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The Role of Insulin in Lens Metabolism*
T H E ROLE O F INSULIN IN LENS METABOLISM* T. G. FARKAS, R. F. IVORY, AND S. J. COOPERSTEIN
Cleveland, Ohio AND J. W .
PATTERSON
Nashville, Tennessee
The uptake of glucose by rat lenses incu bated in Tyrode's solution containing 100 mg. of sugar per 100 cc. is significantly in creased if insulin is injected intravenously prior to removal of the lens.1·2 The results are the same whether the uptake of glucose is measured as glucose disappearing from the medium or as glucose disappearing from the total system of lens plus medium.3 On the other hand, the addition of insulin directly to the incubation medium does not increase the uptake of glucose by lenses ob tained from untreated animals.2-4 These facts suggest either (a) that insulin is ineffective in the form injected and is activated in some way following injection, (b) that in order to be effective insulin re quires the presence of one or more addi tional substances or (c) that insulin does not act directly on the lens, but rather stimu lates the production or release of some other substance which is transported to the lens and affects lens metabolism. Each of these possibilities implies that some organ other than the lens is necessary if the action of insulin on lens metabolism is to be demonstrated. It is the purpose of this paper to report the results of experi ments designed to determine which organ or organs may be required to produce an in crease in glucose uptake following the in jection of insulin. * We wish to express our appreciation for the support received from the National Institute of Arthritis and Metabolic Diseases, of the National Institutes of Health, U. S. Public Health Service Grant Number A-154 and for a fellowship from the National Council to Combat Blindness, New York City, in support of one of the authors (T. G. Farkas). t In the totally eviscerated rats the external jug ular was used. 394
EXPERIMENTAL
Male Sprague-Dawley rats weighing 120170 gm. were anesthetized by the intra venous injection of 6.6 mg. Nembutal/kg. body weight. In each series of experiments the animals were divided into two major groups. Sham operations were performed on a control group and certain organs were re moved from the experimental group. Im mediately following operation all animals were injected subcutaneously with 1.0 ml. of a 30-percent glucose solution. At the same time half of each group received 20 units of insulin by injection into the surgically ex posed inferior vena cava.t One hour after this treatment the animals were decapitated and the lenses were removed and placed in Tyrode's solution containing 100 mg.% of glucose. The glucose uptake was determined as previously described by measuring glu cose disappearance from the medium2 using the Somogyi method.5 The figures given on the bar graphs are the average values and standard deviations. RESULTS
The data are presented in Figures 1 through 6 and the following points may be emphasized: 1. The values for glucose uptake by lenses from sham-operated animals, both with and without insulin injection (fig. 1-6), are in good agreement with those previously re ported for normal animals.1 The increase in glucose uptake observed following the in jection of insulin has been termed the "in sulin effect." 2. Total evisceration, that is the removal of the stomach, intestines, spleen, pancreas, kidneys, adrenals and liver, abolishes the "insulin effect" (fig. 1).
395
ROLE OF INSULIN IN LENS METABOLISM
20
MH INSULIN
- iM NOT INJECTED ■
Cvl} INSULIN Κ·:·1 INJEÇTED
Π 7 Ι NUMBER LÜJ OF LENSE
| INSULIN 1 NOT INJECTEO
-
ΓνΙ] INSULIN L U I INJECTED
ΠΖ] NUMBER LÜJ OF LENSES
-
1.54102 4
1.5
GLUCOSE UPTAKE 1.0 mg./gm. of Itna/tir.
05
1.1310.18
11210.19
.
'ÜM
-
Χ·*·Χ\
-
: : :
El
:xx
ö
SHAM OPERATED
m
■
STOMACH, INTESTINE, SPLEEN, PANCREAS, KIDNEYS, ADRENALS a LIVER REMOVED.
3. Removal of the stomach, intestines, spleen, pancreas, (fig. 2) and in a separate series of experiments the kidneys (fig. 3), does not modify the "insulin effect." 4. Removal of the liver along with the stomach, intestines, spleen and pancreas pro duces different results, for, under these cir cumstances the "insulin effect" is abolished, as it is following complete evisceration (fig. 4)· 5. Following adrenalectomy the glucose uptake of the lens is increased to the level obtained in normal animals following the in jection of insulin (fig. 5). This may be termed the "adrenal effect." The injection of insulin in the adrenalectomized animal produces no further increase in the uptake of Ι Γ 3 INSULIN | w 3 INJECTED
Π
-
Fig. 1 (Farkas, et al.). Effect of evisceration on glucose uptake of rat lenses.
ILIN INJECTEO
GLUCOSE UPTAKE mg./gm. of l»ns/hr.
SHAM OPERATED
Fig. 3 (Farkas, et al.) Effect of nephrectomy on glucose uptake of rat lenses. glucose (fig. 5). Removal of the stomach, intestines, spleen and pancreas along with the adrenals does not modify the "adrenal effect" (fig. 6 ) . DISCUSSION
It is apparent that the adrenals and the liver are the key organs involved in explain ing the results obtained. The stomach, intes tines, spleen, pancreas and kidneys do not modify either the "insulin effect" or the "adrenal effect" and can be eliminated from consideration. Stimulation of the glucose up take of the lens, as observed after adrenalec tomy or the injection of insulin, can be demonstrated only when the liver is intact, indicating that the liver plays a stimulatory role. Conversely, since removal of the 1 INSULIN JINJECTEO
Π 7 Ί NUMBER L U I OF LENSE LENSES
GLUCOSE UPTAKE mg./gm. of Itnt/hr.
KIDNEYS REMOVED
ΓΤΓΊ N |"l0
GLUCOSE UPTAKE mg./gm. of lent / hr.
6 SHAM OPERATED
STOMACH, INTESTINES, SPLEEN & PANCREAS , REMOVED
Fig. 2 (Farkas, et al.). Effect of removal of the stomach, intestines, spleen, and pancreas on glucose uptake of rat lenses.
M OPERATED
LIVER, STOMACH, INTESTINES, SPLEEN a PANCREAS REMOVED.
Fig. 4 (Farkas, et al.). Effect of removal of the liver, stomach, intestines, spleen, and pancreas on glucose uptake of rat lenses.
396
FARKAS, IVORY, COOPERSTEIN AND PATTERSON 1 INSULIN | NOT INJECTED
Κ Ή INSULIN L±J INJECTED
ΓΤ7Ί NUMBER L_J OF LENSES
INSULIN NOT INJECTED
fvJ, INSULIN [ ± J INJECTED
HJ1 NUMBER LIU OF LENSES
l.64±0J2 1.4 5 ±0.36
l.4T±0.39
1.45 ± 0 3 6
1.52 ±0.32
GLUCOSE UPTAKE mo. /em. of ttnt/hr.
GLUCOSE UPTAKE mq. /gm. of Uni / hr.
m SHAM OPERATED
SHAM OPERATED
ADRENALS REMOVED
Fig. S (Farkas, et al.) Effect of adrenalectomy on glucose uptake of rat lenses.
adrenals is accompanied by an increase in glucose uptake, its influence in the intact ani mal must be inhibitory. The adrenals could exert their effect by secreting an inhibitor which either acts di rectly on the lens or prevents the formation or action of the liver stimulator. Although, as noted, removal of the liver along with the adrenals abolishes the "adrenal effect," removal of the liver from an animal with intact adrenals does not decrease the glucose uptake below the normal level. This finding suggests that the adrenal inhibitor and liver stimulator do not act independently on the lens but rather that the adrenal inhibitor either blocks the production of the liver stimulator or neutralizes its action during or after secretion. The "insulin effect" can be demonstrated only if the adrenals and liver are both intact. The simplest explanation consistent with the data is that insulin blocks the action of the adrenal inhibitor and thereby permits the stimulator to exert its effect. This would ex plain why the "adrenal effect" and "insulin effect" are of the same order of magnitude and why an "insulin effect" cannot be demon strated in the absence of the adrenals or the liver. Alternatively, it is possible (a) that insulin increases the secretion of liver stimu lator or potentiates its action, (b) that in sulin stimulates the liver to produce another substance which by itself or acting synergistically with insulin stimulates the lens, or (c)
STOMACH, INTESTINES, SPLEEN,PANCREAS, KIDNEYS ft ADRENALS REMOVED.
Fig. 6 (Farkas, et al.). Effect of removal of the adrenals, stomach, intestines, spleen and pancreas on glucose uptake of rat lenses.
that the liver modifies the injected insulin and thereby makes it effective. These alterna tives, however, require the additional as sumption that after adrenalectomy the en zyme systems of the lens are operating maxi mally and cannot be stimulated further by insulin injection. The results suggest comparisons with the results obtained in the whole animal in which the blood sugar can be lowered either by the injection of insulin or by removal of the adrenal glands. The present findings may contribute to a localization of this effect. SUMMARY
Studies have shown that the glucose up take of isolated rat lenses can be increased either by adrenalectomy or by insulin injec tion prior to removal of the lens. In both cases the increase can be demonstrated only if the liver is intact. The increase following adrenalectomy is of the same order of mag nitude as that following insulin injection, and injection of insulin into an adrenalectomized animal does not further increase the glucose uptake. The removal of the other visceral organs has no effect. Hypotheses to explain these results are offered. Western Reserve University School of Medicine (6). Vanderbilt University School of Medicine
(5).
ROLE OF INSULIN IN LENS METABOLISM
397
REFERENCES
1. Farkas, T. G., and Patterson, J. W.: Insulin and the lens. Am. J. Ophth., 44:341-344, 1957. 2. Macintyre, M. N., Polt, S. S., and Patterson, J. W.: Glucose uptake by isolated normal and diabetic rat lenses. Am. J. Physiol., 186:406-408,1956. 3. Farkas, T. G., Ivory, R. F., and Patterson, J. W. : Unpublished data. 4. Harris, J. E., Hauschildt, J. D., and Nordquist, L. T.: Transport of glucose across the lens sur faces. Am. J. Ophth., 39:161-169, 1955. 5. Somogyi, M.: A new reagent for the determination of sugars. J. Biol. Chem., 160:61-68, 1945. DISCUSSION DR. JOHN F. R. KUCK, JR. (Detroit) : The
essayists are to be commended for the progress made in implicating the liver and adrenals with insulin action and lens glucose metabolism. It is particularly wise, I think, to avoid using diabetic animals as long as information can be obtained otherwise, since severe diabetes brings so many complications that secondary effects on the lens may mask any primary fault in glucose metabolism. The present experiments demonstrate clearly that in the presence of an intact liver either in sulin injection or adrenalectomy will increase lens glucose uptake. This effect occurs after glucose injection. I would like to ask if the glucose injection is necessary to show the phenomenon, and what effect this 300 mg. of glucose has on the blood sugar level and the lens glucose level. If the rise in blood glucose is appreciable, then it is possible that the intact liver serves merely as a converter-storage
system for the removal of excess glucose and its storage as glycogen. This system, of course, is inoperative after hepatectomy. DR. T. G. FARKAS (closing) : Glucose was in
jected into our animals after operation to be able to maintain them alive for one hour, especially after hepatectomy. As we are all aware, the blood glucose level drops rather precipitously after hepatectomy, and the animals could not be kept alive for the dura tion of the experiment without the supplement of glucose. Since the sham-operated animals were -also in jected with the same amount of glucose, and the glucose uptake in these lenses was exactly the same as in normal animals in which glucose was not injected, I do not think the injected amount of glucose plays any role in the phenomenon we did observe.
ULTRASTRUCTURE O F T H E IRIS : AN ELECTRON MICROSCOPIC STUDY* A. J. TOUSIMIS AND BEN S. FlNEÎ Washington, D.C. The electron microscope with its manyfold increase in resolving power over the light microscope and several improved tech niques of specimen preparation have been applied to the study of ocular tissues. Our main interest has been focused on the mam malian iris, the histology of which has here tofore been based almost entirely on the use of conventional techniques.1"3 The inherent limitations of the latter together with new * From the Biophysics and Ophthalmic Pathology Branches, Armed Forces Institute of Pathology, Washington 25, D.C. Supported in part by grant B-1021, National Institutes of Health. t Dr. Fine is a special trainee of the National Institute of Neurological Diseases and Blindness, NIH.
ideas derived from other studies have intro duced a number of questions concerning the finer structure of this tissue: a. Is there a continuous cellular border layer on the anterior surface of the iris? b. Are there specialized endothelial cells on this surface? c. What is the nature of the "thick walls" of the stromal vessels? d. What is the nature of the abundant ground substance of the iris stroma? e. What is the relationship of the pigment epithelium to the dilator muscle? f. Is there an internal limiting membrane on the posterior surface of the iris compara-
Cleveland, Ohio AND J. W .
PATTERSON
Nashville, Tennessee
The uptake of glucose by rat lenses incu bated in Tyrode's solution containing 100 mg. of sugar per 100 cc. is significantly in creased if insulin is injected intravenously prior to removal of the lens.1·2 The results are the same whether the uptake of glucose is measured as glucose disappearing from the medium or as glucose disappearing from the total system of lens plus medium.3 On the other hand, the addition of insulin directly to the incubation medium does not increase the uptake of glucose by lenses ob tained from untreated animals.2-4 These facts suggest either (a) that insulin is ineffective in the form injected and is activated in some way following injection, (b) that in order to be effective insulin re quires the presence of one or more addi tional substances or (c) that insulin does not act directly on the lens, but rather stimu lates the production or release of some other substance which is transported to the lens and affects lens metabolism. Each of these possibilities implies that some organ other than the lens is necessary if the action of insulin on lens metabolism is to be demonstrated. It is the purpose of this paper to report the results of experi ments designed to determine which organ or organs may be required to produce an in crease in glucose uptake following the in jection of insulin. * We wish to express our appreciation for the support received from the National Institute of Arthritis and Metabolic Diseases, of the National Institutes of Health, U. S. Public Health Service Grant Number A-154 and for a fellowship from the National Council to Combat Blindness, New York City, in support of one of the authors (T. G. Farkas). t In the totally eviscerated rats the external jug ular was used. 394
EXPERIMENTAL
Male Sprague-Dawley rats weighing 120170 gm. were anesthetized by the intra venous injection of 6.6 mg. Nembutal/kg. body weight. In each series of experiments the animals were divided into two major groups. Sham operations were performed on a control group and certain organs were re moved from the experimental group. Im mediately following operation all animals were injected subcutaneously with 1.0 ml. of a 30-percent glucose solution. At the same time half of each group received 20 units of insulin by injection into the surgically ex posed inferior vena cava.t One hour after this treatment the animals were decapitated and the lenses were removed and placed in Tyrode's solution containing 100 mg.% of glucose. The glucose uptake was determined as previously described by measuring glu cose disappearance from the medium2 using the Somogyi method.5 The figures given on the bar graphs are the average values and standard deviations. RESULTS
The data are presented in Figures 1 through 6 and the following points may be emphasized: 1. The values for glucose uptake by lenses from sham-operated animals, both with and without insulin injection (fig. 1-6), are in good agreement with those previously re ported for normal animals.1 The increase in glucose uptake observed following the in jection of insulin has been termed the "in sulin effect." 2. Total evisceration, that is the removal of the stomach, intestines, spleen, pancreas, kidneys, adrenals and liver, abolishes the "insulin effect" (fig. 1).
395
ROLE OF INSULIN IN LENS METABOLISM
20
MH INSULIN
- iM NOT INJECTED ■
Cvl} INSULIN Κ·:·1 INJEÇTED
Π 7 Ι NUMBER LÜJ OF LENSE
| INSULIN 1 NOT INJECTEO
-
ΓνΙ] INSULIN L U I INJECTED
ΠΖ] NUMBER LÜJ OF LENSES
-
1.54102 4
1.5
GLUCOSE UPTAKE 1.0 mg./gm. of Itna/tir.
05
1.1310.18
11210.19
.
'ÜM
-
Χ·*·Χ\
-
: : :
El
:xx
ö
SHAM OPERATED
m
■
STOMACH, INTESTINE, SPLEEN, PANCREAS, KIDNEYS, ADRENALS a LIVER REMOVED.
3. Removal of the stomach, intestines, spleen, pancreas, (fig. 2) and in a separate series of experiments the kidneys (fig. 3), does not modify the "insulin effect." 4. Removal of the liver along with the stomach, intestines, spleen and pancreas pro duces different results, for, under these cir cumstances the "insulin effect" is abolished, as it is following complete evisceration (fig. 4)· 5. Following adrenalectomy the glucose uptake of the lens is increased to the level obtained in normal animals following the in jection of insulin (fig. 5). This may be termed the "adrenal effect." The injection of insulin in the adrenalectomized animal produces no further increase in the uptake of Ι Γ 3 INSULIN | w 3 INJECTED
Π
-
Fig. 1 (Farkas, et al.). Effect of evisceration on glucose uptake of rat lenses.
ILIN INJECTEO
GLUCOSE UPTAKE mg./gm. of l»ns/hr.
SHAM OPERATED
Fig. 3 (Farkas, et al.) Effect of nephrectomy on glucose uptake of rat lenses. glucose (fig. 5). Removal of the stomach, intestines, spleen and pancreas along with the adrenals does not modify the "adrenal effect" (fig. 6 ) . DISCUSSION
It is apparent that the adrenals and the liver are the key organs involved in explain ing the results obtained. The stomach, intes tines, spleen, pancreas and kidneys do not modify either the "insulin effect" or the "adrenal effect" and can be eliminated from consideration. Stimulation of the glucose up take of the lens, as observed after adrenalec tomy or the injection of insulin, can be demonstrated only when the liver is intact, indicating that the liver plays a stimulatory role. Conversely, since removal of the 1 INSULIN JINJECTEO
Π 7 Ί NUMBER L U I OF LENSE LENSES
GLUCOSE UPTAKE mg./gm. of Itnt/hr.
KIDNEYS REMOVED
ΓΤΓΊ N |"l0
GLUCOSE UPTAKE mg./gm. of lent / hr.
6 SHAM OPERATED
STOMACH, INTESTINES, SPLEEN & PANCREAS , REMOVED
Fig. 2 (Farkas, et al.). Effect of removal of the stomach, intestines, spleen, and pancreas on glucose uptake of rat lenses.
M OPERATED
LIVER, STOMACH, INTESTINES, SPLEEN a PANCREAS REMOVED.
Fig. 4 (Farkas, et al.). Effect of removal of the liver, stomach, intestines, spleen, and pancreas on glucose uptake of rat lenses.
396
FARKAS, IVORY, COOPERSTEIN AND PATTERSON 1 INSULIN | NOT INJECTED
Κ Ή INSULIN L±J INJECTED
ΓΤ7Ί NUMBER L_J OF LENSES
INSULIN NOT INJECTED
fvJ, INSULIN [ ± J INJECTED
HJ1 NUMBER LIU OF LENSES
l.64±0J2 1.4 5 ±0.36
l.4T±0.39
1.45 ± 0 3 6
1.52 ±0.32
GLUCOSE UPTAKE mo. /em. of ttnt/hr.
GLUCOSE UPTAKE mq. /gm. of Uni / hr.
m SHAM OPERATED
SHAM OPERATED
ADRENALS REMOVED
Fig. S (Farkas, et al.) Effect of adrenalectomy on glucose uptake of rat lenses.
adrenals is accompanied by an increase in glucose uptake, its influence in the intact ani mal must be inhibitory. The adrenals could exert their effect by secreting an inhibitor which either acts di rectly on the lens or prevents the formation or action of the liver stimulator. Although, as noted, removal of the liver along with the adrenals abolishes the "adrenal effect," removal of the liver from an animal with intact adrenals does not decrease the glucose uptake below the normal level. This finding suggests that the adrenal inhibitor and liver stimulator do not act independently on the lens but rather that the adrenal inhibitor either blocks the production of the liver stimulator or neutralizes its action during or after secretion. The "insulin effect" can be demonstrated only if the adrenals and liver are both intact. The simplest explanation consistent with the data is that insulin blocks the action of the adrenal inhibitor and thereby permits the stimulator to exert its effect. This would ex plain why the "adrenal effect" and "insulin effect" are of the same order of magnitude and why an "insulin effect" cannot be demon strated in the absence of the adrenals or the liver. Alternatively, it is possible (a) that insulin increases the secretion of liver stimu lator or potentiates its action, (b) that in sulin stimulates the liver to produce another substance which by itself or acting synergistically with insulin stimulates the lens, or (c)
STOMACH, INTESTINES, SPLEEN,PANCREAS, KIDNEYS ft ADRENALS REMOVED.
Fig. 6 (Farkas, et al.). Effect of removal of the adrenals, stomach, intestines, spleen and pancreas on glucose uptake of rat lenses.
that the liver modifies the injected insulin and thereby makes it effective. These alterna tives, however, require the additional as sumption that after adrenalectomy the en zyme systems of the lens are operating maxi mally and cannot be stimulated further by insulin injection. The results suggest comparisons with the results obtained in the whole animal in which the blood sugar can be lowered either by the injection of insulin or by removal of the adrenal glands. The present findings may contribute to a localization of this effect. SUMMARY
Studies have shown that the glucose up take of isolated rat lenses can be increased either by adrenalectomy or by insulin injec tion prior to removal of the lens. In both cases the increase can be demonstrated only if the liver is intact. The increase following adrenalectomy is of the same order of mag nitude as that following insulin injection, and injection of insulin into an adrenalectomized animal does not further increase the glucose uptake. The removal of the other visceral organs has no effect. Hypotheses to explain these results are offered. Western Reserve University School of Medicine (6). Vanderbilt University School of Medicine
(5).
ROLE OF INSULIN IN LENS METABOLISM
397
REFERENCES
1. Farkas, T. G., and Patterson, J. W.: Insulin and the lens. Am. J. Ophth., 44:341-344, 1957. 2. Macintyre, M. N., Polt, S. S., and Patterson, J. W.: Glucose uptake by isolated normal and diabetic rat lenses. Am. J. Physiol., 186:406-408,1956. 3. Farkas, T. G., Ivory, R. F., and Patterson, J. W. : Unpublished data. 4. Harris, J. E., Hauschildt, J. D., and Nordquist, L. T.: Transport of glucose across the lens sur faces. Am. J. Ophth., 39:161-169, 1955. 5. Somogyi, M.: A new reagent for the determination of sugars. J. Biol. Chem., 160:61-68, 1945. DISCUSSION DR. JOHN F. R. KUCK, JR. (Detroit) : The
essayists are to be commended for the progress made in implicating the liver and adrenals with insulin action and lens glucose metabolism. It is particularly wise, I think, to avoid using diabetic animals as long as information can be obtained otherwise, since severe diabetes brings so many complications that secondary effects on the lens may mask any primary fault in glucose metabolism. The present experiments demonstrate clearly that in the presence of an intact liver either in sulin injection or adrenalectomy will increase lens glucose uptake. This effect occurs after glucose injection. I would like to ask if the glucose injection is necessary to show the phenomenon, and what effect this 300 mg. of glucose has on the blood sugar level and the lens glucose level. If the rise in blood glucose is appreciable, then it is possible that the intact liver serves merely as a converter-storage
system for the removal of excess glucose and its storage as glycogen. This system, of course, is inoperative after hepatectomy. DR. T. G. FARKAS (closing) : Glucose was in
jected into our animals after operation to be able to maintain them alive for one hour, especially after hepatectomy. As we are all aware, the blood glucose level drops rather precipitously after hepatectomy, and the animals could not be kept alive for the dura tion of the experiment without the supplement of glucose. Since the sham-operated animals were -also in jected with the same amount of glucose, and the glucose uptake in these lenses was exactly the same as in normal animals in which glucose was not injected, I do not think the injected amount of glucose plays any role in the phenomenon we did observe.
ULTRASTRUCTURE O F T H E IRIS : AN ELECTRON MICROSCOPIC STUDY* A. J. TOUSIMIS AND BEN S. FlNEÎ Washington, D.C. The electron microscope with its manyfold increase in resolving power over the light microscope and several improved tech niques of specimen preparation have been applied to the study of ocular tissues. Our main interest has been focused on the mam malian iris, the histology of which has here tofore been based almost entirely on the use of conventional techniques.1"3 The inherent limitations of the latter together with new * From the Biophysics and Ophthalmic Pathology Branches, Armed Forces Institute of Pathology, Washington 25, D.C. Supported in part by grant B-1021, National Institutes of Health. t Dr. Fine is a special trainee of the National Institute of Neurological Diseases and Blindness, NIH.
ideas derived from other studies have intro duced a number of questions concerning the finer structure of this tissue: a. Is there a continuous cellular border layer on the anterior surface of the iris? b. Are there specialized endothelial cells on this surface? c. What is the nature of the "thick walls" of the stromal vessels? d. What is the nature of the abundant ground substance of the iris stroma? e. What is the relationship of the pigment epithelium to the dilator muscle? f. Is there an internal limiting membrane on the posterior surface of the iris compara-