931) a n d the LoUy Coustan Memorial F u n d . D.F.S. is the recipient of a
U.S. Public H e a l t h Service Research Career D e v e l o p m e n t Award. R.R., Jr. is the
recipient of a U.S. Public H e a l t h Postdoctoral Fellowship (2 F2 HM 22, 798-o2).
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