Glucose uptake and utilization of isolated lenses from normal and diabetic rats following insulin injection.код для вставкиСкачать
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 126.96.36.199(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.