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International Journal of Pancreatology, 5 (1989) 229-238 $02.00 Copyright �9 1989 by The Humana Press Inc. All rights of any nature whatsoever reserved.

Effect of Alloxan Diabetes on Exocrine Pancreatic Secretion in the Anesthetized Rabbit

C a r m e n A l v a r e z * a n d M a r i a A . L @ e z

Departamento de Fisiologia y Farmacologia, Facultad de Biologia, Universidad de Salamanca, Pza~ ?r S/N, 37008 Salamanca, Spain

Received November 14, 1988; Revised February 16, 1989; Accepted February 20, 1989

Summary

The influence of the endocrine pancreas on the exocrine pancreatic secretion of both electrolytes and enzymes was studied in rabbits made diabetic by alloxan administration. No alterations were observed in the flow of pancreatic juice. Bicarbonate concentrations were considerably increased, whereas chloride concentrations were clearly reduced in the alloxan-diabetic rabbits compared with the control animals. Insulin treatment restored anion levels to normal. Similar, although less pronounced changes were seen in the output values of bicarbonate and chloride. There were no significant differences between the control and alloxan-treated animals in the output of sodium and potassium; however, potassium concentrations exhibited a significant rise both in untreated and insulin-treated diabetic rabbits compared with the controls. Total protein and amylase secretion decreased markedly in the diabetic animals. The secretion of amylase was not brought back to control values by additional administration of insulin. These results suggest that the endocrine pancreas plays an extremely im- portant role in exocrine pancreatic function.

Key Words: Insulin; pancreatic juice; electrolytes; amylase.

I N T R O D U C T I O N

T w o secre to ry c o m p o n e n t s can be d i f f e ren t i a t ed in the pancreas: an endocrine par t and an exocr ine one. M o r p h o l o g i c a l studies have revealed the close con tac t be tween endocr ine and exocr ine cells (1,2). Addi t iona l ly , vascular connec t ions in the f o r m o f an insu lo-ac inar por ta l system have been d e m o n - s t ra ted be tween the islets and the ac inar tissue (3-5) . These m o r p h o l o g i c a l

*Author to whom all correspondence and reprint requests should be addressed.

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230 Alvarez and L@ez

arrays suggest a possible direct influence of islet hormones on the function of the exocrine pancreas (6). The experimental basis for the existence of an islet- acinar axis is strongest for insulin (7).

In humans, diabetes mellitus may be associated with abnormal exocrine pancreatic responses to secretin and cholecystokinin (8-10). Pathologically, in recent-onset Type 1 diabetes the pancreas exhibits acinar cell atrophy sur- rounding insulin-deficient islets (11). The effects of diabetes mellitus have been studied in experimental animals using the B-cell toxins, alloxan, or streptozotocin. The acinar cells located close to the islets (peri-insular regions) of the rat pancreas were seen to contain more zymogen granules than distant cells (tele-insular regions) (12,13). This difference was abolished after injection of alloxan and this abolition was not a direct effect of alloxan on the exocrine pancreas (12). On the other hand, in streptozotocin-induced diabetic rats acinar cells located in the peri-insular regions of the pancreas appear totally devoid of amylase immunolabeling while positive and negative cells are found in the tele-insular regions (14). The presence of amylase in pancreatic tissue (15-23) as well as the basal and stimulated amylase exocrine secretion (19,20,22-24) are severely reduced in diabetic rats. Likewise, in sheep, the volume of pancreatic juice and its protein content decrease markedly during alloxan-induced diabetes (25). Both in the rat and in the sheep, the effects of diabetes can be reversed by in vivo insulin administration (15- 21,25). However, Trimble et al. (26) and later Okabayashi et al. (23) found that protein and enzyme outputs in response to cerulein are considerably reduced in insulin-treated diabetic rat pancreas despite restoration of the pancreatic enzyme contents to control levels. The literature contains no references to the influence of experimentally-induced diabetes on the concentration of the major electrolytes (bicarbonate, chloride, sodium, and potassium) in pancreatic juice.

The aim of the present work was to study the function of the exocrine pancreas in resting conditions in rabbits with alloxan-induced diabetes. We also investigated the effect of insulin treatment both on the content of electrolytes in pancreatic juice and enzyme secretion.

MATERIAL AND METHODS

Animals

Male New Zealand rabbits (1.5-2.5 kg b wt) were used in all experiments. The animals had free access to standard rabbit chow~ They were divided into three groups: A) Group I (untreated rabbits) served as nondiabetic controls. B) Group II rabbits comprised animals in which diabetes was induced by intravenous injection of 75 mg/kg body weight of alloxan monohydrate (Sigma Chemical Co., St. Louis, MO). The diabetic state was confirmed by the positive glucose reaction in urine. Urine samples were collected and analyzed periodically for the presence of glucose using reagent strips (Keto-Diabur Test 5000, Boehringer GmbH, Mannheim, FRG). The rabbits exhibited glycosuria 3-6 d after alloxan injection. C) Group III, these were alloxan-diabetic

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Effect of A lloxan Diabetes on Exocrine Pancreatic Secretion 231

rabbits treated with a subcutaneous injection of 1-5 U of insulin (suspension of porcine protamine insulin, Nordisk, Gentofte, Denmark) given once daily. Treatment with insulin was begun when diabetes had been confirmed and was maintained until the day before the experiments when the rabbits were used to study pancreatic exocrine secretion. The insulin dose given to each animal was adjusted to the amount of urinary glucose excretion and judged to be suitable for avoiding glycosuria.

To examine the chronic effect of diabetes on pancreatic exocrine function, the rabbits were prepared surgically for collection of pancreatic juice 29 d after alloxan administration~ At the time of the experiments, blood samples were taken for glucose measurements from the ear vein before the rabbits were anesthetized.

Collection of Pancreatic Secretion

After a 14-15 h fasting period, but with free access to water, normal and diabetic rabbits were anesthetized by intravenous injection of t .0 g /kg b wt of urethane (Sigma Chemical Co., St. Louis, MO). The animals were then routinely tracheostomized. After performing a median laparotomy, the main pancreatic duct was exposed and cannulated near its entrance to the duod- enum following ligation of the pylorus and cannulation of the bile duct for deviation of bile to the exterior. Samples of pancreatic juice were taken after an equilibration period of 30 min. Pancreatic juice was collected over ice in preweighed plastic tubes in 40-rain batches. Throughout the experiments, the rectal temperature of the animals was kept at 38 _+ 1 ~ by a heating pad.

Assays

The volume of each sample of pancreatic juice was determined by weighing on a fully automatic electronic balance (Type H35, Mettler, Ztirich, Switzer- land) assuming the density of the juice to be 1.0. Bicarbonate concentrations were determined by measuring the total COz content with a Natelson micro- gasometer (Model 600, Scientific Industries, Inc., Bohemia, NY). Chloride concentrations were evaluated by a chloridometer (Model 106, Analytical Control, S.P.A. , Turin, Italy). Sodium and potassium were estimated with an atomic absorption/emission spectrophotometer (Model A-1800, Hitachi, Ltd., Tokyo, Japan). Total protein concentrations were measured by the method of Bradford (27). Amylase activity was determined according to the method of Noelting and Bernfeld (28) as modified by Hickson (29).

Blood glucose was measured with a commercially available kit (BM-Test- Glycemie 20-800R) using a reflectance photometer (Reflolux)o The glycemie test kit and photometer were supplied by Boehringer GmbH, Mannheim, FRG.

Statistical Analysis

Results are expressed as means +_ SEM. One-way analysis of variance followed by the Scheff~ test (30) were applied for comparisons between groups

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of animals. Differences with p values of less than 0.05 were considered statistically significant.

RESULTS

Rabbits given an alloxan injection characteristically developed polydipsia, polyuria, and gtycosuria, and during the first week showed a slight weight loss. The body weights of the animals on the day of alloxan injection were 1.95 _+0.04 kg in the controls (Group I; n = 20), 1.81 +0.09 kg in the diabetic animals (Group II; n = 7), and 2.00-+ 0.06 kg in the insulin-treated diabetic group (Group III; n = 14), and 29 d after the injection they were 2.35 +0.06 kg, 2.01 _+ 0.08 kg, and 2.32 +0.08 kg, respectively. Blood glucose concentrations in the insulin-treated diabetic rabbits at the time of the experiments (6.4-+0.4 mmol /L , n = 8) were similar to those found in the control group (6.1 _4_- 0.4 mmol /L , n = 6). The animals in Group II (alloxan-diabetic rabbits) showed glycosuria, such that it was not considered necessary to check state of hyperglycemia.

No differences were observed in the flow of pancreatic juice between the controls (5.58 _+ 0.53/~L/min, n = 20), treated with alloxan alone (4.95 _+ 0.59 tzL/min, n = 7) and the animals treated with alloxan plus insulin (5.28 +_ 0.56 /zL/min, n = 14). Bicarbonate concentrations were significantly higher in the untreated diabetic rabbits compared to both the controls (p<0 .01 ) and the insulin-treated diabetic animals (p < 0.05) (Fig. 1). The chloride concentrations in the rabbits treated with alloxan alone (Group II) were considerably lower than those found in the controls ( p < 0 . 0 t ) and additional treatment with insulin (Group III) led to chloride concentrations that were not significantly different to those in the control group (Fig~ 1). The changes in the output of pancreatic anions were only statistically significant in the case of chloride secretion of the untreated diabetic group as compared with control values ( p < 0.05) (Fig. 1). Whereas sodium concentrations hardly showed any alterations, the concentrations of potassium exhibited clear rises in both alloxan- treated groups (Groups II and III) compared with the controls (p<0 .01) (Table 1). The outputs of both sodium and potassium were unaffected by alloxan treatment (Group II); neither were they affected by additional administration of insulin (Group III) (Table 1).

Protein concentrations were seen to decrease after alloxan treatment although the changes in this parameter were not significant (Fig. 2). The reduc- tion was more pronounced in output values; thus, the protein output of the untreated diabetic rabbits was significantly lower than that of the control group (p < 0.05) (Fig. 2). Amylase secretion was strongly reduced by alloxan administration (p < 0.01) and was not restored to control values by additional administration of insulin (p<0 .01) (Fig. 2). Furthermore, amylase activity was considerably decreased in both untreated diabetic (p < 0.05) and alloxan plus insulin-treated ( p < 0.01) groups (Fig. 2).

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Effect o f A lloxan Diabetes on Exocrine Pancreatic Secretion 233

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Fig. 1. Bicarbonate and chloride concentrations (top panel) and outputs (bottom panel) in the pancreatic juice in control ([]) , untreated diabetic (E~), and insulin- treated diabetic ([]) rabbits. Bars represent means and SEM. Number of animals in parentheses. Asterisks indicate significant differences between control and untreated diabetic rabbits *p<0.05, **p<0.01, and between untreated diabetic and insulin- treated diabetic rabbits ~ p < 0.05.

Table 1 Sodium and Potassium Concentrations and Outputs in the Pancreatic Juice

in Control, Untreated Diabetic, and Insulin-Treated Diabetic Rabbits a

Control Untreated diabetic Treated diabetic

Sodium concentration, mmol /L 157.6+ 4.8 (17) 162.6_+ 2.5 (7) 158.1 _+ 3,3 (14) output, nmol /min 989.7 + 98.3 (17) 816.3 + 111.5 (7) 817.0_+ 99.1 (14)

Potassium concentration, mmol /L 3.1 +_ 0.2 (16) 4.0_+ 0.2** (6) 4 .0+ 0.1"* (14) output, nmol /min 20.2 + t.7 (16) 21.8 +_ 3.3 (6) 20.6-+ 2.4 (14)

~Values are given as means + SEM with number of animals in parentheses. Asterisks indicate significant differences with respect to control rabbits **p < 0.01.

international Journal o f Pancreatology Volume 5, 1989

234 A lvarez and L @ez

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DISCUSSION

The literature contains conflicting results concerning the effect of experimentally-induced diabetes on pancreatic juice flow. In this sense, the volume of pancreatic secretion has been seen to be strongly reduced in patients with insulin-dependent diabetes (8-10) and in alloxan-diabetic sheep (25), whereas in streptozotocin-treated rats, Berg et at. (20) reported an increase in the fluid secretory rate. Our findings are consistent with those of Sofrankov~i and Dockray (24) in rats rendered diabetic with streptozotocin, which showed a flowrate similar to that observed in control animals.

The changes observed in bicarbonate secretion conflict with the available information, mainly concerning insulin-dependent diabetic patients (8-10). The chloride concentration in pancreatic juice varies inversely to the concentration of bicarbonate (Fig. 1). The alterations found in both parameters are reversed following insulin administration, pointing to a direct effect of al-


Effect of A lloxan Diabetes on Exocrine Pancreatic Secretion 235

loxan-induced diabetes on the secretion of these anions. An explanation for the changes in anion levels would be the occurrence of an increase in pancreatic proton flux to interstitial fluid, and, hence, in bicarbonate flux to pancreatic juice, according to some models proposed for duct cell electrolyte secretion (31-33). Evidence has been advanced to suggest that the sodium/ proton exchange carrier is very efficient in the absence of insulin, at least in muscle cells (34). In untreated diabetic rabbits, this exchanger could be hyper- activated by a higher contribution of electrochemical energy that in animals with normal levels of insulin would be displaced toward other membrane car- riers that require this hormone to be able to function efficiently. According to Kuijpers et al. (35), chloride secretion is regulated secondarily to bicarbonate secretion and thus in the alloxan-diabetic rabbits a decrease might occur in chloride secretion to maintain the charge of the anion. Finally, it is also possible that in the secretory ducts of untreated diabetic animals the exchange of ductal bicarbonate with interstitial chloride, which has been shown to occur in rabbit pancreas (36,37), could be altered. Contrariwise, the rise observed in potassium concentrations in alloxan-diabetic rabbits is not reversed after insulin administration. It is clear that both groups treated with alloxan were subject to more stressful conditions, which probably induced an increase in aldosterone secretion, which in turn could account for the greater concentration of this ion in pancreatic juice.

The changes observed in enzyme secretion in the alloxan-diabetic rabbits are consistent with the results found in previous studies in which the secretion of amylase was seen to be strongly decreased in rats with experimental diabetes (19,20,22-24). However, there are discrepancies concerning the effects of diabetes on total protein secretion. Depending on the authors, in streptozotocin-treated rats the secretion of protein was similar to that observed in the control animals (24), or was enhanced (20), whereas in sheep (25), as in the case of our own animals, this parameter was clearly reduced during alloxan diabetes.

Under our experimental conditions, insulin administration was unable to restore amylase activity and output to normal values. In most of the literature consulted, treatment with insulin has been reported to reverse the effects of chemically induced diabetes on pancreatic enzyme content (15-19,21,23,26) and both its in vitro (19) and in vivo (20) secretion. Thus, these effects of alloxan or streptozotocin on exocrine pancreas appeared to be owing to insulin deficiency. The doses of insulin given to normalize secretion were higher than those used in this study (19) or at least were higher than those required to avoid hyperglycemia (20). In in vitro assays with the pancreases from streptozotocin-diabetic rats treated with insulin, Trimble et al. (26) and Okabay- ashi et al. (23) found that although these organs had normal enzyme contents, the stimulated amylase and protein secretions were reduced. It is interesting to note that the protocols followed by these authors for the administration of insulin were the same as or similar to those used by us; thus, the insulin dose received by each animal depended either on the amount of urinary glucose excretion (23) or on the daily urine volume (26).

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236 A lvarez and L@ez

It is known that insulin has receptors on pancreatic acinar cells (38-41). Insulin regulates acinar protein synthesis, in particular, it promotes the synthesis of amylase (16-18,21,42) and it has been shown that insulin potentiates cholecystokinin- or acetylcholine-induced exocrine secretion (43-45). Owing to the existence of the insulo-acinar portal system, arterial blood supplying the pancreatic islets flows to the acini directly (3-5). As a result of this, under normal physiological conditions, the exocrine tissue around the islets is exposed to concentrations of insulin that are evidently higher than those found in the systemic circulation. Accordingly, in our assays, like those of Trimble et al. (26) and Okabayashi et al. (23), where insulin doses strictly necessary to normalize the general metabolism were employed, it is possible that the doses employed were insufficient to reverse the effects of diabetes on pancreatic amylase secretion.

It seems feasible to conclude that the alloxan-induced diabetes affects both hydroetectrolytic and enzymatic exocrine pancreatic secretion, especially regarding the enzymatic component , where the insuto-acinar portal system might play a major role.

ACKNOWLEDGMENTS

This research was supported by a project from the Comisidn Asesora de Investigacidn Cientifica y T~cnica (PR 81-445).

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