TOXICOLOGY

The

AND

APPLIED

Inhibitory

PHARMACOLOGY

31,

134-142 (1975)

Effect of Cadmium the Isolated Perfused TAGHI GHAFGHAZI’

Department

of Pharmacology

March

Activity

of

AND JOHN H. MENNEAR

and Toxicology,

Received

on the Secretory Rat Pancreas’

27,1974;

Purdue accepted

University,

Lafayette,

Indiana

47907

June 17,1974

The Inhibitory Effect of Cadmium on the Secretory Activity of the Isolated Perfused Rat Pancreas. GHAFGHAZI, T. AND MENNEAR, J. H. (1975). Toxicol. Appl. Pharmacol.31, 134-142. Perfusion of the isolated

rat pancreaswith cadmium (Cd) (1 x 10m3and 5 x 10m4M) inhibits the insulin secretory responseto glucose(300 mg/lOO ml), tolbutamide (40 mg/lOO ml), and potassiumions (30 mEq/liter). Cadmium inhibition of pancreatic secretory activity is immediate in onset, and not reversed by simple washout of the organ with perfusion medium. Perfusion of the

inhibited organ with a combination of glucoseand theophylline results in partial reversalof the inhibition. It is suspectedthat Cd-inducedinhibition of insulinsecretionmay bemediatedthrough interferencewith calcium uptake by the pancreaticbetacell. Cadmium (Cd) has been shown to influence carbohydrate metabolism in a number of species.Voinar (1952) reported that the iv administration of Cd to rabbits resultsin the development of hyperglycemia. Similarly, Caujolle et al. (1964) have reported hyperglycemia in dogs and Ghafghazi and Mennear (1973) reported that a single ip dose of Cd produces elevations in blood glucose concentrations in mice. The hyperglycemic responseof mice to a single injection of Cd is mediated through the adrenal gland sinceadrenalectomy abolished the response(Ghafghazi and Mennear, 1973). The effect of the metal is probably mediated through the adrenal medulla since it has beendemonstrated that Cd is a potent releaserof catecholaminesfrom the bovine adrenal (J. L. Borowitz and D. T. Hart, personal communication). In addition to causing acute hyperglycemia, Cd administration reduces glucose tolerance. This effect on carbohydrate metabolism is not mediated through the adrenal gland since it is not attenuated by adrenalectomy. The observation of decreased concentrations of circulating insulin in Cd-treated mice provides indirect evidence to suggestthat the metal may impair glucose tolerance by reducing the insulin secretory activity of the pancreas (Ghafghazi and Mennear, 1973). Earlier workers presentedevidence to support the possibility of Cd-induced decreased pancreatic secretory activity. Barbieri et al. (1961) showed that Cd accumulates in the pancreas of rabbits and that this accumulation is accompanied by a decreasein the ratio of beta to alpha cells. Also, Havu (1969) described Cd-induced necrotic lesions of pancreatic beta cells in the bony fish Cottus scorpius. Finally, decreasedpancreatic 1 Supported by grants from NIH (AM-14134 and ES 00921) and the Purdue Research Foundation. z Present address: University of Isfahan, Isfahan, Iran. 134 Copyright 0 1975 by Academic Press, Inc. All rights ofreproduction Printed in Great Britain

in any form

reserved.

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function has been reported to be a feature of Itai-Itai disease, the result of chronic Cd intoxication in humans (Murata et al., 1970). The objective of the research reported in this article was to assess the effects of Cd on pancreatic secretory activity. By using the isolated perfused rat pancreas we have demonstrated a direct inhibitory effect of the metal on the ability of the pancreas to secrete insulin. The inhibitory effect was relatively nonspecific since it decreased pancreatic responsiveness to tolbutamide and potassium ions as well as to glucose.

METHODS

Male albino Sprague-Dawley derived rats (300-350 g) were employed for these experiments. The animals were obtained from the Laboratory Supply Company, Indianapolis, and were acclimated to laboratory conditions for at least 1 week prior to experimentation. While in our laboratories the animals were housed in groups of five with free accessto food and water. The isolated perfused rat pancreas preparation described by Grodsky et al. (1963) was employed. Rats were anesthetized with pentobarbital (43 mg/kg, ip). The pancreas, stomach, spleen, and a portion of the duodenum were isolated and placed in a perfusion chamber containing the perfusion medium (Krebs-Ringer bicarbonate buffer supplemented with 1.5 % bovine serum albumin). Perfusion of the preparation was from the abdominal aorta through the portal vein at a flow rate of from 3 to 4 ml/min with a pulse rate of 30/min. Flow was maintained with a multichannel piston-type metering pump (Harvard Instruments, Model 1504). The temperature of the perfusion medium was maintained at 37°C. The perfusion medium was aerated with 95 % oxygen, 5 % carbon dioxide for 20 min prior to the initiation of perfusion, This aeration resulted in a perfusion medium pH of from 7.0 to 7.4. Aeration of the medium was resumed after the initial 15 min of perfusion and continued throughout the remainder of the experiment. Two concentrations of glucosewere usedin the perfusion medium. The low concentration, 60 mg/lOO ml, was employed during an initial IO-min perfusion period used to achieve stabilization of temperature and flow rates. This concentration of glucose does not induce insulin secretion by the perfused organ. The high concentration, 300 mg/lOO ml, was employed to stimulate the pancreata to secrete insulin. Drugs and chemicals under investigation were added to the perfusion medium just prior to use. Concentrations employed are presentedin the Results section. After the stabilization perfusion period, the perfusate was collected at 1-min intervals for determination of immunoreactive insulin (IRI). Insulin was determined as IRI by the immunoassay procedure of Hales and Randle (1963) as available commercially (Amersham/Searle Co., Chicago). This assay is essentially an isotope dilution technique in which IRI in the sample and a known amount of 1Z51-insulinare allowed to react with anti-insulin serum to form an insulin-antibody complex. The complex is recovered by filtration and its radioactivity determined by liquid scintillation spectrophotometry. The amount of 1251-insulinrecovered is inversely proportional to the concentration of IRI in the sample. A preliminary experiment was conducted to determine the influence of Cd on the results of the IRI assay.When Cd (asthe acetate salt) wasadded to the assaymixture at a

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concentration of 1 x lop4 M, a definite interference was noted. At this concentration the metal reduces the amount of 1251-insulin recovered and results in an erroneously high estimate of the amount of IRI in the sample. Lower concentrations of Cd (1 x 10m5 and 1 x 1O-6 M) did not interfere with the assay. Because of the required dilution of the perfusate prior to assay, the final concentration of Cd in the assay mixture was never greater than 1 x lo-’ M. Therefore, Cd interference with the IRI assay was not expected. Concentrations of IRI were expressed as /tU IRl/ml of perfusate. Treatment groups were compared for significance of differences in insulin release by factorial analysis of variance. RESULTS

The results shown in Fig. 1 summarize the effects of Cd perfusion on the ability of the isolated perfused rat pancreas to secreteinsulin in responseto glucose stimulation. In this experiment the pancreata were perfused with either the high concentration of glucose (300 mg/lOO ml) alone, or glucose combined with Cd (1 x 10e4-1 x 10e3M). As reported by earlier workers (Grodsky et al., 1963) perfusion of the pancreata with glucose alone results in a marked and prompt release of insulin. The secretory responseto glucose is characteristically biphasic with the initial peak appearing within Glucosd300ma/lOOml) = f

I 0 Cd(lx10-3M) . Cd(5x10-4hlj

IO

20 MINUTES

30

40

50

6l

OF PERFUSION

FIG. 1. The effect of cadmium perfusion on glucose-stimulated insulin release by the isolated perfused rat pancreas. Each point represents the mean IRI released at indicated intervals by four pancreata. All concentrations of cadmium reduced IRI release (p < 0.05).

3 min. After the initial peak, insulin secretion decreasesslightly, in spite of the fact that glucosestimulation is continued. The secondphaseof insulin secretion is characterized by a gradual increasewhich persistsfor the remainder of the experiment. Concentrations of either 1 x 10e3or 5 x lo-” M Cd in the perfusion medium resulted in a complete inhibition of glucose-inducedinsulin secretion; 1 x lo-” Cd significantly attenuated (p < 0.05) insulin secretion. While glucose is recognized as being the major physiological stimulus to insulin secretion, several other agentshave been shown to induce the releaseof the hormone. The next experiments were conducted to determine the effect of Cd on pancreatic responsivenessto tolbutamide and potassium ions.

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In these experiments the perfusion medium contained the low concentration of glucose (60 mg/lOO ml), tolbutamide (40 mg/lOO ml) or potassium (30 mEq/liter, added as KCl), and Cd (1 x 10m3 or 5 x 10m4 M). The results of the experiments are shown in Figs. 2 and 3.

Tolbutamide

(40mg/lCOml)

f

1

200 l

Tolbutamide

~Cd(lxlO-~M) 0 Cd(5x10-4#)

I

2

3

4

5

6

7

8 9 IO II I2 13 14 I5 16 17 IS 19 20 MINUTES OF PERFUSION

FIG. 2. The effect of cadmium perfusion on tolbutamide-stimulated insulin release by the isolated perfused rat pancreas. Each point represents the mean IRI release at indicated intervals by three pancreata. Both concentrations of cadmium reduced IRI release (p < 0.01).

I \E 3 3 z300-

K(30m I

Era/l.) I

l K 0 KtCd(5x10-4M)

4

MINUTES

OF PERFUSION

FIG. 3. The effect of cadmium perfusion on potassium-stimulated insulin release by the isolated perfused rat pancreas. Each point represents the mean IRI release at indicated intervals from three pancreata. Cadmium reduced IRI release (p ( 0.001).

It can be seenfrom the data in Fig. 2 that perfusion of the pancreata with tolbutamide alone resulted in a definite secretory response.Unlike the responseto glucose,however, tolbutamide induces a monophasic secretion of insulin. In the presence of Cd, tolbutamide failed to stimulate insulin secretion. Both concentrations of the metal produced a complete blockade of pancreatic responsiveness. The data in Fig. 3 shows that potassium ions elicit a monophasic releaseof insulin. The addition of Cd to the perfusion medium did not result in a total blockade of responsivenessto potassium; however, secretion of insulin was significantly reduced.

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GHAFGHAZI AND MENNEAR

During the entire 20-min perfusion period, control pancreata secretednearly threefold more insulin than did the Cd-exposed organs (mean values of 648 ? 21 ng vs 237 of:30 ng; p < 0.001).3 In the preceding three experiments Cd was present in the perfusion medium throughout the entire experimental period. The next experiment was conducted to determine if Cd-induced inhibition of insulin secretion could be reversed by perfusion of the organ with Cd-free buffer. Pancreata were perfused initially with medium containing the high concentration of glucose and Cd (1 x 10e3M). After 15 min of perfusion with this medium, the organs were exposed to Cd-free medium containing the low concentration of glucose. After this second 15-min period the pancreata were challengedfor 30 min with Cd-free buffer

5

IO

15

20

25 MINUTES

30 35 40 OF PERFUSION

45

50

55

60

FIG. 4. Lack of reversalof cadmium-induced pancreatic inhibition by perfusion of the inhibited organ with buffer. Each point represents the mean IRI release, at indicated intervals, from three pancreata.

containing the high concentration of glucose. The results of this experiment are shown in Fig. 4. The organs which had been perfused for the initial 30-min period with iow glucose medium without Cd respondedpromptly and characteristically to the glucosechallenge. Pancreata which had been exposed to Cd during the initial 15-min period, however, were totally unresponsive to the glucosechallenge. Thus, once established,Cd inhibition of pancreatic secretion is not readily reversible by simple perfusion with Cd-free medium for as long as 45 min. The final experiments were conducted to determine the effect of theophylline on Cd-induced inhibition of insulin secretion. The rationale for these experiments was based upon the work of Brisson et al. (1972). These workers have shown that glucose fails to stimulate insulin secretion from isolated pancreatic islets when the islets are incubated in a calcium-free medium. The addition of theophylline to the calcium-free medium, however, restores the responsivenessof the islets to glucose. Furthermore, 3 The amount of total insulin release was converted to nanograms (25 PU of IRI = 1 ng).

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1

MINUTES

OF PERFUSION

FIG. 5. Effect of theophylline on cadmium-induced pancreatic inhibition. Three pancreata were used in each treatment group. Cadmium, theophylline plus glucose released significantly more IRI during the initial 20 min of the experiment than did cadmium plus glucose (p < 0.01). These two groups were not different during the final 40 min of the experiment.

the release of insulin in response to the glucose-theophylline combination was accompanied by a release of calcium. This observation may indicate that the action of theophylline is mediated through a redistribution of intracellular calcium, shifting the ion from a storage pool into an active pool. The first approach was to perfuse pancreata with a combination of glucose (300 mg/lOO ml), Cd (5 x 10S4 M), and theophylline (5 x 10m3 M) throughout the entire perfusion period. The results of this experiment are shown in Fig. 5.

FIG. 6. Effect of theophylline on established cadmium-induced pancreatic inhibition. Three pancreata were used in each treatment group. Theophylline plus glucose released more IRI from the cadmium exposed pancreata than did glucose alone (p K 0.01).

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As noted in earlier experiments, perfusion of the pancreata with Cd and glucose resulted in a total blockade of the insulin secretion in response to glucose. Pancreata that were perfused with glucose and theophylline (in the absence of Cd) responded with a remarkable release of insulin. This response to the combination was far in excess of any responses observed when pancreata were perfused with glucose alone. Interestingly, pancreata that were simultaneously exposed to glucose, Cd, and theophylline exhibited a definite secretory response. However, this response was relatively short lived, lasting for less than 30 min. After 30 min all pancreata of this treatment group were totally unresponsive. The second approach employed was to establish Cd blockade and then challenge it with a combination of glucose and theophylline. The results of this experiment are shown in Fig. 6. As in the earlier experiment (Fig. 4) the pancreata that were exposed to Cd (initial 12 min of perfusion) and then challenged with glucose were completely unresponsive to the stimulus. Inhibited pancreata which were challenged with the glucose and theophylline combination, however, exhibited a definite release of insulin. Insulin secretion occurred promptly after the initiation of perfusion with the combination and persisted throughout the remainder of the experiment. DISCUSSION Singhal et al. (1974) have studied the effects of chronic Cd administration on several parameters of carbohydrate metabolism in rats. These workers have found that after 45 days of repeated Cd administration rats exhibited persistent hyperglycemia as well as increased activities of the four-key, rate-limiting gluconeogenic enzymes (pyruvate carboxylase, phosphopyruvate carboxylase, hexosediphosphatase, and glucose-6phosphatase) in both liver and kidney. Although these workers did not measure serum insulin, their results offer indirect evidence of decreased pancreatic function since the activities of these enzymes are increased by the absence of insulin. Earlier studies conducted in our laboratories (Ithakissios et al., 1974) offer direct evidence that chronic Cd administration inhibits pancreatic secretory activity. In these experiments, rats were administered Cd on alternate days for a total of 70 doses. The glucose responsiveness of the isolated perfused pancreata from the treated rats was markedly reduced when compared to that of age-matched control rats. The results of the present in vitro experiments show that Cd can directly inhibit beta cell responsiveness to glucose, tolbutamide, and potassium ions. The ability of Cd to inhibit insulin secretion from the isolated perfused pancreas preparation is immediate in onset and, once established, is not reversed by up to 45 min of perfusion with Cd-free medium. The effect of Cd can, however, be partially antagonized or reversed by theophylline. This effect of theophylline (albeit only partial) suggests a possible mechanism for the inhibitory effect of Cd. Glucose-induced release of insulin from beta cells is believed to be the end result of a series of cellular events. Glucose (or one of its metabolites) is thought to act as a signal for both the uptake of extracellular calcium and the inhibition of calcium efflux from beta cells. When the concentration of calcium in the cytosol reaches a critical level, an interaction between the ion and the microtubular-microfilamentous

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system leads to emiocytosis of the insulin secretory granules (Malaisse-Lagae et al., 1971; Brisson et al., 1972). Pancreatic islets are not responsive to glucose in the absence of calcium. When theophylline is added to the calcium-free medium, however, responsiveness to glucose is restored. It has been suggested that theophylline acts in this preparation by inducing an intracellular translocation of calcium, shifting it from the vacuolar system to the cytosol (Brisson et al., 1972). If Cd prevents the uptake of calcium by the beta cells, the end result would be insensitivity to glucose. Further, theophylline, by virtue of its suspected ability to induce an intracellular translocation of calcium, should restore the responsiveness of the beta cells to glucose. This was the case in our experiments. The short-lived nature of the effect of theophylline in this experiment was not unexpected. If calcium uptake is blocked by Cd, the intracellular concentration of the ion prior to the application of the theophylline would be the limiting factor in the duration of the effect of theophylline since calcium ej?ux is known to accompany insulin secretion (Brisson et al., 1972). Thus, the ability of theophylline to partially reverse the inhibitory effect of Cd suggests that Cd-induced inhibition of glucose-stimulated insulin secretion may be mediated, at least in part, through an inhibition of calcium uptake by beta cells. Such an interaction between Cd and calcium has been suggested earlier. Toda (1973), working with aortic strips, has presented evidence to suggest that Cd reduces the permeability of this smooth muscle preparation to calcium. We are currently conducting experiments to assess this possible mechanism of inhibitory action. Finally, it should be pointed out that, as employed in the present experiments, the isolated perfused rat pancreas preparation is not particularly sensitive to Cd inhibition. It is likely that the 1 x 1O-4 M concentration of Cd, a concentration which produced only minimal inhibition (Fig. l), is far in excess of concentrations achieved in the chronic in uioo experiments. We believe that the inability of lower concentrations of Cd to inhibit pancreatic secretory activity in these experiments is a reflection of the fact that the beta cells were exposed to the metal for short periods of time (up to 60 min). We were unable to employ longer perfusion periods during these experiments because of the development of pancreatic edema which was generally noticeable after approximately 90 min of perfusion. Becauseof this difficulty, our future experiments on the pancreatic effects of Cd will employ either isolated or perifused4pancreatic islets. REFERENCES BARBIERI, L., COLOMBI, R. AND STRANEO, G. (1961). Modificazioni istologiche delle isole pancreatiche de1 coniglio dopo intossicazione cronica da cadmio. Folia Med. (Naples)44, 1120-l 133. BRISSON, G. R., MALAISSE-LAGAE, F. AND MALAISSE, W. J. (1972). The stimulus-secretion coupling of glucose-induced insulin release. VII. A proposed site of action for adenosine-3’,5’-cyclic monophosphate. J. C/in. Invest. 51, 232-241. CAUJOLLE, F., CHANH, P. H., MAMY, G. AND PATLE, L. T. (1964). Comparative action of zinc and cadmium. Agressologie 3, 263-268. J The acinar tissue is digested away and the medium flows around the surface of the remaining tissue.

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T. AND MENNEAR, J. H. (1973). Effects of acute and subacute cadmium administration on carbohydrate metabolism in mice. Toxicol. Appl. Pharmacol. 26, 231-240. GRODSKY, G. M., BATTS, A. A., BENNETT, L. L., VCELLA, C., MCWILLIAMS, N. B. AND SMITH, D. F. (1973).Effects of carbohydrateson secretionof insulin from isolatedrat pancreas. GHAFGHAZI,

Amer. J. Physiol. 205, 638-644. C. D. AND RANDLE, P. J. (1963). Immuno-assayof insulin with insulin antibody precipitate. Biochem. J. 88, 137-146. HAVU, N. (1969). Sulfhydryl inhibitors and pancreatic islet tissue.Acta Endocrinol. Sappl. 139,1-231. ITHAKISSIOS, D. S., GHAFGHAZT, T., MENNEAR, J. H. AND KESSLER, W. V. (1974).Effect of multiple dosesof cadmiumon glucosemetabolismand insulinsecretionin the rat. Toxicol. Appl. PharmacoL 31, 143-149. MALAISSE-LAGAE, F., GREDER, M. H., MALAISSE, W. J. AND LACY, P. E. (1971).The stimulus coupling of glucose-inducedinsulin release.IV. The effect of vincristine and deuterium oxide on the microtubular systemof the pancreaticbeta cell. J. Ceil. Biof. 49, 530-535. MURATA, I., HIRONO, T., SAEKI, Y. AND NAKAGA, WA. S. (1970). Cadmium enteropathy, renal osteomalacia(“Itai-Itai” diseasein Japan).Ball. Sot. Znt. Chin. 1, 3442. SINGHAL, R. L., MERALI, Z., KACEW, S. AND SUTHERLAND, D. J. B. (1974). Persistence of cadmium-inducedmetabolicchangesin liver and kidney. Science 183,10941096. TODA, N. (1973).Influence of cadmiumon contractile response of isolatedaortasto stimulatory agents.Amer. J. Physiol. 225, 350-355. VOINAR, A. C. (1952). The existenceand role of cadmium in the organismof animalsand man. In Vsesioaznaia Konferentsiia Pomikroelementam 1st Moscow, 1950, pp. 580-583. Mikroelementy v. Zhizni Rasteniii Zhivotnykh, Moscow. HALES,