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The inhibitory effect of dehydroascorbic acid on insulin secretion from mouse pancreatic islets
TOXICOLOGY
AND
APPLIED
PHARMACOLOGY
50, 57-65 (1979)
The Inhibitory Effect of Dehydroascorbic Acid on Insulin Secretion from Mouse Pancreatic Islets’ LARRY A. PENCE~ AND JOHN H. MENNEAR~ Department
of Pharmacology and Toxicology, Purdue University, West Lafayette, Indiana 47907
Received November 9, 1978; accepted February 26, 1979
The Inhibitory Effect of Dehydroascorbic Acid on Insulin Secretion from Mouse Pancreatic Islets. PENCE, L. A., AND MENNEAR, J. H. (1979). Toxicol. Appl. ‘Pharmacol. 50, 57-65. Intravenous doses of 200 mg/kg dehydroascorbic acid (DHA) produced hyperglycemia, hypoinsulinemia, and decreased glucose tolerance in mice. This effect of DHA is mediated, at least in part, through a direct inhibition of pancreatic insulin release. Exposure of isolated pancreatic islets to a concentration of 2.0 mg/dl DHA reduces the responsiveness of the islets to both glucose (300 mg/dl) and tolbutamide (6 f 10-3 M). Exposure of isolated islets to DHA in a high concentration of o-glucose (300 mg/dl) partially protected them against the inhibitory effect of DHA. Exposure of islets to 4.0 mg/dl of DHA causes a leakage of insulin. Similarly, islets isolated from mice which had been treated with 300 mg/ kg DHA iv exhibited increased insulin release in the presence of only 60 mg/dl glucose. Intravenous administration of either 200 or 300 mg/kg DHA prior to islet isolation results in increased insulin secretion in response to 300 mg/dl glucose. The results show that the pancreatic effects of DHA are similar to those of the diabetogen, alloxan.
Dehydroascorbic acid (DHA), the reversible oxidation product and one of the transport forms of ascorbic acid, has been reported to produce alloxan-like effects on pancreatic tissue. Patterson (1949, 1950) reported that iv injection of DHA induces diabetes mellitus in rats. The daily administration of 60 to 80 mg/rat for 3 days resulted in permanent hyperglycemia while a single dose of 1.4 g/rat produced blood glucose changes similar to those elicited by the administration of
alloxan (transient hyperglycemia followed by marked hypoglycemia and finally permanent hyperglycemia). In vitro experiments employing DHA conducted on the toadfish pancreatic islet model also revealed effects similar to those produced by alloxan. Exposure of these islets to DHA increased their permeability to mannitol, a monosaccharide which does not normally enter cells, and caused a leakage of insulin from the islets (Pillsbury et al., 1973). These results are identical to those reported for alloxan (Cooperstein et al., 1969). Since DHA might be considered to be a diabetogen and it is a normal metabolite of ascorbic acid, its effects and occurrence in humans are of interest. Chatterjee et al. (1975) have studied the effects of large daily
r This research was supported by Grants AM-14134 and ES-01 123 from the National Institutes of Health and the Juvenile Diabetes Foundation. * Present address: Hazleton Laboratories America, Inc., 9200 Leesburg Turnpike, Vienna, Va. 22180. 3 Present address: Travenol Laboratories, 6301 Lincoln Ave, Morton Grove, Ill. 60053. 5-l
0041-008X/79/1ooO57-09$02.00/0 Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in Great Britain
58
PENCE AND MENNEAR
doses of ascorbic acid on serum DHA and glucose in normal humans. The oral administration of 4.0 g of ascorbic acid per day had no effect on either parameter. However, in humans who were eating a diet containing a high cereal content, the administration of the ascorbic acid produced elevated serum concentrations of DHA which were associated with hyperglycemia. These workers also reported that the DHA content of sera from diabetic humans is nearly lOO-fold greater than that of sera from nondiabetics (1.0-2.6 vs 0.01-0.05 mg/dl). Similarly, prediabetic humans (who subsequently developed overt diabetes mellitus) have elevated serum DHA concentrations. These results suggest the provocative possibility of a relationship between DHA and the etiology of diabetes mellitus. On the other hand, Mann (1974) has suggested that diabetic microangiopathies may be related to a reduced ability of DHA to enter diabetic tissues. He has shown that concentrations of glucose from 100 to 800 mg/dl inhibit DHA uptake by human red blood cells. This inhibition, if widespread, could lead to localized areas of scurvy and abnormalities in collagen metabolism. Mann and Newton (1975) have also demonstrated that insulin facilitates the uptake of DHA into tissues such as muscle and blood vessels. It is possible that hyperglycemia and hypoinsulinemia (or increased peripheral resistance to insulin) which accompany diabetes reduces the cellular uptake of DHA. Thus, earlier research suggests that there may be a relationship between ascorbic acid metabolism, DHA disposition, and the development of diabetes mellitus per se as well as subsequent complications. The nature of this relationship, i.e., causative or preventative, is unclear. The experiments reported in this communication were conducted to assess the effects of DHA on one aspect of the overall problem, the insulin secretory response of the pancreatic B cell. The results indicate that DHA directly inhibits the responsiveness of the pancreas to glucose.
METHODS Male Swiss albino mice (Laboratory Supply Company, Indianapolis, Ind.) weighing 25 to 30 g were used throughout this study. Animals were housed in groups of 10 with free access to food (Wayne Lab Blox) and water for at least 1 week prior to experimentation. In experiments in vivo, DHA was administered iv (100-300 mg/kg) in volume doses of 2.0ml/kg. Solutions were prepared in redistilled water immediately prior to use. The glucose challenge test was conducted by administering o-glucose (2.0 g/kg) ip. Solutions of glucose were prepared, in redistilled water, at least 1 hr prior to administration. Serum glucose concentrations were determined by a glucose oxidase method (Beckman glucose analyzer) and serum insulin was assayed as immunoreactive insulin (IRI) by use of a commercially available kit (Amersham/Searle). Blood samples were obtained by the orbital sinus puncture technique and because of the volume of serum required (0.1 ml) for the IRI assay, each animal was bled only once. For experiments in vitro, pancreatic islets were isolated by the collagenase method of Lacy and Kostianovsky (1967). Immediately after isolation, the islets were incubated for 30min at 37°C in Krebs-Ringer bicarbonate buffer (pH 7.4) supplemented with 0.3% bovine serum albumin and 60 mg/dl o-glucose. After this stabilization period, groups of three similarly sized islets were exposed to ascorbic or dehydroascorbic acids (2.0-4.0 mg/dl) in 2.0 ml of incubation medium at 0°C by the method of Fung and Mennear (1974). This lower temperature was used because the half-life of DHA in solution is short when maintained at physiological temperatures and pH (Pillsbury et al., 1973). Control islets also were exposed to 0°C for the 30-min period. After the exposure period, the islets were rinsed and transferred to 2.0 ml of DHA-free medium containing either o-glucose (60 or 300 mg/dl) or tolbutamide (6 x 1O-J M) at 37°C. Incubation was continued for 90 or 360 min with a gas phase of 95% oxygen and 5 % carbon dioxide. All incubations were carried out in a Dubnoff metabolic shaker (60 oscillations/min). Aliquots of the incubation medium were taken at various intervals and assayed for IRI. At the termination of each experiment, all incubation vessels were inspected to ensure the presence of three islets. The results of these experiments were statistically analyzed by analysis of variance followed, when appropriate, by application of the Newman-Keuls range test for determining differences between means. The means and SE are based on three or more independent replications of.each experiment.
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RESULTS Initial experiments were conducted in an attempt to produce diabetes or permanent hyperglycemia in mice by the intravenous injection of DHA. Daily administrations of doses as high as 300 mg/kg produced periods of transient hyperglycemia; however, none of the animals developed diabetes. In fact, the most prominent effect noted after two or three injections of the 300 mg/kg dose was intense central nervous system stimulation which was characterized by increased motor activity, clonic convuisions, respiratory arrest, and death. Experiments were designed to determine if the observed hyperglycemia was associated with changes in serium insulin concentrations. Mice were given 200 mg/kg DHA iv with blood samples being taken immediately prior to and 45, 60, 90, and 165 min after treatment. The results of this experiment are shown in Fig. 1. Control mice responded to the experimental manipulation with a slight hyperglycemic response (left panel) which was evident at the 45-min interval. The administration of DHA produced a more pronounced hyperglycemic response which persisted throughout the experimental period. Serum IRI concentrations (center panel) remained relatively constant in control animals throughout the experimental period. The serum IRI concentrations in DHA-treated mice were significantly lower than those of control animals at each of the test periods despite the fact that serum glucose concentrations were elevated. This effect is even more vividly apparent when insulinogenic indices (ratio of IRI in pU/ml to glucose in mg/dl) are compared (Seltzer ‘and Harris, 1964). Decreases in insulinogenic indexes imply decreased pancreatic insulin secretory activity. The insulinogenic indexes (right panel) of control mice ranged from 0.16 to 0.23 during the experiment while those of DHAtreated animals ranged from only 0.07 to 0.13.
ACID
AND
INS”L,N
59
The inhibition of pancreatic insulin secretion by DHA was even more pronounced when mice were challenged with a glucose load (2.0 g/kg, ip). Animals were given the 200 mg/kg dose of DHA 45 min prior to the injection of glucose. Serum glucose and IRI concentrations were then determined at 15, 45, and 120 min after the glucose injection. The results of this experiment are shown in Fig. 2. In control mice, the injection of glucose produced a prompt hyperglycemia which was evident 15 min after injection (60 min into the experiment). Serum glucose concentrations returned to normal values within 45 min of glucose administration. The administration of DHA produced the expected hyperglycemia which was evident at the 45-min interval (immediately prior to the injection of glucose). Fifteen minutes after the glucose injection, serum glucose concentrations were nearly 600 mg/dl. Mean serum glucose concentration returned to control values by the 165-min interval. The hyperglycemia observed in control animals was accompanied by a prompt and sharp increase in serum IRI (increasing from 30 to 107 @J/ml in 15 min). Serum IRI levels were significantly depressed by DHA immediately prior to glucose injection but did rise in response to the glucose load. However, this increase was neither as rapid nor as great in magnitude as in the control animals. Insulinogenic indexes of the DHAtreated mice were significantly lower than control values at the 45-, 60-, and 90-min intervals. The results of these in tko experiments indicate that DHA reduces pancreatic insulin secretion in response to glucose stimulation. The design of the experiments does not, however, allow us to make a determination of whether the effect is a direct pancreatic action or secondary to some other effect of DHA. For this reason, experiments were conducted to assess the effects of DHA on insulin secretion from the isolated pancreatic islet.
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tip/)*)
AND
3Al13V3~0NnbWl
3so3n79
MENNEAR
WlY3S
wnas
DEHYDROASCORBIC
XXlNI
lI”‘/fin)
ACID
3lN3bONllnSNI
NIlnSNI
3hl13V3MONnVWl
(iP/b~u)
3So3nio
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wn83s
AND
INSULIN
61
62
PENCE AND MENNEAR
EFFECT
TABLE 1 OF L-ASCORBIC AND L-DEHYDROASCORBICACIDS ON INSULIN RELEASE FROM ISOLATED MOUSE PANCREATIC ISLETS IRI + SEC (pU/islet)
Mean
Treatment’
Control Control L-ascorbic acid (2 mg/dl) L-Dehydroascorbic acid (2 m&W Control L-Dehydroascorbic acid (2 m&W
Insulin secretagogue* Glucose Glucose Glucose Glucose
(60 mg/dl) (300 mg/dl) (300 mg/dl) (300 mg/dl)
Tolbutamide Tolbutamide
(6 x 1O-3 M) (6 x 1O--3M)
11.3k1.4 30.4kO.6 29.2k 1.8 9.6k 1.3 32.1+ 1.9 15.0+ 1.5
’ Islet treatment conducted at 0°C for 30 min. * Post-treatment incubation was conducted at 37°C for 90 min. Islets were stimulated with either glucose (60 or 300 mg/dl) or tolbutamide (6 x 10m3M). c Total microunits of immunoreactive insulin released per islet after 90 min of incubation. Values represent mean + SE for three sets of islets.
The results summarized in Table 1 show the comparative effects of ascorbic and dehydroascorbic acids (2.0 mg/dl) on the responsiveness of mouse pancreatic islets to glucose. Control islets which were exposed to a nonstimulating concentration of glucose (60 mg/dl) released only minimal amounts of IRI during the 90-min incubation period. The exposure of control islets to the stimulating concentration of glucose (300 mg/ dl) resulted in a nearly threefold increase in IRI release. The exposure of islets to ascorbic acid prior to glucose stimulation failed to alter the amount of IRI released during the incubation period. When islets were exposed to DHA prior to glucose stimulation, however, they released only 9.6 pU-IRI/ islet, a value which was similar to the release from control islets exposed to the nonstimulating concentration of glucose. The results of this experiment indicate that DHA inhibits glucose-stimulated insulin secretion, at least in part, through a direct action on the pancreatic p cell. In order to determine if this inhibitory effect is specific for glucose, a second in vitro experiment was conducted in which the B cell stimulant, tolbutamide, was employed as the insulin secretagogue. The results of this experiment are also shown in Table 1.
Control islets responded to 90 min of exposure to tolbutamide (6 x 1O-3 M) with a release of 32.1 PU-IRI/islet. Exposure of islets to DHA prior to tolbutamide reduced IRI release to only 15 pU/islet (p < 0.05). Therefore, the inhibitory effect of DHA on IRI release from isolated islets is not specific for glucose. To further explore the pancreotoxic effects of dehydroascorbic acid, it was of interest to assess the interaction between DHA and glucose in the isolated islet preparation. To this end, islets were exposed to DHA in the presence of either 60 or 300 mg/dl glucose. Islets were then rinsed and incubated at 37°C in the presence of 300 mg/dl glucose. The results of this experiment are shown in Fig. 3. Islets which had been exposed to 300 mg/dl glucose (but no DHA) at 0°C responded to glucose at 37°C with a relase of 41 /tU-IRI/ islet over the 90-min incubation period. Again, the islets which were exposed to DHA in the presence of only 60 mg/dl glucose exhibited inhibition of glucose stimulation, releasing only 6.1 PU-lRI/islet. The presence of the 300 mg/dl concentration of glucose during DHA exposure, however, afforded partial protection against the inhibitory effect of DHA. These islets released signifi-
63
DEHYDROASCORBIC ACID AND INSULIN
MINUTES
OF
INCUBATION
3. Antagonistic effect of D-glucose on the inhibitory action of dehydroascorbic acid on insulin release. Groups of three pancreatic islets were exposed to DHA (2.0 mg/dl) in the presence of either 60 or 300 mg/dl o-glucose at 0°C for 30 min. Control islets were not exposed to DHA. All islets were then exposed to 300 mg/dl o-glucose at 37°C.. Values represent mean+SE /IU-IRI released per islet. Significant (p < 0.05) differences from control were detected 90 min for the DHA+300 mg/dl o-glucose groulj and at 60 and 90 min for the DHA+60 mg/dl D-glucose group. If SE is smaller than the size of the symbol it will not appear on the figure. FIG.
in vitro
0
60
120 MINUTES
cantly more IRI (22.4 pU/islet) than did islets exposed to DHA in the presence of the low concentration of glucose. It should be pointed out, however, that this protective effect was only partial since these islets released significantly less IRI than did control islets. The next experiment was conducted to explore the effect of a higher concentration of DHA followed by an extended incubation period. In this experiment islets were exposed to 2.0 and 4.0 mg/dl DHA prior to a 6-hr incubation in 60 mg/dl glucose. The results of this experiment are shown in Fig. 4. Islets which had been exposed to the 2.0 mg/dl concentration of DHA were identical to control islets in their response to the nonstimulating concentration of glucose. Total IRI release for both groups was minimal over the 6-hr period and no significant differences were detected. Islets which were exposed to the 4.0 mg/dl concentration of DHA exhibited a prompt and marked release of IRI throughout the entire incuba-
I 160 240 OF INCUBATION
300
c 36(
FIG. 4. Effect of dehydroascorbic acid on nonstimulated insulin release from isolated mouse pancreatic islets. Groups of islets were exposed to 2.0 or 4.0 mg/dl DHA in the presence of 60 mg/dl o-glucose at 0°C for 30 min. They were then incubated at 37°C for 360 min in the absence of DHA. Islets exposed to the high concentration of DHA released more (~~0.05) IRI than did controls or islets exposed to the low concentration of DHA. If SE is smaller than the size of the symbol it will not appear on the figure.
64
PENCE AND MENNEAR
tion period. At the conclusion of the experiment, these islets had released nearly 70 pUIRI/islet despite the low concentration of glucose in the incubation medium. Fung and Mennear (1974) reported that exposure of mouse islets to a high concentration of alloxan resulted in an increase in the release of IRI. In the case of alloxan, the release required 4.5 hr of incubation to be produced. These workers suggested that this in vitro effect corresponded to the second phase of the in vivo reaction to alloxan administration, i.e., hyperinsulinemia and hypoglycemia. In our experiments, we have never observed hypoglycemia when DHA was administered to intact mice. It is possible that our inability to produce diabetic mice through the use of DHA is a reflection of our inability to administer doses sufficient to produce pancreatic damage. For this reason, a final experiment was conducted in which mice were treated with intravenous doses of 100, 200, and 300 mg/kg DHA 60 min prior to sacrifice and isolation of pancreatic islets. These islets were then incubated for 90 min in a nonstimulating concentration of glucose.
-
FIG. 6. Glucose stimulated insutm release from pancreatic islets isolated from dehydroascorbic acid-treated mice. Mice were treated with intravenous DHA 60 min prior to sacrifice and islet isolation. Groups of islets were incubated at 37°C in the presence of 300 mgjdl D-glucose. Islets from mice treated with either 200 or 300 mg/kg DHA released significantly more IRI (pcO.05) than did controls. If SE is smaller than the size of the symbol it will not appear on the figure.
The results of this experiment are shown in Fig. 5. Islets isolated from mice which had received either 100 or 200 mg/kg DHA released the same, minimal, amount of IRI in the presence of 60 mg/dl glucose as did islets isolated from control mice. Islets from mice which had been treated with the 300 mg/kg dose of DHA, however, released significantly more IRI at each of the time intervals than any other group. When islets from DHA-pretreated mice were exposed to the 300 mg/dl concentration of glucose, both the 200 and the 300 mg/kg groups released more IRI than did controls (Fig. 6).
I 0
30 MINUTES
OF
60 INCUBATION
90
FIG. 5. Nonstimulated insulin release from pancreatic islets isolated from dehydroascorbic acidtreated mice. Mice were treated with intravenous DHA 60 min prior to sacrifice and islet isolation. Groups of islets were incubated at 37°C in the presence of 60 mg/dl n-glucose. Islets from mice treated with 300 mg/kg DHA released significantly (p < 0.05) more IRI than controls. If SE is smaller than the size of the symbol it will not appear on the figure.
DISCUSSION The results of these experiments have demonstrated several similarities between the in vitro actions of DHA and alloxan. Earlier workers (Tomita et al., 1974) have reported that alloxan inhibits the responsiveness of isolated pancreatic islets to glucose and tolbutamide. Like alloxan, lower concentrations of DHA also inhibit islet responsiveness
DEHYDROASCORBIC
to both
secretogogues. Further, both the in vivo and in vitro pancreotoxic effects of alloxan and DHA can be antagonized by glucose (Sutherland and Rall, 1958; EspositoAvella and Mennear, 1973; Tomita et al., 1974). This observation may indicate that the pancreotoxic action of DHA is mediated through an action at the level of the glucose receptor. This observed inhibitory effect of DHA on glucose responsiveness of isolated islets is consistent with in U~ZJOhyperglycemia and decreased glucose tolerance. Inhibition of pancreatic responsiveness is not, however, consistent with the earlier report of Pillsbury et al. (1973) of DHA-induced leakage of insulin from isolated toadfish pancreatic islets. It is possible that the 2.0 mg/dl concentration of DHA is not sufficient to produce leakage from mouse islets or that the 90-min incubation period is not long enough for the effect to manifest itself. The exploration of this hypothesis demonstrated that higher concentrations of DHA like alloxan resulted in the increased insulin release, even in the presence of nonstimulating concentrations of glucose. This action of DHA could be a prelude to the induction of the diabetic state. This possibility is difficult to assess completely in the present experiments, however, because the administration of 300 mg/kg DHA to mice prior to the islet isolation resulted in IRI release in the presence of low glucose but is not a diabetogenic treatment in our hands. It is possible that this effect is an artifact produced by the combination of a pancreatic effect of DHA and the collagenase treatment during islet isolation. Finally, it is noteworthy to point out that the concentration of DHA employed in the experiments in vitro, 2.0 mg/dl, is within the range of serum concentrations of the vitamin reported for diabetic sera (Chatterjee et al. 1975). Since DHA does decrease pancreatic insulin secretory activity in mouse islets, its potential role in the development of diabetes mellitus should be studied further. Also, additional experiments on the serum concen-
ACID
AND
65
INSULIN
trations of DHA in diabetic and non diabetic humans should be conducted. Chatterjee’s experiments were conducted in India and it is possible that ethnic or dietary peculiarities accounted for the findings reported.
REFERENCES A. K., NANDI, B. K., N. (1975). Synthesis and some major functions of vitamin C in animals. Ann, N. Y.
CHATTERJEE, AND
1. B.,
MAJUMDER,
SUBRAMANIAN,
Acad.
Sci. 258, 24-47.
S. J., WATKINS, D., and LAZAROW, A. (1969). The effect of alloxan on islet tissue permeability. In The Stractrtre and Metabolism of the Pancreatic Islets (S. E. Broline, B. Hellman, and H. Knutson, eds.), pp. 398. Pergamon, Oxford. ESPOSITO-AVELLA, M., AND MENNEAR, J. H. (1973). Studies on the protective effect of diphenylhydantoin against alloxan diabetes in mice. Proc. Sot. COOPERSTEIN,
Exp.
Biol.
Med.
142, 82-85.
W., AND MENNEAR, J. H. (1974). Effects of alloxan on insulin release by isolated mouse pancreatic islets. Proc. Sot. Exp. Biol. Med. 146,
FUNG,
949-952.
P. E., AND KOSTIANOVSKY, M. (1967). Method for the isolation of intact islets of Langerhans from the rat pancreas. Diabetes 16, 35-39. MANN, G. V. (1974). Hypothesis: The role of vitamin C in diabetic angiopathy. Perspect. Biol. Med.
LACY,
17,210-212.
G. V., AND NEWTON, P. (1975). The membrane transport of ascorbic acid. Ann. IV. Y. Acad. Sri.
MANN,
258, 243-25
I.
J. W. dehydroascorbic PATTERSON, J. W. dehydroascorbic PATTERSON,
J. Biol. .PILLSBURY,
(1949). The diabetogenic effects of acid. Endocrinology 45, 344. (1950). The diabetogenic effect of acid and dehydroisoascorbic acid.
Chem. 183, 81-88. S., WATKINS, D.,
AND
COPPERSTEIN,
S. J.
(1973). Effect of dehydroascorbic acid on permeability of pancreatic islet tissue in vitro. J. Pharmacol. Exp. Ther. 185, 713-718. SELTZER, H. S., AND HARRIS, V. L. (1964). Exhaustion of insulinogenic reserve in maturity onset diabetic patients during prolonged and continuous hyperglycemic stress. Diabetes 13, 6-l 3. SUTHERLAND, E. W., AND RALL, T. W. (1958). Fractionation and characterization of a cyclic adenine ribonucleotide formed by tissue particles. J. Biol. Chem. 232, 1065-1076. TOMITA, T., LACY, P. E., MATSCHINSKY, F. M., AND MCDANIEL, M. L. (1974). The effect of alloxan on insulin secretion from isolated rat islets perifused in vitro. Diabetes 23, 517-524.
AND
APPLIED
PHARMACOLOGY
50, 57-65 (1979)
The Inhibitory Effect of Dehydroascorbic Acid on Insulin Secretion from Mouse Pancreatic Islets’ LARRY A. PENCE~ AND JOHN H. MENNEAR~ Department
of Pharmacology and Toxicology, Purdue University, West Lafayette, Indiana 47907
Received November 9, 1978; accepted February 26, 1979
The Inhibitory Effect of Dehydroascorbic Acid on Insulin Secretion from Mouse Pancreatic Islets. PENCE, L. A., AND MENNEAR, J. H. (1979). Toxicol. Appl. ‘Pharmacol. 50, 57-65. Intravenous doses of 200 mg/kg dehydroascorbic acid (DHA) produced hyperglycemia, hypoinsulinemia, and decreased glucose tolerance in mice. This effect of DHA is mediated, at least in part, through a direct inhibition of pancreatic insulin release. Exposure of isolated pancreatic islets to a concentration of 2.0 mg/dl DHA reduces the responsiveness of the islets to both glucose (300 mg/dl) and tolbutamide (6 f 10-3 M). Exposure of isolated islets to DHA in a high concentration of o-glucose (300 mg/dl) partially protected them against the inhibitory effect of DHA. Exposure of islets to 4.0 mg/dl of DHA causes a leakage of insulin. Similarly, islets isolated from mice which had been treated with 300 mg/ kg DHA iv exhibited increased insulin release in the presence of only 60 mg/dl glucose. Intravenous administration of either 200 or 300 mg/kg DHA prior to islet isolation results in increased insulin secretion in response to 300 mg/dl glucose. The results show that the pancreatic effects of DHA are similar to those of the diabetogen, alloxan.
Dehydroascorbic acid (DHA), the reversible oxidation product and one of the transport forms of ascorbic acid, has been reported to produce alloxan-like effects on pancreatic tissue. Patterson (1949, 1950) reported that iv injection of DHA induces diabetes mellitus in rats. The daily administration of 60 to 80 mg/rat for 3 days resulted in permanent hyperglycemia while a single dose of 1.4 g/rat produced blood glucose changes similar to those elicited by the administration of
alloxan (transient hyperglycemia followed by marked hypoglycemia and finally permanent hyperglycemia). In vitro experiments employing DHA conducted on the toadfish pancreatic islet model also revealed effects similar to those produced by alloxan. Exposure of these islets to DHA increased their permeability to mannitol, a monosaccharide which does not normally enter cells, and caused a leakage of insulin from the islets (Pillsbury et al., 1973). These results are identical to those reported for alloxan (Cooperstein et al., 1969). Since DHA might be considered to be a diabetogen and it is a normal metabolite of ascorbic acid, its effects and occurrence in humans are of interest. Chatterjee et al. (1975) have studied the effects of large daily
r This research was supported by Grants AM-14134 and ES-01 123 from the National Institutes of Health and the Juvenile Diabetes Foundation. * Present address: Hazleton Laboratories America, Inc., 9200 Leesburg Turnpike, Vienna, Va. 22180. 3 Present address: Travenol Laboratories, 6301 Lincoln Ave, Morton Grove, Ill. 60053. 5-l
0041-008X/79/1ooO57-09$02.00/0 Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in Great Britain
58
PENCE AND MENNEAR
doses of ascorbic acid on serum DHA and glucose in normal humans. The oral administration of 4.0 g of ascorbic acid per day had no effect on either parameter. However, in humans who were eating a diet containing a high cereal content, the administration of the ascorbic acid produced elevated serum concentrations of DHA which were associated with hyperglycemia. These workers also reported that the DHA content of sera from diabetic humans is nearly lOO-fold greater than that of sera from nondiabetics (1.0-2.6 vs 0.01-0.05 mg/dl). Similarly, prediabetic humans (who subsequently developed overt diabetes mellitus) have elevated serum DHA concentrations. These results suggest the provocative possibility of a relationship between DHA and the etiology of diabetes mellitus. On the other hand, Mann (1974) has suggested that diabetic microangiopathies may be related to a reduced ability of DHA to enter diabetic tissues. He has shown that concentrations of glucose from 100 to 800 mg/dl inhibit DHA uptake by human red blood cells. This inhibition, if widespread, could lead to localized areas of scurvy and abnormalities in collagen metabolism. Mann and Newton (1975) have also demonstrated that insulin facilitates the uptake of DHA into tissues such as muscle and blood vessels. It is possible that hyperglycemia and hypoinsulinemia (or increased peripheral resistance to insulin) which accompany diabetes reduces the cellular uptake of DHA. Thus, earlier research suggests that there may be a relationship between ascorbic acid metabolism, DHA disposition, and the development of diabetes mellitus per se as well as subsequent complications. The nature of this relationship, i.e., causative or preventative, is unclear. The experiments reported in this communication were conducted to assess the effects of DHA on one aspect of the overall problem, the insulin secretory response of the pancreatic B cell. The results indicate that DHA directly inhibits the responsiveness of the pancreas to glucose.
METHODS Male Swiss albino mice (Laboratory Supply Company, Indianapolis, Ind.) weighing 25 to 30 g were used throughout this study. Animals were housed in groups of 10 with free access to food (Wayne Lab Blox) and water for at least 1 week prior to experimentation. In experiments in vivo, DHA was administered iv (100-300 mg/kg) in volume doses of 2.0ml/kg. Solutions were prepared in redistilled water immediately prior to use. The glucose challenge test was conducted by administering o-glucose (2.0 g/kg) ip. Solutions of glucose were prepared, in redistilled water, at least 1 hr prior to administration. Serum glucose concentrations were determined by a glucose oxidase method (Beckman glucose analyzer) and serum insulin was assayed as immunoreactive insulin (IRI) by use of a commercially available kit (Amersham/Searle). Blood samples were obtained by the orbital sinus puncture technique and because of the volume of serum required (0.1 ml) for the IRI assay, each animal was bled only once. For experiments in vitro, pancreatic islets were isolated by the collagenase method of Lacy and Kostianovsky (1967). Immediately after isolation, the islets were incubated for 30min at 37°C in Krebs-Ringer bicarbonate buffer (pH 7.4) supplemented with 0.3% bovine serum albumin and 60 mg/dl o-glucose. After this stabilization period, groups of three similarly sized islets were exposed to ascorbic or dehydroascorbic acids (2.0-4.0 mg/dl) in 2.0 ml of incubation medium at 0°C by the method of Fung and Mennear (1974). This lower temperature was used because the half-life of DHA in solution is short when maintained at physiological temperatures and pH (Pillsbury et al., 1973). Control islets also were exposed to 0°C for the 30-min period. After the exposure period, the islets were rinsed and transferred to 2.0 ml of DHA-free medium containing either o-glucose (60 or 300 mg/dl) or tolbutamide (6 x 1O-J M) at 37°C. Incubation was continued for 90 or 360 min with a gas phase of 95% oxygen and 5 % carbon dioxide. All incubations were carried out in a Dubnoff metabolic shaker (60 oscillations/min). Aliquots of the incubation medium were taken at various intervals and assayed for IRI. At the termination of each experiment, all incubation vessels were inspected to ensure the presence of three islets. The results of these experiments were statistically analyzed by analysis of variance followed, when appropriate, by application of the Newman-Keuls range test for determining differences between means. The means and SE are based on three or more independent replications of.each experiment.
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RESULTS Initial experiments were conducted in an attempt to produce diabetes or permanent hyperglycemia in mice by the intravenous injection of DHA. Daily administrations of doses as high as 300 mg/kg produced periods of transient hyperglycemia; however, none of the animals developed diabetes. In fact, the most prominent effect noted after two or three injections of the 300 mg/kg dose was intense central nervous system stimulation which was characterized by increased motor activity, clonic convuisions, respiratory arrest, and death. Experiments were designed to determine if the observed hyperglycemia was associated with changes in serium insulin concentrations. Mice were given 200 mg/kg DHA iv with blood samples being taken immediately prior to and 45, 60, 90, and 165 min after treatment. The results of this experiment are shown in Fig. 1. Control mice responded to the experimental manipulation with a slight hyperglycemic response (left panel) which was evident at the 45-min interval. The administration of DHA produced a more pronounced hyperglycemic response which persisted throughout the experimental period. Serum IRI concentrations (center panel) remained relatively constant in control animals throughout the experimental period. The serum IRI concentrations in DHA-treated mice were significantly lower than those of control animals at each of the test periods despite the fact that serum glucose concentrations were elevated. This effect is even more vividly apparent when insulinogenic indices (ratio of IRI in pU/ml to glucose in mg/dl) are compared (Seltzer ‘and Harris, 1964). Decreases in insulinogenic indexes imply decreased pancreatic insulin secretory activity. The insulinogenic indexes (right panel) of control mice ranged from 0.16 to 0.23 during the experiment while those of DHAtreated animals ranged from only 0.07 to 0.13.
ACID
AND
INS”L,N
59
The inhibition of pancreatic insulin secretion by DHA was even more pronounced when mice were challenged with a glucose load (2.0 g/kg, ip). Animals were given the 200 mg/kg dose of DHA 45 min prior to the injection of glucose. Serum glucose and IRI concentrations were then determined at 15, 45, and 120 min after the glucose injection. The results of this experiment are shown in Fig. 2. In control mice, the injection of glucose produced a prompt hyperglycemia which was evident 15 min after injection (60 min into the experiment). Serum glucose concentrations returned to normal values within 45 min of glucose administration. The administration of DHA produced the expected hyperglycemia which was evident at the 45-min interval (immediately prior to the injection of glucose). Fifteen minutes after the glucose injection, serum glucose concentrations were nearly 600 mg/dl. Mean serum glucose concentration returned to control values by the 165-min interval. The hyperglycemia observed in control animals was accompanied by a prompt and sharp increase in serum IRI (increasing from 30 to 107 @J/ml in 15 min). Serum IRI levels were significantly depressed by DHA immediately prior to glucose injection but did rise in response to the glucose load. However, this increase was neither as rapid nor as great in magnitude as in the control animals. Insulinogenic indexes of the DHAtreated mice were significantly lower than control values at the 45-, 60-, and 90-min intervals. The results of these in tko experiments indicate that DHA reduces pancreatic insulin secretion in response to glucose stimulation. The design of the experiments does not, however, allow us to make a determination of whether the effect is a direct pancreatic action or secondary to some other effect of DHA. For this reason, experiments were conducted to assess the effects of DHA on insulin secretion from the isolated pancreatic islet.
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ACID
3lN3bONllnSNI
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INSULIN
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62
PENCE AND MENNEAR
EFFECT
TABLE 1 OF L-ASCORBIC AND L-DEHYDROASCORBICACIDS ON INSULIN RELEASE FROM ISOLATED MOUSE PANCREATIC ISLETS IRI + SEC (pU/islet)
Mean
Treatment’
Control Control L-ascorbic acid (2 mg/dl) L-Dehydroascorbic acid (2 m&W Control L-Dehydroascorbic acid (2 m&W
Insulin secretagogue* Glucose Glucose Glucose Glucose
(60 mg/dl) (300 mg/dl) (300 mg/dl) (300 mg/dl)
Tolbutamide Tolbutamide
(6 x 1O-3 M) (6 x 1O--3M)
11.3k1.4 30.4kO.6 29.2k 1.8 9.6k 1.3 32.1+ 1.9 15.0+ 1.5
’ Islet treatment conducted at 0°C for 30 min. * Post-treatment incubation was conducted at 37°C for 90 min. Islets were stimulated with either glucose (60 or 300 mg/dl) or tolbutamide (6 x 10m3M). c Total microunits of immunoreactive insulin released per islet after 90 min of incubation. Values represent mean + SE for three sets of islets.
The results summarized in Table 1 show the comparative effects of ascorbic and dehydroascorbic acids (2.0 mg/dl) on the responsiveness of mouse pancreatic islets to glucose. Control islets which were exposed to a nonstimulating concentration of glucose (60 mg/dl) released only minimal amounts of IRI during the 90-min incubation period. The exposure of control islets to the stimulating concentration of glucose (300 mg/ dl) resulted in a nearly threefold increase in IRI release. The exposure of islets to ascorbic acid prior to glucose stimulation failed to alter the amount of IRI released during the incubation period. When islets were exposed to DHA prior to glucose stimulation, however, they released only 9.6 pU-IRI/ islet, a value which was similar to the release from control islets exposed to the nonstimulating concentration of glucose. The results of this experiment indicate that DHA inhibits glucose-stimulated insulin secretion, at least in part, through a direct action on the pancreatic p cell. In order to determine if this inhibitory effect is specific for glucose, a second in vitro experiment was conducted in which the B cell stimulant, tolbutamide, was employed as the insulin secretagogue. The results of this experiment are also shown in Table 1.
Control islets responded to 90 min of exposure to tolbutamide (6 x 1O-3 M) with a release of 32.1 PU-IRI/islet. Exposure of islets to DHA prior to tolbutamide reduced IRI release to only 15 pU/islet (p < 0.05). Therefore, the inhibitory effect of DHA on IRI release from isolated islets is not specific for glucose. To further explore the pancreotoxic effects of dehydroascorbic acid, it was of interest to assess the interaction between DHA and glucose in the isolated islet preparation. To this end, islets were exposed to DHA in the presence of either 60 or 300 mg/dl glucose. Islets were then rinsed and incubated at 37°C in the presence of 300 mg/dl glucose. The results of this experiment are shown in Fig. 3. Islets which had been exposed to 300 mg/dl glucose (but no DHA) at 0°C responded to glucose at 37°C with a relase of 41 /tU-IRI/ islet over the 90-min incubation period. Again, the islets which were exposed to DHA in the presence of only 60 mg/dl glucose exhibited inhibition of glucose stimulation, releasing only 6.1 PU-lRI/islet. The presence of the 300 mg/dl concentration of glucose during DHA exposure, however, afforded partial protection against the inhibitory effect of DHA. These islets released signifi-
63
DEHYDROASCORBIC ACID AND INSULIN
MINUTES
OF
INCUBATION
3. Antagonistic effect of D-glucose on the inhibitory action of dehydroascorbic acid on insulin release. Groups of three pancreatic islets were exposed to DHA (2.0 mg/dl) in the presence of either 60 or 300 mg/dl o-glucose at 0°C for 30 min. Control islets were not exposed to DHA. All islets were then exposed to 300 mg/dl o-glucose at 37°C.. Values represent mean+SE /IU-IRI released per islet. Significant (p < 0.05) differences from control were detected 90 min for the DHA+300 mg/dl o-glucose groulj and at 60 and 90 min for the DHA+60 mg/dl D-glucose group. If SE is smaller than the size of the symbol it will not appear on the figure. FIG.
in vitro
0
60
120 MINUTES
cantly more IRI (22.4 pU/islet) than did islets exposed to DHA in the presence of the low concentration of glucose. It should be pointed out, however, that this protective effect was only partial since these islets released significantly less IRI than did control islets. The next experiment was conducted to explore the effect of a higher concentration of DHA followed by an extended incubation period. In this experiment islets were exposed to 2.0 and 4.0 mg/dl DHA prior to a 6-hr incubation in 60 mg/dl glucose. The results of this experiment are shown in Fig. 4. Islets which had been exposed to the 2.0 mg/dl concentration of DHA were identical to control islets in their response to the nonstimulating concentration of glucose. Total IRI release for both groups was minimal over the 6-hr period and no significant differences were detected. Islets which were exposed to the 4.0 mg/dl concentration of DHA exhibited a prompt and marked release of IRI throughout the entire incuba-
I 160 240 OF INCUBATION
300
c 36(
FIG. 4. Effect of dehydroascorbic acid on nonstimulated insulin release from isolated mouse pancreatic islets. Groups of islets were exposed to 2.0 or 4.0 mg/dl DHA in the presence of 60 mg/dl o-glucose at 0°C for 30 min. They were then incubated at 37°C for 360 min in the absence of DHA. Islets exposed to the high concentration of DHA released more (~~0.05) IRI than did controls or islets exposed to the low concentration of DHA. If SE is smaller than the size of the symbol it will not appear on the figure.
64
PENCE AND MENNEAR
tion period. At the conclusion of the experiment, these islets had released nearly 70 pUIRI/islet despite the low concentration of glucose in the incubation medium. Fung and Mennear (1974) reported that exposure of mouse islets to a high concentration of alloxan resulted in an increase in the release of IRI. In the case of alloxan, the release required 4.5 hr of incubation to be produced. These workers suggested that this in vitro effect corresponded to the second phase of the in vivo reaction to alloxan administration, i.e., hyperinsulinemia and hypoglycemia. In our experiments, we have never observed hypoglycemia when DHA was administered to intact mice. It is possible that our inability to produce diabetic mice through the use of DHA is a reflection of our inability to administer doses sufficient to produce pancreatic damage. For this reason, a final experiment was conducted in which mice were treated with intravenous doses of 100, 200, and 300 mg/kg DHA 60 min prior to sacrifice and isolation of pancreatic islets. These islets were then incubated for 90 min in a nonstimulating concentration of glucose.
-
FIG. 6. Glucose stimulated insutm release from pancreatic islets isolated from dehydroascorbic acid-treated mice. Mice were treated with intravenous DHA 60 min prior to sacrifice and islet isolation. Groups of islets were incubated at 37°C in the presence of 300 mgjdl D-glucose. Islets from mice treated with either 200 or 300 mg/kg DHA released significantly more IRI (pcO.05) than did controls. If SE is smaller than the size of the symbol it will not appear on the figure.
The results of this experiment are shown in Fig. 5. Islets isolated from mice which had received either 100 or 200 mg/kg DHA released the same, minimal, amount of IRI in the presence of 60 mg/dl glucose as did islets isolated from control mice. Islets from mice which had been treated with the 300 mg/kg dose of DHA, however, released significantly more IRI at each of the time intervals than any other group. When islets from DHA-pretreated mice were exposed to the 300 mg/dl concentration of glucose, both the 200 and the 300 mg/kg groups released more IRI than did controls (Fig. 6).
I 0
30 MINUTES
OF
60 INCUBATION
90
FIG. 5. Nonstimulated insulin release from pancreatic islets isolated from dehydroascorbic acidtreated mice. Mice were treated with intravenous DHA 60 min prior to sacrifice and islet isolation. Groups of islets were incubated at 37°C in the presence of 60 mg/dl n-glucose. Islets from mice treated with 300 mg/kg DHA released significantly (p < 0.05) more IRI than controls. If SE is smaller than the size of the symbol it will not appear on the figure.
DISCUSSION The results of these experiments have demonstrated several similarities between the in vitro actions of DHA and alloxan. Earlier workers (Tomita et al., 1974) have reported that alloxan inhibits the responsiveness of isolated pancreatic islets to glucose and tolbutamide. Like alloxan, lower concentrations of DHA also inhibit islet responsiveness
DEHYDROASCORBIC
to both
secretogogues. Further, both the in vivo and in vitro pancreotoxic effects of alloxan and DHA can be antagonized by glucose (Sutherland and Rall, 1958; EspositoAvella and Mennear, 1973; Tomita et al., 1974). This observation may indicate that the pancreotoxic action of DHA is mediated through an action at the level of the glucose receptor. This observed inhibitory effect of DHA on glucose responsiveness of isolated islets is consistent with in U~ZJOhyperglycemia and decreased glucose tolerance. Inhibition of pancreatic responsiveness is not, however, consistent with the earlier report of Pillsbury et al. (1973) of DHA-induced leakage of insulin from isolated toadfish pancreatic islets. It is possible that the 2.0 mg/dl concentration of DHA is not sufficient to produce leakage from mouse islets or that the 90-min incubation period is not long enough for the effect to manifest itself. The exploration of this hypothesis demonstrated that higher concentrations of DHA like alloxan resulted in the increased insulin release, even in the presence of nonstimulating concentrations of glucose. This action of DHA could be a prelude to the induction of the diabetic state. This possibility is difficult to assess completely in the present experiments, however, because the administration of 300 mg/kg DHA to mice prior to the islet isolation resulted in IRI release in the presence of low glucose but is not a diabetogenic treatment in our hands. It is possible that this effect is an artifact produced by the combination of a pancreatic effect of DHA and the collagenase treatment during islet isolation. Finally, it is noteworthy to point out that the concentration of DHA employed in the experiments in vitro, 2.0 mg/dl, is within the range of serum concentrations of the vitamin reported for diabetic sera (Chatterjee et al. 1975). Since DHA does decrease pancreatic insulin secretory activity in mouse islets, its potential role in the development of diabetes mellitus should be studied further. Also, additional experiments on the serum concen-
ACID
AND
65
INSULIN
trations of DHA in diabetic and non diabetic humans should be conducted. Chatterjee’s experiments were conducted in India and it is possible that ethnic or dietary peculiarities accounted for the findings reported.
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CHATTERJEE, AND
1. B.,
MAJUMDER,
SUBRAMANIAN,
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Sci. 258, 24-47.
S. J., WATKINS, D., and LAZAROW, A. (1969). The effect of alloxan on islet tissue permeability. In The Stractrtre and Metabolism of the Pancreatic Islets (S. E. Broline, B. Hellman, and H. Knutson, eds.), pp. 398. Pergamon, Oxford. ESPOSITO-AVELLA, M., AND MENNEAR, J. H. (1973). Studies on the protective effect of diphenylhydantoin against alloxan diabetes in mice. Proc. Sot. COOPERSTEIN,
Exp.
Biol.
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142, 82-85.
W., AND MENNEAR, J. H. (1974). Effects of alloxan on insulin release by isolated mouse pancreatic islets. Proc. Sot. Exp. Biol. Med. 146,
FUNG,
949-952.
P. E., AND KOSTIANOVSKY, M. (1967). Method for the isolation of intact islets of Langerhans from the rat pancreas. Diabetes 16, 35-39. MANN, G. V. (1974). Hypothesis: The role of vitamin C in diabetic angiopathy. Perspect. Biol. Med.
LACY,
17,210-212.
G. V., AND NEWTON, P. (1975). The membrane transport of ascorbic acid. Ann. IV. Y. Acad. Sri.
MANN,
258, 243-25
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J. W. dehydroascorbic PATTERSON, J. W. dehydroascorbic PATTERSON,
J. Biol. .PILLSBURY,
(1949). The diabetogenic effects of acid. Endocrinology 45, 344. (1950). The diabetogenic effect of acid and dehydroisoascorbic acid.
Chem. 183, 81-88. S., WATKINS, D.,
AND
COPPERSTEIN,
S. J.
(1973). Effect of dehydroascorbic acid on permeability of pancreatic islet tissue in vitro. J. Pharmacol. Exp. Ther. 185, 713-718. SELTZER, H. S., AND HARRIS, V. L. (1964). Exhaustion of insulinogenic reserve in maturity onset diabetic patients during prolonged and continuous hyperglycemic stress. Diabetes 13, 6-l 3. SUTHERLAND, E. W., AND RALL, T. W. (1958). Fractionation and characterization of a cyclic adenine ribonucleotide formed by tissue particles. J. Biol. Chem. 232, 1065-1076. TOMITA, T., LACY, P. E., MATSCHINSKY, F. M., AND MCDANIEL, M. L. (1974). The effect of alloxan on insulin secretion from isolated rat islets perifused in vitro. Diabetes 23, 517-524.