Download Onset of changes in glucose transport across ocular barriers

Onset of Changes in Glucose Transport Across Ocular
Barriers in Streptozotocin-lnduced Diabetes
Joseph DiMattio, Jose A. Zadunaisky, and Norman Altszuler
In a previous study, measurements were made of facilitated and passive transport of glucose, using
[3H]-3-0-methyl-D-glucose and |l4C]-L-glucose, respectively, across blood-aqueous and blood-vitreous
barriers in long-term streptozotocin-diabetic rats. It was found that passive transport was increased,
while facilitated transport was decreased, possibly due to saturation of the transport system. The present
study examines the appearance of these changes in glucose transport at various times following streptozotocin (STZ) injection. Passive transport, as indicated by the L-glucose rate constant, began to
increase at about 10 days following induction of diabetes, stabilized at the elevated rates at 50-60 days
and persisted during the 170-day period of observation. Rate constants for (3H]-3-0-methyl-D-glucose
transport decreased within 1 day following induction of diabetes. Prevention of hyperglycemia by insulin
treatment upon onset of diabetes prevented the latter changes ruling out a direct effect of STZ. Glucose
infusion into normal rats produced a similar decrease in 3-O-methylglucose transport constants suggesting
that hyperglycemia was responsible for the early decrease in facilitated transport found in the diabetic
rats. It is speculated that increased passive transport of glucose may reflect an early loss in ocular
barrier integrity. The later decrease in carrier facilitated transport cannot be explained by hyperglycemia
alone and, thus, a loss in carrier function is suggested. Despite a decrease in facilitated transport,
absolute glucose entry rates are increased in the diabetic due to elevated plasma glucose, which serves
as an inward driving force, due to the significantly increased entry of glucose by the passive route.
Invest Ophthalmol Vis Sci 25:820-826, 1984
Materials and Methods
D-glucose and the nonmetabolizable analogue
3-O-methylglucose are transported across the bloodaqueous and blood-vitreous barriers of the rat by carrier-mediated facilitated diffusion.1'2 By contrast,
movement of L-glucose is by simple diffusion, and its
rate of transport is a measure of passive permeability.
We have found previously that rats with long-term
(6 months), streptozotocin-induced diabetes exhibited
significantly increased rates of passive transport across
the aqueous and vitreous barriers.3 The diabetic animals also showed a decreased rate of transport of
3-O-methylglucose, which was attributed to saturation
of transport sites due to the prevailing hyperglycemia
and possibly a decrease in carrier function. The present
study explores the development of the changes in glucose transport in relation to the onset and progression
of the streptozotocin-induced diabetic state.
All experiments were carried out in male albino
Sprague-Dawley rats 200-300 g, under sodium pentobarbital anesthesia (50 mg/kg, ip) with methods conforming to the ARVO Resolution on the Use of Animals in Research. Streptozotocin (65 mg/kg) was dissolved in citrate buffer solution (0.09 M in Saline,
pH 9.5) immediately before injection into a tail vein.
Plasma glucose was elevated within 24 hr and remained
elevated, as determined by periodic monitoring of urine
or blood using respectively, Keto-Diastix (Miles Laboratories; Elkhart, IN) or glucose oxidase,4 using the
Beckman Glucose Analyzer (Beckman Instruments;
Fullerton, CA).
The animals received no insulin except in some
acute experiments as indicated, and in such cases all
animals had demonstrated urinary glucose prior to
insulin administration. All other animals had plasmaglucose levels higher than 300 mg/dl at the time ocular
transport was studied.
Each experiment was performed using double-labeling with [3H]-3-O-methyl-D-glucose and [14C]-Lglucose, thus allowing the determination of transport
rates of both D- and L-glucose in each test animal.
The procedure for determining blood ocular transport
rate has been outlined in detail previously.1 In sum-
From the Department of Physiology and Biophysics and the Department of Pharmacology, New York University School of Medicine,
New York, New York.
Supported by grant EY-04418 and training grant EY-07009 from
the National Institutes of Health, Bethesda, Maryland.
Submitted for publication: July 1, 1983.
Reprint requests: Joseph DiMattio, Department of Physiology and
Biophysics and the Department of Pharmacology, New York University School of Medicine, 550 First Avenue, New York, NY 10016.
820
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CHANGES IN GLUCOSE TRANSPORT IN STZ-DIADETES / DiMorrio er ol.
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Table 1. Acute effects of streptozocin-induced diabetes on ocular glucose transport in rats
Blood-aqueous
Blood-vitreous
Entry rate constant Kt (min~[)
Entry rate constant /'(i (min l)
Days
post-STZ
Plasma glucose
(mg/dl)
3-O-methylglucose
L-glucose
3-O-methylglucose
L-glucose
Control (14)*
154 + 18f
0.067 ± 0.006
0.013 + 0.002
0.042 + 0.004
0.007 + 0.001
1
1
340
362
0.051
0.046
0.014
0.013
0.033
0.030
0.007
0.008
2
2
2
387
360
480
0.061
0.038
0.053
0.011
0.015
0.015
0.026
0.030
0.031
0.005
0.009
0.006
3
3
426
539
0.048
0.046
0.013
0.013
0.032
0.028
0.008
0.008
5
460
0.052
0.014
0.035
0.006
7
7
484
510
0.044
0.059
0.013
0.017
0.033
0.037
0.008
0.011
9
440
0.058
0.015
0.041
0.008
444 ± 76f
0.050 + 0.007
0.013 + 0.002
0.033 + 0.004
0.008 + 0.002
< 0.001
< 0.001
N.S.*
< 0.001
N.S.
Mean + SD
P
i P > 0.05; statistical analysis based on Student's /-test.
* Number of experiments.
t Mean + SDM.
mary, a bolus injection of labeled material is introduced
into a cannulated femoral vein of an anesthetized
Sprague Dawley rat. From 0-18 min, small samples
of blood (100 MD are withdrawn at about 2-min intervals and replaced with saline. At 18 min, a final
sample of blood is obtained, the eyes are quickly removed and measured volumes (10-25 fx\) of aqueous
and vitreous humor are obtained. The 14C and 3 H in
each sample of plasma, aqueous humor, and vitreous
humor is counted by liquid scintillation spectrometry
with appropriate corrections for channel spillover and
quenching performed with computer assistance to yield
concentrations of label in DPM/ml. The plasma concentration (Cp) data are plotted with time and an equation of the form:
Cp = A
Be" blt
Ce"b2t
(Eq.l)
is graphically fit to the data. The constants A, B, C,
b{ and b2 are determined from a best fit to the data.
Ocular concentrations obtained at 18 min, along with
the determined plasma constants that define the blood
function and previously determined steady state values
were analyzed to give the transport constants, K;, using
a model described previously.1'3 The model accounts
for passive, active and bulk transport of fluid into the
ocular compartments, but it can be simplified for
L-glucose and 3-O-methylglucose transport to give the
following equation:
dC,
dt
— K.jC p
(Eq.2)
where CA and Cp refer to concentrations of test substance in aqueous (or vitreous) and plasma, respectively. For both L-glucose and 3-O-methyl-D-glucose
steady state values of aqueous to plasma and vitreous
to plasma concentration ratios approach 1.0, thus K;
= KQ and consequently only K; is used here. Evidence
for this has been presented previously.' Rate constants
for entry and exit, K; and K<, (min"1), respectively,
are determined by a computer solution to the above
equation and reflect a fractional change in ocular compartment concentration occurring per unit of time.
Thus, the rate constants measure how fast the ocular
compartment concentration of labeled substance is
changing and since neither tracer is utilized in the
body the rate constants assess barrier function and
integrity. Absolute mass transport entry rates are directly proportional to both the transport rate constant,
K; and the prevailing plasma glucose concentration, Cp.
Results
As shown in Table 1, plasma glucose levels rose
significantly as early as 1 day after streptozotocin injection. This is similar to observations reported by
others in rats.5"7 One day after streptozotocin, there
also is a significant decrease in 3-O-methylglucose
transport constants for both ocular barriers. This early
decrease probably reflects decreased availability of
transport carrier rather than a direct impairment of
transport inasmuch as a similar decrease in transport
is produced in normal rats made hyperglycemic by
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INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / July 1984
Vol. 25
Table 2. Ocular glucose transport following insulin treatment within '7 days of streptozotocin-induced diabetes
3-O-methylglucose
Plasma glucose
mg/dl
132 + 21§
111 + 12
Control normal (3)J
STZ-Diabetic + insulin (6)||
n~1)*
KA0
0.071 + 0.005
0.078 + 0.005
* Rate of transport across blood-aqueous barrier.
t Rate of transport across blood-vitreous barrier,
j Number of animals.
L-glucose
Ky(min~l)f
KA( 'min-')
Kv (min-')
0.044 -h 0.004
0.047 -1- 0.005
0.007 + 0.002
0.011 + 0.003
0.014 + 0.002
O.O18 + O.OO5
§ Mean + SDM.
|| Animals received last insulin injection about 3 hr prior to experiment.
glucose administration.1 Indeed, in normal animals at
plasma glucose concentrations of 300, 400, and 500
mg/dl, the respective 3-O-methylglucose transport rate
constants would be 0.049, 0.039, and 0.035 min" 1 for
the aqueous barrier and 0.038, 0.031, and 0.024 min" 1
for the vitreous barrier, and these are similar to the
values found in the diabetic rats. It should be noted
that glucose entry rate, which is directly dependent on
both the transport constant, K; and plasma glucose
concentration, C p , is nevertheless significantly elevated
as early as 1 day after streptozotocin since the decrease
in rate constant is more than offset by the increase in
plasma glucose concentration in the diabetic animal.
The possibility of a direct effect of streptozotocin
on glucose transport also is deemed unlikely inasmuch
as short-term diabetic animals that received insulin to
restore normal glucose levels did not show the decrease
in facilitated glucose transport (Table 2). These animals, which exhibited hyperglycemia for several days,
were given insulin, usually 5-10 U/kg, using a 1:1
mixture of Lente and regular (Iletin-Lilly; Indianapolis,
IN). The dose of insulin was adjusted as needed until
glucosuria disappeared (1-2 days) and ocular glucose
transport measurements were made within the following 2 days.
Passive transport of glucose showed little change in
the first 10 days following streptozotocin. Table 1 shows
the individual values obtained on specific days and
Table 3. Ocular glucose transport at various periods of streptozotocin-induced diabetes in rats
Blood-aqueous transport Kt (min ')
Days
Plasma glucose
gm/dl
3-O-methylglucose
0
1-10
11-20
21-30
31-40
51-60
80-90
152-172
(16)*
(11)
(7)
(5)
(7)
(5)
(5)
(11)
134 + 20f
420 + 61
454 + 41>
464 + 30
451 + 4 0
490 + 37
475 + 55
510 + 50
0.062
+ 0.003
0.055
+ 0.004
0.048
+ 0.004
0.045
+ 0.005
0.068
+ 0.004
P
L-glucose
0.013
+ 0.002
P
D/L transport
ratio
5.35
+ 0.18
0.050
+ 0.006
0.049
+ 0.004
0.058
+ 0.003
<0.001
<0.001
<0.01
N.S4
<0.001
<0.001
<0.001
0.014
+ 0.001
0.017
+ 0.002
0.019
+ 0.002
0.020
+ 0.003
0.023
+ 0.003
0.023
+ 0.003
0.022
+ 0.003
N.S.
<0.005
<0.001
<0.001
<0.001
<0.001
<0.001
3.36
2.90
+ 0.22
3.08
+ 0.19
3.18
+ 0.18
2.37
+ 0.24
2.06
+ 0.23
2.04
+ 0.26
Blood-vitreous transport K, (min~\;
3-O-methylglucose
0.042
+ 0.004
P
L-glucose
0.007
+ 0.001
P
D/L transport
ratio
6.17
+ 0.41
0.033
+ 0.003
0.037
+ 0.002
<0.001
<0.01
0.008
+ 0.002
0.038
+ 0.003
0.039
+ 0.004
0.037
+ 0.002
0.033
+ 0.003
0.028
+ 0.004
N.S.
N.S.
<0.02
<0.001
<0.001
0.007
+ 0.002
0.009
+ 0.002
0.011
+ 0.001
0.012
+ 0.002
0.012
+ 0.002
0.012
+ 0.001
N.S.
N.S.
<0.02
<0.001
<0.001
<0.001
<0.001
4.77
+ 0.23
5.13
+ 0.31
3.96
+ 0.38
3.66
+ 0.39
2.98
+ 0.25
2.61
+ 0.36
2.43
+ 0.29
* Number of animals; data for days 159-172 taken from previous study (1).
t Mean + SDM.
% P> 0.05; all statistical comparisons are made with control normal animals
(days 0).
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CHANGES IN GLUCOSE TRANSPORT IN STZ-DIADETES / DiMorrio er ol.
No. 7
.07
823
BL000-AQUEOUS TRANSPORT
3- 0 - Mtthyl -D-Qluco«t •
.06 -
L-GIUCOME2
.05
.04
(mkT1)
.03
.02
.01
NO
10
20
30
DAYS
40
50
AFTER
60
70
80
90
100
140
160 ISO
STREPTOZOTOCIN
Fig. 1. Rates of facilitated and passive transport of glucose across the aqueous humor in rats in normal (N) state and at various times
following injection of streptozotocin (at 0 time). The actual mean valves ± SD and statistical evaluations are shown in Table 3. The contribution
of passive transport was subtracted from the observed facilitated transport and such corrected facilitated transport is shown by the dotted
points in each bar and taken as a measure of facilitation of transport.
the mean value for the 10-day period, which is not
significantly different from normal. However, passive
transport is seen to increase thereafter, as shown in
Table 3. It is increased significantly in the 11-20-day
period and continues to rise slowly until it attains relatively stable values at 50-60 days and remains elevated
for the remainder of the experimental period. These
changes are due to the diabetic state because control
normal rats kept under similar experimental conditions
and similar time period did not show any changes in
L-glucose transport across either the blood-aqueous
or vitreous barriers (data not shown).
Table 3 also shows the values for 3-O-methylglucose
transport during the course of the 172-day period of
observation. As already discussed, the initial decrease
in facilitated transport is attributed to saturation of
glucose transport due to the hyperglycemia. There is
a brief upswing in the transport rate at 21-40 days.
This change occurs concomitantly with the rise in passive transport, which probably contributes to the observed rises. However, beginning at 40-50 days, the
facilitated transport decreases again and remains low
for the duration of the study, despite the persistent
elevation in passive transport.
Table 3 also gives the ratio of D/L glucose transport
rates. It is evident that in the normal rat, glucose transport occurs largely by facilitated diffusion. The D/L
ratio decreases in the diabetic due to a decrease in
D-glucose and increase in L-glucose transport.
Figures 1 and 2 depict graphically the glucose transport rates at the two ocular barriers. The pattern of
changes in transport of 3-O-methylglucose and L-glucose are clearly evident. Since passive transport is
available to both sugars, subtraction of passive transport
from the observed transport of 3-O-methylglucose may
give a more appropriate measure of facilitated glucose
transport. Such corrected transport rates are shown by
the line drawn across the bar graphs. It can be seen
that the rise in measured facilitated transport, observed
or corrected, occurring at 21-40 days is not sustained,
and, indeed, the rates decline despite the continued
increased rates of passive transport. The corrected
transport rates in the final period are significantly lower
(P < 0.001) than those at 30-40 days suggesting a
progressive impairment of carrier function.
Discussion
A main finding in this study is that passive glucose
transport begins increasing soon after the onset of diabetes. The rise begins at about 10 days following induction of diabetes, continues slowly up to 50-60 days,
and stabilizes at the elevated values beyond that period.
The increase in permeability to glucose occurs at both
the aqueous and vitreous barriers. This is in agreement
with the observations that there is greater passive penetration of fluorescein into the vitreous humor in diabetic humans than in normal subjects.8'9 It is of in-
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INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / July 1984
Vol. 25
BLOOD -VITREOUS
TRANSPORT
S-O-Mtthyl-D-GlucottO
L-Glucott E2
(min"1)
H 0
20
30
40
50
60
70
80
90
100
140
160
160
OAYS AFTER STREPTOZOTOCIN
Fig. 2. Rates of facilitated and passive transport of glucose across the vitreous humor. Legends same as in Figure 1.
terest that although hyperglycemia occurs within hours
after STZ injection, significant increases in passive
transport of glucose begin to occur only after 10 days.
The reasons for the slow changes in permeability to
glucose are not known. Conceivably, this may involve
development of structural changes or biochemical adaptations, but these possibilities need to be explored.
These altered rates of permeability to L-glucose may
reflect changes either in the vascular supply to the eye
or in the epithelial barriers of the eye compartments.
It is unlikely that the increases in passive transport of
glucose into the eye compartments involve only
changes in vascular permeability. This is reasoned from
the fact that the formation and composition of the
aqueous and vitreous fluids appears to be regulated by
tight barriers of epithelium. At the blood-aqueous barrier, the double layered ciliary epithelium and tight
junctions of the unpigmented layer block passive
movement of molecules, including glucose. Although
the epithelial tissues bordering on the aqueous do not
completely envelop the aqueous, they do provide a
major protective barrier. On the other hand, the vascular input to aqueous secretion is derived from capillaries at the ciliary process, but these capillaries are
well-fenestrated allowing free, passive movement and,
therefore, it is unlikely that they would contribute to
further increase aqueous barrier permeability. In the
case of the iris, however, the barrier between the blood
vessels in the stroma and the aqueous is achieved by
a tight endothelium lining of the capillaries as well as
by the restraint provided by the posterior epithelium
of the iris.10 It is conceivable that changes in permeability to glucose could occur in these capillaries and,
thereby, contribute to the observed increase in permeability to glucose between blood and the aqueous,
but, to date, there is no evidence for this possibility.
Similarly, the retinal pigment epithelium regulates
the environment of the outer retina and forms a barrier
of tight junctions between the fenestrated vessels of
the choroid and the retina. Thus, changes in permeability of this epithelial barrier could contribute to the
increased permeability of the vitreous barrier. The inner
retina also is supplied with vessels with tight junctions
and a change in their permeability could possibly be
a contributory factor.
The mechanism whereby persistent hyperglycemia
may alter cellular function remains unclear. If tissue
concentration of glucose exceeds that of the plasma,
it could exert an osmotic effect as has been suggested
by Kinoshita et al." However, in the diabetic, the
retina-plasma ratio of glucose concentration is probably unchanged from the normal, which may argue
against an osmotic effect. Another explanation for tissue damage involves increased polyol pathway activity
seen in diabetes.12 The hyperglycemia would be expected to provide more glucose precursor for the polyol
pathway, resulting in increased tissue accumulation of
less permeable glucitol (sorbitol) and a parallel increase
in water content as observed in the lens. However, it
is not clear at present that the "osmotic theory" fully
explains sugar cataractogenesis since a number of other
plausible hypotheses have been put forth including the
suggestion that decreased availability of NADPH could
lead to a limited ability of the lens to dispose of damaging peroxides.13 Moreover, the decrease in glucose
metabolism observed in lens exposed to high carbo-
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CHANGES IN GLUCOSE TRANSPORT IN 5TZ-DIADETES / DiMorrio er ol.
hydrate concentrations could contribute to alterations
in sodium-dependent water movement across the lens
epithelium.13 Despite uncertainty of the mechanism,
our work suggests that early permeability changes tend
to enhance glucose entry into the ocular compartments
and expose intraocular tissues to damaging high glucose
concentrations.
Another possible mechanism for the altered permeability may involve nonenzymatic glycosylation,
which has been shown to occur in vivo in a variety of
proteins including hemoglobin, red cell membrane
protein, collagen, lens crystallin, serum proteins and
nerve myelin.14
Facilitated transport of glucose also appears to show
changes in the diabetic animals, although these are
more difficult to interpret. There are several factors
that influence the measurement of transport of (3H)
3-O-methylglucose. Since the tracer uses the same
transport system as endogenous D-glucose, it will be
affected by the prevailing plasma glucose concentration.
Indeed, as we have reported previously,1 increasing
plasma glucose levels in normal rats by glucose infusion
decreased the rate of transport of 3-O-methylglucose
to about 60% of control level. The observed decrease
in tracer transport in the diabetic rats reported here
may have a similar explanation.
Another complication in the measurement of facilitated transport is the extent of contribution of the
passive movement of glucose. We presume that an
increase in passive movement or permeability to glucose also would increase transport of 3-O-methylglucose. Since we made independent measurements of
glucose permeability, using (l4C)-L-glucose, we can
subtract these rates from those obtained using 3-Omethylglucose and obtain a more reliable measure of
facilitated transport. Such values are represented in
Figures 1 and 2 by the solid lines.
Facilitated transport of glucose, total or corrected,
is shown to decrease immediately upon induction of
diabetes, and this result is attributed to the hyperglycemia and saturation of the transport system. In the
10-40-day period, facilitated transport is seen to rise
somewhat. This may be due, in part, to the concomitant rise in passive transport. With time, 3-O-methylglucose transport declines to initial diabetic values
and upon subtracting the passive transport, the corrected facilitated transport is lower than at the onset
of diabetes. This finding is viewed as suggestive of a
decline in carrier facilitated transport in long-term diabetes. Nevertheless, since plasma glucose concentration, which is the driving force for glucose entry, is
more than three times normal as early as 1 day poststreptozotocin, the mass rate of glucose entry is still
significantly elevated and a small loss in carrier function
825
will have little consequence in determining intraocular
glucose concentration.
Although the preceding discussion has emphasized
the possible role of hyperglycemia and increased permeability to glucose as contributing to the complications of long-term diabetes, other factors may also
play a role, such as changes in the vasculature,15"17
with particular emphasis on thickening of the basement
membrane.18"20 Such thickening in small blood
vessels21'22 and kidneys23'24 is well documented, and
this finding has been associated with increased permeability. Basement membrane thickening in diabetics
is generally a slow process and is a major factor in the
microangiopathy of kidney, but it is uncertain if it has
much influence on the function of other organs.20 Presumably, the increase in permeability seen in the present study can lead eventually to basement membrane
abnormalities and altered membrane transport functions. It is of interest that in the tissues most affected
in chronic diabetes, eg, nervous tissue, kidney, blood
vessels, transport of glucose is not dependent on insulin25 and, thus, may be influenced largely by the
prevailing plasma glucose.2126
Regulation of glucose transport in the eye and a
possible role of insulin in this process is still unsettled.27
The early studies by Ross 2829 and Harris30 proposed
an effect of insulin on glucose transport in the lens,
but others 3132 have questioned these findings. The
present study shows that the carrier facilitated glucose
transport is decreased in the long-term diabetic animal.
Since the magnitude of this defect is greater at the
later stage of the diabetes than in the early stage, it
would appear that it is not directly related to the lack
of insulin. Thus, the role played by insulin in glucose
transport in the eye, if any, requires better substantiation than has been available thus far.
Key words: glucose transport in eye, diabetic rats, facilitated
glucose transport, passive glucose transport
Acknowledgment
The skillful technical assistance of Jack Streitman is acknowledged gratefully.
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INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / July 1984
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