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Glucose uptake and utilization of isolated lenses from normal and diabetic rats following insulin injection.

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Glucose Uptake and Utilization of Isolated Lenses
from Normal and Diabetic Rats Following
Insulin Injection'
T. G . FARKAS, R. F. IVORY AND J. W. PATTERSON
Western Reserve University, Cleveland, Ohio and Vanderbilt University,
Nashville, Tennessee
It has been reported that the intravenous At the end of the incubation period the
injection of insulin into normal or diabetic lens was homogenized in its own incubarats causes a significant increase in the tion medium to which 0.1 ml of 10%
glucose disappearance from the media in ZnSOl had been added. The ZnS04 was
which their isolated lenses are incubated. neutralized with equivalent amounts of
These results could be explained by two saturated Ba(OH)2 and the homogenate
possible mechanisms. (1) Insulin might was made up to 3 ml final volume with
accelerate the glucose transport mechan- distilled water. The homogenate was
ism without altering the rate of glucose centrifuged for 15 minutes at 2,500 rpm.
utilization by the lens, in which case there
would be an accumulation of glucose with- Aliquots were removed from the clear
in the lens. ( 2 ) Insulin might increase supernatant for glucose determination
the glucose utilization inside the lens (Somogyi, '45a, '45b). The difference beeither by direct action on metabolic path- tween the glucose content of the 0 time
ways or by increasing available substrate control and that of the experimental samfollowing an accelerated glucose transport. ple represents the amount of glucose
These two possibilities can be differenti- utilized by the lens during the incubation
ated by comparing the glucose disappear- period. Lenses obtained from non-injected
ance from the medium with the glucose animals and carried through the procedure
disappearance from the total system of described above served as normal controls.
lens plus medium. This paper reports the
Male Sprague-Dawley rats weighing
results of such studies.
about 150 gm were made diabetic by the
intravenous injection of 36 mg of alloxan
EXPERIMENTAL
and 360 mg dehydroascorbic acid/kg of
Glucose uptake or glucose disappear- body weight. The blood sugar content of
ance from the medium was determined as the injected animals was determined 60
previously described (Macintyre et al.,
'56). Glucose utilization or glucose dis- and 84 hours following the injection of
dehydroascorbic acid and alloxan. Only
appearance from the total system of lens those animals which had a blood sugar
plus medium was determined as follows:
of 300 mg % or higher on two succesMale Sprague-Dawley rats weighing level
sive determinations were used in the exabout 150 gm were injected with 20 LI perimen t s.
crystalline insulin intravenously. One hour
The lenses were handled in the same
after the injection the animals were de- manner as described for the normal lenses.
capitated and the lenses removed and Glucose was determined by the glucose
weighed. One lens was homogenized in
oxidase method. Fructose was determined
0.20 ml of Tyrode's solution containing
100 mg % of glucose and 0.1 ml of 10% according to Kendrick ('52).
ZnS04<oserveas the 0 time control. The
'This investigation was supported by a PHS
Other lens Of the pair was 'IXubated for
research grant A-154 National Institute of Artwo hours at 37°C in o.20 ml of Tyrode
thritis and Metabolic Diseases, Public Health
solution containing 100 mg % glucose. Service.
235
236
T. G. FARKAS, R. F. IVORY AND J. W. PATTERSON
RESULTS
Table 1 presents the results obtained
with lenses from normal rats. Glucose
uptake and glucose utilization are both
significantly increased in lenses obtained
from rats that had received insulin injections. For lenses from normal rats the
measurement of glucose disappearance
from the medium gives the same results as
the measurement of glucose disappearance
from the total system of lens plus medium.
The values obtained for glucose uptake are
in good agreement with those previously
reported (Macintyre et al., '56; Farkas and
Patterson, '57).
Table 2 represents the results obtained
with lenses from diabetic rats. Disappearance of reducing sugar from the media is
not comparable with glucose utilization in
these lenses. Lenses from diabetic rats
utilize 25% less sugar than the lenses of
normal animals. Reducing sugar uptake
and utilization are both significantly increased in lenses obtained from diabetic
rats following insulin injection and utilization is restored to the normal level by this
procedure.
Table 3 presents some analyses for reducing sugars in isolated lenses from diabetic rats. Glucose and fructose are both
present in the diabetic lens. The sum of
the fructose and glucose content determined by methods specific for each (2.63
mg/gm of lens) was in good agreement
with the total reducing sugar measured
directly. The rate of disappearance of the
reducing sugars upon incubation was 2.32
mg/gm of lens/hr.
DISCUSSION
When isolated normal rat lenses are
incubated in glucose containing medium,
the disappearance of glucose from the medium is a reflection of the glucose utilization since the free sugar content of these
lenses is below the level detectable by our
methods. Diabetic lenses contain appreciable amounts of free reducing sugar, a
large portion of which is fructose, as reported by Kuch and Kinsey ('57). The
free reducing sugar pool is depleted at an
initial rate of 2.32 mg/gm/hr. which is
over twice the rate of disappearance from
the total system of lens plus medium. This
suggests the passage of free sugar from
the lens into the medium during incubation and such passage would account for
part of the decreased sugar disappearance
from the medium observed with diabetic
lenses. Nevertheless reducing sugar dis-
TABLE 1
Effect of insulin on reducing sugar uptake and utilization o f normal rat lenses
Glucose uptake mg/gm/hr.
Glucose utilization mg/gm/hr.
Without insulin
With insulin
P value
1.104.0.10(14)l
1.11&0.32(32)
1.64&0.13(33)
1.54f 0.45(29)
< 0.001
< 0.001
Number of lenses.
TABLE 2
Effect o f insulin on reducing sugar uptake and utilization of diabetic rat lenses
Without insulin
Reducing sugar uptake mg/gm/hr.
Reducing sugar utilization mg/gm/hr.
With insulin
0.42-1-0.25(15)1
0.84-1- 0.17(20)
0.9050.31(11)
1.0550.26(19)
P value
< 0.001
< 0.001
Number of lenses.
TABLE 3
Sugar content of lenses from diabetic rats
Glucose mg/gm
Fructose mg/gm
Sum
1 Number of lenses.
0 Time
After 15 minute
incubation
A15
minutes
1.11 f0.35(46)'
0.725 0.33(22)
1.33f 0.44(23)
2.05
0.39
0.19
0.58
1.5220.45(47)
2.63
GLUCOSE UPTAKE AND UTILIZATION O F LENSES
237
appearance from lens and medium de- permit localization of the insulin action to
creases in diabetes and is restored to nor- a specific step in these pathways.
mal levels following insulin injection.
It has been postulated previously that
LITERATURE CITED
the diabetic lens suffers from a lack of
glucose and this glucose yarn- Farkas, T. G.,and J. W. Patterson 1957 Insulin
is the
Of cataract
formation in diabetic lenses. Our results
indicate that this is not the case since
there is a large quantity of glucose present
inside the diabetic lens. This suggests that
the major defect in diabetes is in the metabolic pathways of glucose utilization rather
than in the transport mechanism.
Similarly, the results demonstrate that
insulin causes a major increase in the
activity of these metabolic pathways. The
information presently available does not
and the lens. Am. J. Ophth., 44: 341-344.
Kendrick, A. B. 1952 Determination of fructose
in presence of glucose in blood and urine by
use of diphenylamine. Fed. Proc., 11: 239.
Kuch, F. Jr., and E. Kinsey 1957 Personal
communication.
Macintyre, M. N.,s. s. Pelt and J. w. Patterson
1956 Glucose uptake by isolated
and
diabetic rat lenses. Am. J. Physiol., 186: 406408.
Somogyi, M. 1945a A new reagent for the determination of sugars. J. Biol. Chem., 260: 6169.
___ 1945b Determination of blood sugar.
Ibid., 160: 69-73.
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injections, lenses, utilization, following, isolated, uptake, norman, insulin, rats, diabetic, glucose
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