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Regulation of pancreatic polypeptide secretion in the isolated perfused human pancreas
Regulation of Pancreatic Polypeptide Secretion in the Isolated Perfused Human Pancreas F. Charles Brunicardi, MD, Paul Druck, MD, You Su Sun, MD, Brooklyn, New York, Dariush Elahi, PhD, Boston, Massachusetts, Ronald L. Gingerich, PhD, St. Louis, Missouri, and Dana K. Andersen, MD, Brooklyn, New York
Pancreatic polypeptide is a 36 amino acid polypeptide (4,200 molecular weight) originally isolated as a contaminant from insulin preparations by Kimmel et al [I] and independently by Lin and Chance [2]. Pancreatic polypeptide is localized exclusively to histologically specific cells within the pancreatic islets, called pancreatic polypeptide cells, which are found predominantly in the pancreatic head and uncinate process [3,4]. Pancreatic polypeptide cells comprise 10 to 20 percent of the entire pancreatic islet cell population, and are therefore second only to B cells in total number [5]. Plasma levels ofimmunoreactive pancreatic polypeptide range from 50 to 200 pg/ml (12.5 to 50 pmol/ liter) in man and dog in the basal state and increase promptly after feeding to levels of 400 to 1,200 pg/ ml. The release of pancreatic polypeptide is thought to be mediated by both neural and enteric hormonal factors, although the identity of the principal hormonal mediator or mediators of pancreatic polypeptide release remains uncertain [3-7]. Pancreatic polypeptide has been suggested to have an inhibitory role on pancreatic exocrine secretion, choleresis, and on plasma levels of motilin, and recent studies in this laboratory indicate that pancreatic polypeptide may serve as an important mediator of the hepatic response to insulin in the regulation of glucose metabolism [3,4,8,9]. To assess factors mediating islet-cell hormone release, we recently reported the development of a model of the isolated perfused human pancreas [10,11]. In this study, the pancreatic polypeptide response of human pancreatic grafts, harvested in a manner consistent with that of segmental pancreas transplantation, was assessed. The effects of glucose, physiologic concentrations of insulin and the From the Departments of Surgery and Medicine, State University of New York, Health Science Center, Brooklyn, New York; the Department of Medicine, Beth Israel Hospital, Boston, Massachusetts; and the Department of PediatriCs, Washington University School of Medicine, St. Louis, Missouri. Supported in part by the Foundation for Surgical Education and Investigation, Inc., Brooklyn, New York and Grant AM-30336 from the National Institutes of Health, Bethesda, Maryland. Requests for reprints should be addressed to Dana K. Andersen, MD, Department of Surgery, Box 40, State University of New York Health Science Center, 450 Clarkson Avenue, Brooklyn, New York 11203. Presented at the 28th Annual Meeting of the Society for Surgery of the Alimentary Tract, Chicago, Illinois, May 12 and 13, 1987.
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enteric hormone gastric inhibitory polypeptide, and splanchnic nerve stimulation, were examined in freshly harvested distal pancreatic segments perfused with a single-pass perfusion system. The in vitro neural, hormonal, and glycemic mediation of pancreatic polypeptide cell responses was assessed, and an analysis of the interactions of these factors on both stimulation and inhibition of islet cell secretion was carried out.
Material and Methods Pancreases were obtained from 18 cadaveric organ donors after brain death due to trauma (6 donors) or subarachnoid hemorrhage (12 donors). The donors ranged from 14 to 62 years old; 10 were male and 8 were female. There was no history of pancreatic disease in any of the donors; however, they all received a multitude of preharvest medications. After renal harvesting, the pancreas was freed of surrounding structures and mobilized according to the technique described by Kelly et al [12]. After the colon had been mobilized, pancreatic resection began with the excision of the gastrolineal and lineorenal ligaments and transection of the short gastric vessels. The pancreas and spleen were mobilized from the retroperitoneum and lifted medially, thus exposing the posterior surface of the pancreas. The splenic artery, splenic vein, and splanchnic neural trunk were identified and isolated along the posterior surface of the body of the pancreas. The splenic artery was cannulated with a 14 gauge catheter and the pancreas was perfused in situ with cold lactated Ringer's solution containing 70 mg/dl (3.9 mM) glucose and i percent human serum albumin (New York Blood Center, New York, NY). The gland was transected across the body, whereupon the distal segment with the spleen was placed in an ice bath of Ringer's solution. While the cold perfusion continued, the spleen was dissected from the pancreas at the hilum with careful ligation of all vessels. The pancreatic duct was cannulated with an 18 gauge catheter and the cut surface was oversewn. In 13 preparations, the splenic vein was cannulated with Silastic | tubing (6 mm outer diameter) containing multiple drain holes. In six preparations, sudden cardiac arrest of the donor necessitated rapid excision of the pancreatic specimen with immediate submersion into an iced Ringer's solution bath, where subsequent preparation took place. The total dissection time
63
Brunicardi et al
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Figure 1. Effect of age on mean basal immunoreactlve pancreatic polypeptide secretion for each pancreas preparatiork The linear correlation 4- 95 percent confidence limits Is also shown (correlation coefficient 0.576, n = 18, p <0.01).
was 20 to 25 minutes, during which the gland was kept at 4~ The gland was then transported in iced lactated Ringer's solution to the laboratory where single-pass perfusion was performed on the perfusion apparatus (Ambec Two/Ten Perfuser~, MX International, Aurora, CO). The perfusion media was Krebs-Ringer's bicarbonate buffer containing 3.9 raM glucose, 1 percent human serum albumin, and 3 percent T-70 dextran (Sigma Chemical, St. Louis, MO). The media was gassed with 95 percent oxygen and 5 percent carbon dioxide and heated to 37~ After rewarming and equilibration with a 30 minute basal perfusion, sequential 15 minute test periods were performed, separated by 10 minute basal periods. Total length of perfusion was 180 • 9 minutes and ranged from 59 to 240 minutes. Flow rates were adjusted to maintain a perfusion pressure of 50 to 60 cm of water, and ranged from 1 to 1.2 ml/g/min. Aliquots of venous effluent (6 to 9 ml) containing 500 KIU/ml of aprotinin (Trasylol| FBA Pharmaceuticals, New York, NY) were collected on ice by gravity drainage (5 perfusions) or by splenic vein catheter drainage (13 perfusions), immediately assayed for glucose, and then frozen at minus 20 ~C for subsequent radioimmunoassay of hormones. Test periods consisted of perfusion with 16.7 mM (300 mg/dl) glucose, 14 test periods; 1 nM (5 ng/ml) porcine gastric inhibitory polypeptide (Quadralogic Technologies, Vancouver, BC, Canada), 18 test periods; 20 ~U/ml porcine insulin (Lilly Research Laboratories, Indianapolis, IN), 5 test periods; 16.7 mM glucose plus gastric inhibitory polypeptide, 14 test periods; 20 ~U/ml insulin plus gastric inhibitory polypeptide, 10 test periods; 20 ~U/ml insulin plus 16.7 mM glucose, 7 test periods; 20 #U/ml insulin plus 16.7 mM glucose plus gastric inhibitory polypeptide, 4 test periods; bipolar electrical stimulation (10 cycles/second, 5 ms, 10 volts, model 104-A Laboratory Stimulator | American Electronic Labs, Colmar, PA) of the splanchnic neural fibers, 6 test periods; or splanchnic nerve stimulation plus 16.7 mM glucose plus gastric inhibitory polypeptide, 5 test periods, in random order. Not all preparations received every test
64
substance or intervention; comparisons were made only when appropriate control periods were able to be completed in each perfusion. The hormonal perfusates were prepared fresh immediately before each study and delivered by way of a sidearm into the arterialport. Perfusate and effluent glucose levelswere determined by the glucose oxidase method (Beckman Glucose Analyzer~, Beckman Instruments, Fullerton, CA) and immunoreactive pancreatic polypeptide was measured by a double antibody radioimmunoassay as previously described [13].The lower limit of detection with this assay was 10 pg/ml, and intraassay and interassay coefficients of variation were less than 10 percent and 12 percent, respectively. Statisticalanalyses were performed by comparing test versus control periods using the paired Student's t test.p <0.05 was considered significant. The integrated pancreatic polypeptide response was calculated as the weighted mean increase or decrease in pancreatic polypeptide above or below the basal value, using the trapezoidal rule. The integrated area was then divided by the total time of the period (15 minutes) to determine the mean difference from basal value in pg/g/min for pancreatic polypeptide secretion. Comparisons between test periods were carried out by paired analyses of test periods performed during individual perfusions. Data are presented as mean ~=standard error of the mean, unless otherwise stated. This study was reviewed and approved by the Institutional Review Board of SUNY Health Science Center at Brooklyn. Informed consent was obtained from next of kin of each donor subject.
Results Basal immunoreactive pancreaticpolypeptide secretion was 52 + 6 pg/g/min for the 18 pancreas preparations and ranged from 16 to 100 pg/g/min. Although no correlation could be found between basal secretory rate and donor gender, preharvest medications, cause of death, or length of cold ischemia, there was a significantcorrelationbetween the age of the donor and basal immunoreactive pancreatic polypeptide secretion (Figure 1). The pancreaticpolypeptide response to 16.7 m M glucose solutionisshown in Figure 2. Basal pancreatic polypeptide secretion was 67 d: 9.5 pg/g/min and decreased by 5 :~ 2.3 pg/g/min in response to hyperglycemia (14 testperiods,p <0.05). The data are depicted graphicallyas percent of basal due to the large variation in basal and stimulated values observed in the 18 preparations. Calculations of augmentation or inhibitionof responses,and statistical significancethereof, were performed on the actual integrated response of the hormone during the time intervalexamined. Immunoreactive pancreatic polypeptide responses to gastricinhibitorypolypeptide (5 ng/ml) solution infused alone or in combination with 16.7 m M glucose solution are shown in Figure 3. In the presence of gastric inhibitory polypeptide alone, The American Journal of Surgery
Regulation of Pancreatic Polypeptide Secretion
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Figure 2. Immunoreactlve pancreatic polypeptlde ( IR= PP) response (mean • standard error of the mean) response to 16.7 mM glucose solution in 14 test periods. Upper left, the glucose squarewave created by increasing the glucose concentration from a basal value of 70 mg/dl to a value of 300 mg/dl for 15 minutes. Lower left, the pancreatic polypeptide response, expressed as percentage of basal value. Basal immunoreactiVe pancreatic polypeptide secretion was 67 J,- 9.5 pg/g/min. Right, graphs show Immunoreactive insulin, glucagon, and somstostatln response to 16. 7 mM glucose solution.
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immunoreactive pancreatic polypeptide concentration increased to 142 4- 8 percent of the basal value and maintained an average integrated secretion of 11 * 1.4 pg/g/min above the basal value for the entire test period (18 test periods, p <0.001). In the presence of 16.7 mM glucose solution, gastric inhibitory polypeptide perfusion also resulted in a prompt immunoreactive pancreatic polypeptide response, to 148 + 10 percent of the basal value, but subsequent secretion of immunoreactive pancreatic polypeptide decreased below the response seen with gastric inhibitory polypeptide alone. The average integrated immunoreactive pancreatic polypeptide response to gastric inhibitory polypeptide plus glucose infusion was 6 :~ 2.5 pg/g/min above the basal value (14 test periods, p <0.01). Although this was significantly greater than the immunoreactive pancreatic polypeptide response to glucose alone, it was also significantly less than the immunoreactive pancreatic polypeptide response to gastric inhibitory polypeptide alone (nine test periods, p <0.025). The immunoreactive pancreatic polypeptide response to bipolar electrical stimulation (10 volts, 5 ms, 10 Hz) of the splanchnic neural fibers during combined perfusion with 5 ng/ml of gastric inhibitory polypeptide and 16.7 mM glucose solution is shown in Figure 4. The basal immunoreactive pancreatic polypeptide secretion rate of 47 4- 15.7 pg/g/ rain increased by 26 4- 12.5 pg/g/min for the entire perfusion period, which represented a 16 • 2.2 percent augmentation of the immunoreactive pancreatic polypeptide response to 16.7 mM glucose solution plus gastric inhibitory polypeptide alone (five test periods, p <0.005). During splanchnic nerve stimulation alone, the basal immunoreactive pancreatic polypeptide secretion rate of 49 • 10.2 pg/g/
Volume155,January1988
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Figure 3. Effect of gastric Inhibitory polypeptide ( GIP) on Immunoreactive pancreatic polypeptlde ( /R-PP) secretion. The immunoreactlve pancreatic polypeptlde responses to $ ng/ml gastric Inhibitory polypepUde alone ( open squares, broken line), 5 ng/ml gastric inhibitory polypeptlde plus 16.7 mM glucose solution (open circles, solid line), and perfuslon of 16.7 mM glucose solution alone (solid circles, solid line) are shown for 14 test periods (mean .;- standard error of the mean). Basal Immunoreacflve pancreatic polypeptide secretion was 43 -I- 5. 7 pg/g/mln for gastric inhibitory polypeptide, 56 + 13 pg/g/mln for glucose plus gastric Inhibitory polypeptlde, and 67 :}: 9.5 pg/g/ rain for glucose.
min increased an average of 3 ~- 1.3 pg/g/min for the entire perfusion period (six test periods, p <0.05). The effects of physiologic levels (20 ~U/ml) of insulin on basal immunoreactive pancreatic polypeptide secretion, and on the immunoreactive pancreatic polypeptide response to 16.7 mM glucose solution, I nM gastric inhibitory polypeptide solution, or combined perfusion with both were evaluated in a series of studies. The effects of infusing 20 ~U/ml insulin on the immunoreactive pancreatic polypeptide response to combined perfusion with
65
Brunicardi et al
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Figure 4. Effect of splanchnlc nerve stimulation on immunoreacllve pancreatic polypeptide ( IR.PP) secretion. The immunoreactive pancreatic polypepUde responses to electrical stimulation of the splanchnic neural fibers (NS) during perfusion with 5 ng/mi gastric Inhibitory polypeptide ( GIP) plus 16.7 mM glucose solution (open triangles) and perfusion with 5 ng/nl gastric Inhibitory polypeptide plus 16.7 mM glucose solution alone (closed triangles) are shown f o r five lest periods (mean 4standard error of the mean). Basal immunoreactive pancreatic polypeptide secretion was 47 + 15.7 pg/g/mln for glucose, gastric inhibltory polypeptide, and splanchnlc neural stlmulation, and 44 4- 8.5 pg/g/mln for glucose and gastric inhibitory polypeptide.
16.7 mM glucose plus gastric inhibitory polypeptide solutions are shown in Figure 5, During 20 #U/ml insulin infusion, the basal immun0reactive pancreatic polypeptide secretion of 47 4- 14 pg/g/min increased to 150 4- 10 percent of the basal value, and maintained an integrated response for the entire perfusion period of 17 4- 8 pg/g/min above the basal value. This represented a 37 4- 8.9 percent increase over the immunoreactive pancreatic polypeptide response observed during perfusion with 16.7 mM glucose solution plus gastric inhibitory polypeptide alone (four test periods, p <0.025). Perfusion with 20 ~U/ml insulin plus 16.7 mM glucose solution also augmented the immunoreactive pancreatic polypeptide response by 16 4- 4.2 percent above that seen with 16.7 mM glucose solution alone (seven test periods, p <0.05). Perfusion with 20 uU/ml insulin plus 1 nM gastric inhibitory polypeptide solution resulted in an increase in the integrated immunoreactive pancreatic polypeptide response of 18 4- 3.3 pg/g/min above that observed with 1 nM gastric inhibitory polypeptide solution alone (10 test periods, p <0.025). Perfusion with 20 uU/ml insulin alone also increased immunoreactive pancreatic polypeptide levels by 5 4- 1.5 pg/g/min from a basal value of 53 4- 6.8 pg/g/min (five test periods, p
66
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Figure 5. Effect of Insulin infusion on immunoreactive pancreatic polypeptlde ( IR-PP) secretion. The immunoreactlve pancreatic polypeptlde responses to combined infusion of 20 #U/ml insulin (INS), 5 ng/ml gastric Inhibitory polypeptide (GIP), and 16. 7 mM glucose solution (open triangles) and perfuston with 5 ng/ml gastric inhibilory polypeptide plus 16.7 mM glucose solution alone ( closed triangles) are shown for five test periods ( mean • standard error of the mean). Basal Immunoreactive pancreatic polypeptlde secretion was 47 4- 14 pg/g/min for glucose, gastric Inhibitory polypeptide, and Insulin and 44 • 8.5 pg/g/mln for glucose plus gastric Inhibitory polypeptide.
c u r r e n t u n d e r s t a n d i n g of islet p h y s i o l o g y [10,11,14]. We have therefore examined the hormonal and neural regulation of pancreatic polypeptide secretion in this model, and have compared the pancreatic polypeptide cell responses to those of the 8, ~, and ~ cells reported in our previous studies using identical stimuli. The basal secretory rate of pancreatic polypeptide was 52 4- 6 pg/g/min, which represented a relatively low rate of secretion in comparison to those of insulin (1,000 to 1,500 ~U/g/min), glucagon (80 to 120 pg/g/min), and somatostatin (80 to 130 pg/g/ min) seen in this model. Only the distal body and tail of the pancreas were perfused, however; and although the pancreatic polypeptide cells are localized predominantly in the head and uncinate process of the pancreas, the presence of immunoreactive pancreatic polypeptide in the splenic venous effluent in these 18 glands indicates that functioning pancreatic polypeptide cells are nonetheless distributed throughout the pancreas [5,13,15]. In our studies, the basal rate of in vitro immunoreactive pancreatic polypeptide secretion correlated positively with the age of the pancreatic donor. An agerelated increase in plasma levels of immunoreactive pancreatic polypeptide has been previously noted, but it has been unclear whether these higher levels of circulating pancreatic polypeptide in older subjects are the result of increased synthesis or reduced clearance of pancreatic polypeptide [3,4]. Our data, therefore, provide direct evidence for an intrinsic age-related enhancement of basal pancreatic polypeptide secretion.
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Regulation of Pancreatic Polypeptide Secretion
Previous studies on the immunoreactive pancreatic polypeptide response to hyperglycemia have been contradictory [7,13,16-24]. In response to 16.7 mM glucose perfusion, immunoreactive pancreatic polypeptide secretion was modestly but significantly inhibited, glucagon release was significantly suppressed, and insulin and somatostatin release were strongly stimulated [11,14]. Although glucose alone appears to have only a modest role in the release of pancreatic polypeptide, hyperglycemia may mediate the pancreatic polypeptide response to other stimuli either through neural, endocrine, or paracrine mechanisms. When perfused alone, or in combination with other agents, glucose exerted a consistently inhibitory effect on immunoreactive pancreatic polypeptide secretion. This occurred during concomitant increases in the secretion Of insulin and somatostatin, which suggests that/3-cell secretion, b-cell secretion, or both may serve as a tonic inhibitor of the pancreatic polypeptide cell responses to stimuli [10,14]. Although our data cannot identify which of the islet cells is the principal paracrine mediator of pancreatic polypeptide release, our findings confirm the parallel responses of glucagon and pancreatic polypeptide release and the reciprocal relationship of pancreatic polypeptide secretion to that of insulin and somatostatin. The immunoreactive pancreatic polypeptide response to feeding and to oral but not intravenous glucose suggests that pancreatic polypeptide release is mediated by one or more enteric hormonal agents. Cholecystokinin, secretin, gastrin, vasoactive intestinal peptide, bombesin, and gastric inhibitory polypeptide have all been implicated as possible enteric secretagogues of pancreatic polypeptide, bu t only cho!ecystokinin has been demonstrated to have a convincing stimulatory effect when infused in viv0 [6,7,25]. In our study, perfusion with 5 ng/ml gastric inhibitory polypeptide stimulated immunoreactive pancreatic polypeptide secretion significantly, which is consistent with the stimulatory effects of gastric inhibitory polypeptide seen during in situ canine pancreas perfusion [7]. Therefore, our data indicate that gastric inhibitory polypeptide may serve as a secretagogue for pancreatic polypeptide. The splanchnic innervation of the pancreas has been shown to play an important regulatory role in islet cell secretion [26]. Pancreatic polypeptide release was augmented by bipolar electrical stimulation of the sp!anchnic neural bundle alone or during perfusion with gastric inhibitory polypeptide during hyperglycemia, possibly by direct stimulation of the pancreatic polypeptide cell or by an indirect influence on other islet cell secretions. In our previous studies [11], splanchnic neural stimulation during hyperglycemia with or without gastric inhibitory polypeptide resulted in augmented glucagon
Volume 155, January 1988
secretion, a response that parallels that of the pan9creatic polypeptide cell, whereas insulin and somatostatin secretion were inhibited [11,14]. Perfusion with a low physiologic concentration of insulin consistently resulted in an enhancement of immunoreactive pancreatic polypeptide secretion, either in the basal state or during concomitant perfusion with gastric inhibitory polypeptide, 16.7 mM glucose solution, or both, In the presence of 20 ~U/ ml insulin, insulin secretion of 2,000 ~U/ml and somatostatin secretion were significantly reduced, and glucagon secretion was strongly stimulated [10,14]. These findings confirm that the endocrine pancreas is especially sensitive to ambient levels of insulin, and suggest that paracrine mechanisms play a strong role in the regulation of islet cell secretion [27,28]. Although it is possible to ascribe the changes in pancreatic polypeptide secretion to a direct effect of infused insulin o n t h e pancreatic polypeptide cell, a recent morphologic study by Bonner-Weir and Orci [29] supports the concept of a discreet compartmentalization of the islet, with arterial inflow directed preferentially to the central/~-cell core followed by perfusion with the fl-cell products to the peripherally located a, 6, and pancreatic polypeptide cells. Our results seen in the isolated perfused human pancreas support such a theory. When the level of insulin secretion is decreased, as during splanchnic nerve stimulation or with low levels of inflow insulin, the pancreatic polypeptide cell responds by increasing its secretion of pancreatic polypeptide. This effect could represent a direct effect of insulin on the pancreatic polypeptide cell Or may be mediated by an indirect effect of insulin on ()ther islet cells, such as the somatostatin-secreting 5 cell, which may in turn regulate pancreatic polypeptide cell function. Further studies are required to clarify these mechanisms, but our data are nonetheless consistent with a centrifugal Pattern of blood flow within the islet and with the essentiality of the relationship of/3 cells to pancreatic polypeptide cell function. Summary The isolated perfused human pancreas was used as a model to assess factors mediating the pancreatic polypeptide cell response to glucose, insulin, gastric inhibitory polypeptide, and splanchnic nerve stimulation. Pancreases obtained from 18 cadaveric organ donors were isolated and perfused by way of the splenic artery utilizing a Krebs bicarbonate buffer in a single-pass perfusion system. Hormonal stimulation and inhibition of pancreatic polypeptide secretion were assessed, as was the influence of direct electrical stimulation of celiac neural fibers innervating the pancreas. In this in vitro human model, pancreatic polypeptide cell secretion was inhibited by hyperglycemia, although the presence of gastric inhibitory polypeptide augmented the
67
Brunicardi et al
pancreatic polypeptide cell response. Perfusion with low levels of insulin and splanchnic nerve stimulation augmented the response of the pancreatic polypeptide cell to hyperglycemia and gastric inhibitory polypeptide. Since the immunoreactive pancreatic polypeptide response was augmented when insulin and somatostatin release was inhibited by perfusion insulin or nerve stimulation, we conclude that the pancreatic polypeptide cell is regulated by the ambient degree of somatostatin release, insulin release, or both. These findings support a centrifugal pattern of intraislet blood flow. References 1. Kimmet JR, Pollock HG, Hazelwood RL. Isolation and characterization of chicken insulin. Endocrinol 1968; 83: 132330. 2. Lin TM, Chance RE. Spectrum gastrointestinal actions of a new bovine pancreas polypeptide. Gastroenterology 1972; 62: 852. 3. Hazelwood RL. Synthesis, storage, secretion, and significance of panci'eatlc polypeptide in vertebrates. In: Cooperstein SJ, Watkins D, eds. The islets of Langerhans. Biochemistry, physiology , and pathology; New York, Academic Press, 1981: 275-318. 4. Floyd JC Jr. Human pancreatic polypeptide. In: Buchanan KD, ed. Clinics in endocrinology and metabolism. Vol. 8. Philadelphia, WB Saunders, 1977: 379-94. 5. Stefan Y, Orci L, Malaisse-Legae F, Perrelet A, Patel Y, Unger RH. Quantitation of endocrine cell content in the pancreas of nondiabetic and diabetic humans. Diabetes 1982; 31! 694-700. 6. Gingerich RL. Survey of potential pancreatic polypeptide Secretagogues !abstr). Diabetes 1977; 26: 375. 7. Adrian TE, Bloom SR, Hermanson K, Iverson d. Pancreatic polypeptide, glucagon and insulin secretion from the isolated perfused canine pancreas. Diabetologia 1978; 14:413= 7. 8. Bloom SR, Adrian TE, Greenburg GR, et al. Effects of pancreatic POlypeptide infusion in man (abstr). Gastroenterology 1978; 74: 1012. 9. Sun YS, Brunicardi FC, Druck P, et al. Reversal of abnormal glucose metabolism in chronic pancreatitis by administration of pancreatic polypeptide. Am J Surg 1986; 151: 13040. 10. Brunicardi FC, Goulet RJ, Sun YS, Berlin SA, Elahi D, Andersen DK. Neural and hormonal regulation of insulin and glucagon secretion in the isolated perfused human pancreas. Surg Forum 1984; 35: 214-7. 11. Brunicardi FC, Sun YS, DrUck P, Goulet RJ, Elahi D, Andersen DK. Splanchnic neural regulation of insulin and glucagon secretion in the isolated perfused human pancreas. Am J Surg !987; 153: 34-40. 12. Kelly WD, Lillihei RC, Merkel FK, Idezuki Y, Goetz FC. AIIotransplantation of the pancreas and duodenum al0ng with the kidney in diabetic nephropathy. Surgery 1967; 61: 82737. 13. Gingerich RL, Lacy PE, Chance RE, Johnson MG. Regional pancreatic concentration and in vitro secretion of canine pancreatic polypeptide, insulin, and glucagon. Diabetes 1978; 27: 96-101. 14. Brunicardi FC, Druck P, Sun YS, Elahi D, Yamada T, Andersen DK: Neural and hormonal regulation of s0matostatin release in the isolated perfused human pancreas (abstr). Gastroenterology 1986; 90: 1360. 15. Gersell DJ, Gingerich RL, Greider MH. Regional distribution and concentration of pancreatic polypeptide in the human and canine pancreas. Diabetes 1979; 28:11-5.
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16. Weir GC, Samols E, Loo S, Patel YC, Gabbay KH. Somatostatin and pancreatic polypeptide secretion. Diabetes 1979; 28: 35-40. 17. Schusdziarra V, Stapelfe!dt W, Klier M, Mair V, Pfeiffer EF. Effect of physiological increments of blood glucose on plasma somatostatin and pancreatic polypeptide levels in dogs. Regul Pept 1981; 2:211-8. 18. Marco J, Hedo JA, Villanueva ME Control of pancreatic polypeptide secretion by glucose in man. J Clin Endocrinol Metabol 1978; 46: 140-5. 19. Villaneuva ML, Hedo JA, Castillo,Olivares J, Marco J. Effect of exogenous hyperglycemia on human pancreatic polypeptide secretion. Diabetologia 1978; 15: 278-9. 20. Prinz RA, El Sabbagh H, Adrian TE, et al. Neural regulation of pancreatic polypeptide release. Surgery 1983; 94:1011-7. 21. Adrian TE, Bloom SR, Besterman HS, Barnes AJ, Cooke TJ, Faber RG. Mechanism of pancreatic polypeptide release in man. Lancet 1977; 1: 161-3. 22. Floyd JC, Fajans SS, Pek S, Chance RE. A newly recognized pancreatic polypeptide: plasma levels in health and disease. Recent Prog Horm Res 1977; 33: 519-70. 23. Tsuda K, Seino Y, Mori K. Effect of truncal vagotomy on pancreatic polypeptide response after intravenous glucose administration. Regul Pept 1981; 1: 347-52. 24. Sire AA, Vinik AI, VanTonder SV. Pancreatic polypeptide responses to oral and intravenous glucose in man. Am J Gastroenterol 1979; 71: 183-5. 25. Lonovics J, Guzman S, Devitt P, et al. Release of pancreatic polypeptide in humans by infusion of cholecystokinin. Gastroenterology 1980; 79: 817:-22. 26. WoodsSC, Porte D Jr. Neural control of the endocrine pancreas. Physiol Rev 1974; 54: 596-619. 27. Elahi D, Nagulesparan M, Hershcopf R, et al. Feedback inhibition of insulin secretion by insulin. Relation to the hyperinsulinemia of obesity: N Engl J Med 1982; 306:1196-202. 28. Rappaport AM, Ohira S, Coddling JA, et al. Effects on insulin output and on pancreatic blood flow of exogenous insulin infusion into an in situ isolated portion of the pancreas. Endocrinology 1972; 91: 168-76. 29. Bonner-Weir S, Orci L. New perspectives on the microvasculature of the islets of Langerhans in the rat. Diabetes 1982; 31: 883-9.
Discussion
Aaron S. Fink (Los Angeles, CA): Dr. Brunicardi, what effect did splanchnic nerve stimulation alone, in the presence of a physiologic level of glucose, have on pancreatic polypeptide release? Furthermore, were you able to either enhance or block any of the effects of your tested secretogogues, by adding cholinergic blockers such as atropine? I-Iaile T. Debas (San Francisco, CA): Dr. Brunicardi, it is very imaginative to use organs from the cadaveric organ donors to perform appropriate studies in human subjects. I suspect it is the only way we are going to be able to confirm observations made in animal studies. At this level of nerve stimulation there are mixed nerves (cholinergic, adrenergic, and peptidegeric). Did you consider selective blocking to stimulate and define exact mechanisms? I am a bit confused by your results showing that hyperglycemia, when added to gastric inhibitory polypeptide stimulation, inhibits pancreatic polypeptide release. I believe your laboratory first described that gastric inhibitory polypeptide requires a certain level of hyperglycemia to release insulin. You showed insulin released as pancreatic polypeptide. Therefore, in the experiments in which you combined hyperglycemia with gastric inhibitory polypep-
The American Journal of Surgery
Regulation of Pancreatic Polypeptide Secretion
tide, you should see a release of insulin. So the results should be to augment, not inhibit. Finally, most of the pancreatic polypeptide cells are in the head and the uncinate process of the pancreas. Do you think these studies reflect the actual regulation of pancreatic polypeptide, which is mostly in the other part of the gland? James R. Upp, Jr. (Galveston, TX): Dr. Brunicardi, in your study basal pancreatic polypeptide levels in the older patients were elevated. Did you see increased stimulation of pancreatic polypeptide in those patients? Also, the response to gastric inhibitory polypeptide seems to be different in vivo than in your in vitro model. What do you think the different mechanisms are?
Gerald M. Larson (Louisville, KY): Dr. Brunicardi, you have clearly shown that nerve stimulation alters the release of several of these peptides, and we have also shown that surgical sympathectomy leads to changes in pancreatic polypeptide levels in the dog. What is the implication of this for the transplanted pancreas? In most transplants all nerves to the pancreas are cut. What significance does that have for the endocrine function of the pancreas down the road? Certainly insulin metabolism seems to be improved, but just what impact would you predict that denervation of the pancreas would have on hormone balance? F. Charles Brunicardi (closing): Dr. Fink, in additional studies we have shown that splanchnic nerve stimulation as well as perfusion with insulin alone during euglycemia both augment pancreatic polypeptide release. In an extensive study in this model in which we have looked at the neural regulation of all four islet hormones, cholinergic stimulation increased pancreatic polypeptide secretion, whereas a-adrenergic cell stimulation inhibited pancreatic polypeptide secretion and/~-cell stimulation had very little effect. Drs. Bonnet-Weir and Orci have shown that the arteriole penetrates the ~-cell mass first. Therefore, secretogogues reach the B cell first and its produces are carried peripherally to the outer cells, which are the a, ~, and pancreatic polypeptide cells. Therefore, Dr. Debas, our data are consistent with the hypothesis that insulin secretion actually inhibits pancreatic polypeptide secretion. When you administer gastric inhibitory polypeptide dur-
Volume 155, January 1988
ing hyperglycemia, you expect a marked increase of insulin release. Pancreatic polypeptide release should be inhibited compared with the effects seen during perfusion of gastric inhibitory polypeptide with euglycemia alone, and this is precisely what we saw. The response seen with somatostatin release paralleled that of insulin. Therefore, fl-cell secretion influences somatostatin release, both of which in turn influence the a and the pancreatic polypeptide cells. We were pleasantly surprised to find a pancreatic polypeptide response in this preparation, since we used only the body and tail of the pancreas. It is true that 90 to 95 percent of pancreatic polypeptide cell mass is located in the head of the pancreas, with 5 to 10 percent remaining in the tail. We found that in comparison to levels of the other islet hormones, the pancreatic polypeptide levels were relatively low. I can't tell you whether the tail of the pancreas differs from the head of the pancreas in terms of intraislet interactions. Dr. Upp, in vivo studies indicate that basal pancreatic polypeptide levels increase with age. When we compared the age of the donors with the basal pancreatic polypeptide response, we found a highly significant correlation. This seems to indicate that the increase in pancreatic polypeptide secretion one sees with aging is due to increased pancreatic polypeptide secretion and not decreased pancreatic polypeptide clearance. You asked about the effects of gastric inhibitory polypeptide in vivo versus in vitro. As we know from the studies from your own lab, there have been many possible enteric secretagogues that have been suggested for pancreatic polypeptide release, including cholecystokinin. In our model, the insulin and somatostatin responses to gastric inhibitory polypeptide were consistent with the findings of in vivo studies. However, for glucagon and pancreatic polypeptide, the stimulation by gastric inhibitory polypeptide was contradictory to in vivo results. The fact that our glands were denervated and that there was a lack of inflow hormones in the perfusate may have contributed to this finding. Dr. Larson, our findings may be relevant to the function of the transplanted pancreas. There are very few groups who are looking at all four islet hormones in the transplanted pancreas. We suspect that although the transplanted pancreas responds to stimuli, it lacks the finetuned neural regulation of the in situ pancreas.
69
Pancreatic polypeptide is a 36 amino acid polypeptide (4,200 molecular weight) originally isolated as a contaminant from insulin preparations by Kimmel et al [I] and independently by Lin and Chance [2]. Pancreatic polypeptide is localized exclusively to histologically specific cells within the pancreatic islets, called pancreatic polypeptide cells, which are found predominantly in the pancreatic head and uncinate process [3,4]. Pancreatic polypeptide cells comprise 10 to 20 percent of the entire pancreatic islet cell population, and are therefore second only to B cells in total number [5]. Plasma levels ofimmunoreactive pancreatic polypeptide range from 50 to 200 pg/ml (12.5 to 50 pmol/ liter) in man and dog in the basal state and increase promptly after feeding to levels of 400 to 1,200 pg/ ml. The release of pancreatic polypeptide is thought to be mediated by both neural and enteric hormonal factors, although the identity of the principal hormonal mediator or mediators of pancreatic polypeptide release remains uncertain [3-7]. Pancreatic polypeptide has been suggested to have an inhibitory role on pancreatic exocrine secretion, choleresis, and on plasma levels of motilin, and recent studies in this laboratory indicate that pancreatic polypeptide may serve as an important mediator of the hepatic response to insulin in the regulation of glucose metabolism [3,4,8,9]. To assess factors mediating islet-cell hormone release, we recently reported the development of a model of the isolated perfused human pancreas [10,11]. In this study, the pancreatic polypeptide response of human pancreatic grafts, harvested in a manner consistent with that of segmental pancreas transplantation, was assessed. The effects of glucose, physiologic concentrations of insulin and the From the Departments of Surgery and Medicine, State University of New York, Health Science Center, Brooklyn, New York; the Department of Medicine, Beth Israel Hospital, Boston, Massachusetts; and the Department of PediatriCs, Washington University School of Medicine, St. Louis, Missouri. Supported in part by the Foundation for Surgical Education and Investigation, Inc., Brooklyn, New York and Grant AM-30336 from the National Institutes of Health, Bethesda, Maryland. Requests for reprints should be addressed to Dana K. Andersen, MD, Department of Surgery, Box 40, State University of New York Health Science Center, 450 Clarkson Avenue, Brooklyn, New York 11203. Presented at the 28th Annual Meeting of the Society for Surgery of the Alimentary Tract, Chicago, Illinois, May 12 and 13, 1987.
Volume 155, January 1988
enteric hormone gastric inhibitory polypeptide, and splanchnic nerve stimulation, were examined in freshly harvested distal pancreatic segments perfused with a single-pass perfusion system. The in vitro neural, hormonal, and glycemic mediation of pancreatic polypeptide cell responses was assessed, and an analysis of the interactions of these factors on both stimulation and inhibition of islet cell secretion was carried out.
Material and Methods Pancreases were obtained from 18 cadaveric organ donors after brain death due to trauma (6 donors) or subarachnoid hemorrhage (12 donors). The donors ranged from 14 to 62 years old; 10 were male and 8 were female. There was no history of pancreatic disease in any of the donors; however, they all received a multitude of preharvest medications. After renal harvesting, the pancreas was freed of surrounding structures and mobilized according to the technique described by Kelly et al [12]. After the colon had been mobilized, pancreatic resection began with the excision of the gastrolineal and lineorenal ligaments and transection of the short gastric vessels. The pancreas and spleen were mobilized from the retroperitoneum and lifted medially, thus exposing the posterior surface of the pancreas. The splenic artery, splenic vein, and splanchnic neural trunk were identified and isolated along the posterior surface of the body of the pancreas. The splenic artery was cannulated with a 14 gauge catheter and the pancreas was perfused in situ with cold lactated Ringer's solution containing 70 mg/dl (3.9 mM) glucose and i percent human serum albumin (New York Blood Center, New York, NY). The gland was transected across the body, whereupon the distal segment with the spleen was placed in an ice bath of Ringer's solution. While the cold perfusion continued, the spleen was dissected from the pancreas at the hilum with careful ligation of all vessels. The pancreatic duct was cannulated with an 18 gauge catheter and the cut surface was oversewn. In 13 preparations, the splenic vein was cannulated with Silastic | tubing (6 mm outer diameter) containing multiple drain holes. In six preparations, sudden cardiac arrest of the donor necessitated rapid excision of the pancreatic specimen with immediate submersion into an iced Ringer's solution bath, where subsequent preparation took place. The total dissection time
63
Brunicardi et al
0o 9
~0- ~
a
~
D
~
605O3O-
:ii ,o
2o
2o
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Figure 1. Effect of age on mean basal immunoreactlve pancreatic polypeptide secretion for each pancreas preparatiork The linear correlation 4- 95 percent confidence limits Is also shown (correlation coefficient 0.576, n = 18, p <0.01).
was 20 to 25 minutes, during which the gland was kept at 4~ The gland was then transported in iced lactated Ringer's solution to the laboratory where single-pass perfusion was performed on the perfusion apparatus (Ambec Two/Ten Perfuser~, MX International, Aurora, CO). The perfusion media was Krebs-Ringer's bicarbonate buffer containing 3.9 raM glucose, 1 percent human serum albumin, and 3 percent T-70 dextran (Sigma Chemical, St. Louis, MO). The media was gassed with 95 percent oxygen and 5 percent carbon dioxide and heated to 37~ After rewarming and equilibration with a 30 minute basal perfusion, sequential 15 minute test periods were performed, separated by 10 minute basal periods. Total length of perfusion was 180 • 9 minutes and ranged from 59 to 240 minutes. Flow rates were adjusted to maintain a perfusion pressure of 50 to 60 cm of water, and ranged from 1 to 1.2 ml/g/min. Aliquots of venous effluent (6 to 9 ml) containing 500 KIU/ml of aprotinin (Trasylol| FBA Pharmaceuticals, New York, NY) were collected on ice by gravity drainage (5 perfusions) or by splenic vein catheter drainage (13 perfusions), immediately assayed for glucose, and then frozen at minus 20 ~C for subsequent radioimmunoassay of hormones. Test periods consisted of perfusion with 16.7 mM (300 mg/dl) glucose, 14 test periods; 1 nM (5 ng/ml) porcine gastric inhibitory polypeptide (Quadralogic Technologies, Vancouver, BC, Canada), 18 test periods; 20 ~U/ml porcine insulin (Lilly Research Laboratories, Indianapolis, IN), 5 test periods; 16.7 mM glucose plus gastric inhibitory polypeptide, 14 test periods; 20 ~U/ml insulin plus gastric inhibitory polypeptide, 10 test periods; 20 ~U/ml insulin plus 16.7 mM glucose, 7 test periods; 20 #U/ml insulin plus 16.7 mM glucose plus gastric inhibitory polypeptide, 4 test periods; bipolar electrical stimulation (10 cycles/second, 5 ms, 10 volts, model 104-A Laboratory Stimulator | American Electronic Labs, Colmar, PA) of the splanchnic neural fibers, 6 test periods; or splanchnic nerve stimulation plus 16.7 mM glucose plus gastric inhibitory polypeptide, 5 test periods, in random order. Not all preparations received every test
64
substance or intervention; comparisons were made only when appropriate control periods were able to be completed in each perfusion. The hormonal perfusates were prepared fresh immediately before each study and delivered by way of a sidearm into the arterialport. Perfusate and effluent glucose levelswere determined by the glucose oxidase method (Beckman Glucose Analyzer~, Beckman Instruments, Fullerton, CA) and immunoreactive pancreatic polypeptide was measured by a double antibody radioimmunoassay as previously described [13].The lower limit of detection with this assay was 10 pg/ml, and intraassay and interassay coefficients of variation were less than 10 percent and 12 percent, respectively. Statisticalanalyses were performed by comparing test versus control periods using the paired Student's t test.p <0.05 was considered significant. The integrated pancreatic polypeptide response was calculated as the weighted mean increase or decrease in pancreatic polypeptide above or below the basal value, using the trapezoidal rule. The integrated area was then divided by the total time of the period (15 minutes) to determine the mean difference from basal value in pg/g/min for pancreatic polypeptide secretion. Comparisons between test periods were carried out by paired analyses of test periods performed during individual perfusions. Data are presented as mean ~=standard error of the mean, unless otherwise stated. This study was reviewed and approved by the Institutional Review Board of SUNY Health Science Center at Brooklyn. Informed consent was obtained from next of kin of each donor subject.
Results Basal immunoreactive pancreaticpolypeptide secretion was 52 + 6 pg/g/min for the 18 pancreas preparations and ranged from 16 to 100 pg/g/min. Although no correlation could be found between basal secretory rate and donor gender, preharvest medications, cause of death, or length of cold ischemia, there was a significantcorrelationbetween the age of the donor and basal immunoreactive pancreatic polypeptide secretion (Figure 1). The pancreaticpolypeptide response to 16.7 m M glucose solutionisshown in Figure 2. Basal pancreatic polypeptide secretion was 67 d: 9.5 pg/g/min and decreased by 5 :~ 2.3 pg/g/min in response to hyperglycemia (14 testperiods,p <0.05). The data are depicted graphicallyas percent of basal due to the large variation in basal and stimulated values observed in the 18 preparations. Calculations of augmentation or inhibitionof responses,and statistical significancethereof, were performed on the actual integrated response of the hormone during the time intervalexamined. Immunoreactive pancreatic polypeptide responses to gastricinhibitorypolypeptide (5 ng/ml) solution infused alone or in combination with 16.7 m M glucose solution are shown in Figure 3. In the presence of gastric inhibitory polypeptide alone, The American Journal of Surgery
Regulation of Pancreatic Polypeptide Secretion
300
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Figure 2. Immunoreactlve pancreatic polypeptlde ( IR= PP) response (mean • standard error of the mean) response to 16.7 mM glucose solution in 14 test periods. Upper left, the glucose squarewave created by increasing the glucose concentration from a basal value of 70 mg/dl to a value of 300 mg/dl for 15 minutes. Lower left, the pancreatic polypeptide response, expressed as percentage of basal value. Basal immunoreactiVe pancreatic polypeptide secretion was 67 J,- 9.5 pg/g/min. Right, graphs show Immunoreactive insulin, glucagon, and somstostatln response to 16. 7 mM glucose solution.
150
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immunoreactive pancreatic polypeptide concentration increased to 142 4- 8 percent of the basal value and maintained an average integrated secretion of 11 * 1.4 pg/g/min above the basal value for the entire test period (18 test periods, p <0.001). In the presence of 16.7 mM glucose solution, gastric inhibitory polypeptide perfusion also resulted in a prompt immunoreactive pancreatic polypeptide response, to 148 + 10 percent of the basal value, but subsequent secretion of immunoreactive pancreatic polypeptide decreased below the response seen with gastric inhibitory polypeptide alone. The average integrated immunoreactive pancreatic polypeptide response to gastric inhibitory polypeptide plus glucose infusion was 6 :~ 2.5 pg/g/min above the basal value (14 test periods, p <0.01). Although this was significantly greater than the immunoreactive pancreatic polypeptide response to glucose alone, it was also significantly less than the immunoreactive pancreatic polypeptide response to gastric inhibitory polypeptide alone (nine test periods, p <0.025). The immunoreactive pancreatic polypeptide response to bipolar electrical stimulation (10 volts, 5 ms, 10 Hz) of the splanchnic neural fibers during combined perfusion with 5 ng/ml of gastric inhibitory polypeptide and 16.7 mM glucose solution is shown in Figure 4. The basal immunoreactive pancreatic polypeptide secretion rate of 47 4- 15.7 pg/g/ rain increased by 26 4- 12.5 pg/g/min for the entire perfusion period, which represented a 16 • 2.2 percent augmentation of the immunoreactive pancreatic polypeptide response to 16.7 mM glucose solution plus gastric inhibitory polypeptide alone (five test periods, p <0.005). During splanchnic nerve stimulation alone, the basal immunoreactive pancreatic polypeptide secretion rate of 49 • 10.2 pg/g/
Volume155,January1988
-,
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.
T
140
9 Glucose o Glucose/GIP o GIP
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Figure 3. Effect of gastric Inhibitory polypeptide ( GIP) on Immunoreactive pancreatic polypeptlde ( /R-PP) secretion. The immunoreactlve pancreatic polypeptlde responses to $ ng/ml gastric Inhibitory polypepUde alone ( open squares, broken line), 5 ng/ml gastric inhibitory polypeptlde plus 16.7 mM glucose solution (open circles, solid line), and perfuslon of 16.7 mM glucose solution alone (solid circles, solid line) are shown for 14 test periods (mean .;- standard error of the mean). Basal Immunoreacflve pancreatic polypeptide secretion was 43 -I- 5. 7 pg/g/mln for gastric inhibitory polypeptide, 56 + 13 pg/g/mln for glucose plus gastric Inhibitory polypeptlde, and 67 :}: 9.5 pg/g/ rain for glucose.
min increased an average of 3 ~- 1.3 pg/g/min for the entire perfusion period (six test periods, p <0.05). The effects of physiologic levels (20 ~U/ml) of insulin on basal immunoreactive pancreatic polypeptide secretion, and on the immunoreactive pancreatic polypeptide response to 16.7 mM glucose solution, I nM gastric inhibitory polypeptide solution, or combined perfusion with both were evaluated in a series of studies. The effects of infusing 20 ~U/ml insulin on the immunoreactive pancreatic polypeptide response to combined perfusion with
65
Brunicardi et al
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l
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160
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Time (minutes)
Figure 4. Effect of splanchnlc nerve stimulation on immunoreacllve pancreatic polypeptide ( IR.PP) secretion. The immunoreactive pancreatic polypepUde responses to electrical stimulation of the splanchnic neural fibers (NS) during perfusion with 5 ng/mi gastric Inhibitory polypeptide ( GIP) plus 16.7 mM glucose solution (open triangles) and perfusion with 5 ng/nl gastric Inhibitory polypeptide plus 16.7 mM glucose solution alone (closed triangles) are shown f o r five lest periods (mean 4standard error of the mean). Basal immunoreactive pancreatic polypeptide secretion was 47 + 15.7 pg/g/mln for glucose, gastric inhibltory polypeptide, and splanchnlc neural stlmulation, and 44 4- 8.5 pg/g/mln for glucose and gastric inhibitory polypeptide.
16.7 mM glucose plus gastric inhibitory polypeptide solutions are shown in Figure 5, During 20 #U/ml insulin infusion, the basal immun0reactive pancreatic polypeptide secretion of 47 4- 14 pg/g/min increased to 150 4- 10 percent of the basal value, and maintained an integrated response for the entire perfusion period of 17 4- 8 pg/g/min above the basal value. This represented a 37 4- 8.9 percent increase over the immunoreactive pancreatic polypeptide response observed during perfusion with 16.7 mM glucose solution plus gastric inhibitory polypeptide alone (four test periods, p <0.025). Perfusion with 20 ~U/ml insulin plus 16.7 mM glucose solution also augmented the immunoreactive pancreatic polypeptide response by 16 4- 4.2 percent above that seen with 16.7 mM glucose solution alone (seven test periods, p <0.05). Perfusion with 20 uU/ml insulin plus 1 nM gastric inhibitory polypeptide solution resulted in an increase in the integrated immunoreactive pancreatic polypeptide response of 18 4- 3.3 pg/g/min above that observed with 1 nM gastric inhibitory polypeptide solution alone (10 test periods, p <0.025). Perfusion with 20 uU/ml insulin alone also increased immunoreactive pancreatic polypeptide levels by 5 4- 1.5 pg/g/min from a basal value of 53 4- 6.8 pg/g/min (five test periods, p
66
I00
i -4
0
4,
8,
, 12
= 16
,
210
'
Time (minutes)
Figure 5. Effect of Insulin infusion on immunoreactive pancreatic polypeptlde ( IR-PP) secretion. The immunoreactlve pancreatic polypeptlde responses to combined infusion of 20 #U/ml insulin (INS), 5 ng/ml gastric Inhibitory polypeptide (GIP), and 16. 7 mM glucose solution (open triangles) and perfuston with 5 ng/ml gastric inhibilory polypeptide plus 16.7 mM glucose solution alone ( closed triangles) are shown for five test periods ( mean • standard error of the mean). Basal Immunoreactive pancreatic polypeptlde secretion was 47 4- 14 pg/g/min for glucose, gastric Inhibitory polypeptide, and Insulin and 44 • 8.5 pg/g/mln for glucose plus gastric Inhibitory polypeptide.
c u r r e n t u n d e r s t a n d i n g of islet p h y s i o l o g y [10,11,14]. We have therefore examined the hormonal and neural regulation of pancreatic polypeptide secretion in this model, and have compared the pancreatic polypeptide cell responses to those of the 8, ~, and ~ cells reported in our previous studies using identical stimuli. The basal secretory rate of pancreatic polypeptide was 52 4- 6 pg/g/min, which represented a relatively low rate of secretion in comparison to those of insulin (1,000 to 1,500 ~U/g/min), glucagon (80 to 120 pg/g/min), and somatostatin (80 to 130 pg/g/ min) seen in this model. Only the distal body and tail of the pancreas were perfused, however; and although the pancreatic polypeptide cells are localized predominantly in the head and uncinate process of the pancreas, the presence of immunoreactive pancreatic polypeptide in the splenic venous effluent in these 18 glands indicates that functioning pancreatic polypeptide cells are nonetheless distributed throughout the pancreas [5,13,15]. In our studies, the basal rate of in vitro immunoreactive pancreatic polypeptide secretion correlated positively with the age of the pancreatic donor. An agerelated increase in plasma levels of immunoreactive pancreatic polypeptide has been previously noted, but it has been unclear whether these higher levels of circulating pancreatic polypeptide in older subjects are the result of increased synthesis or reduced clearance of pancreatic polypeptide [3,4]. Our data, therefore, provide direct evidence for an intrinsic age-related enhancement of basal pancreatic polypeptide secretion.
The American J o u r n a l
of Surgery
Regulation of Pancreatic Polypeptide Secretion
Previous studies on the immunoreactive pancreatic polypeptide response to hyperglycemia have been contradictory [7,13,16-24]. In response to 16.7 mM glucose perfusion, immunoreactive pancreatic polypeptide secretion was modestly but significantly inhibited, glucagon release was significantly suppressed, and insulin and somatostatin release were strongly stimulated [11,14]. Although glucose alone appears to have only a modest role in the release of pancreatic polypeptide, hyperglycemia may mediate the pancreatic polypeptide response to other stimuli either through neural, endocrine, or paracrine mechanisms. When perfused alone, or in combination with other agents, glucose exerted a consistently inhibitory effect on immunoreactive pancreatic polypeptide secretion. This occurred during concomitant increases in the secretion Of insulin and somatostatin, which suggests that/3-cell secretion, b-cell secretion, or both may serve as a tonic inhibitor of the pancreatic polypeptide cell responses to stimuli [10,14]. Although our data cannot identify which of the islet cells is the principal paracrine mediator of pancreatic polypeptide release, our findings confirm the parallel responses of glucagon and pancreatic polypeptide release and the reciprocal relationship of pancreatic polypeptide secretion to that of insulin and somatostatin. The immunoreactive pancreatic polypeptide response to feeding and to oral but not intravenous glucose suggests that pancreatic polypeptide release is mediated by one or more enteric hormonal agents. Cholecystokinin, secretin, gastrin, vasoactive intestinal peptide, bombesin, and gastric inhibitory polypeptide have all been implicated as possible enteric secretagogues of pancreatic polypeptide, bu t only cho!ecystokinin has been demonstrated to have a convincing stimulatory effect when infused in viv0 [6,7,25]. In our study, perfusion with 5 ng/ml gastric inhibitory polypeptide stimulated immunoreactive pancreatic polypeptide secretion significantly, which is consistent with the stimulatory effects of gastric inhibitory polypeptide seen during in situ canine pancreas perfusion [7]. Therefore, our data indicate that gastric inhibitory polypeptide may serve as a secretagogue for pancreatic polypeptide. The splanchnic innervation of the pancreas has been shown to play an important regulatory role in islet cell secretion [26]. Pancreatic polypeptide release was augmented by bipolar electrical stimulation of the sp!anchnic neural bundle alone or during perfusion with gastric inhibitory polypeptide during hyperglycemia, possibly by direct stimulation of the pancreatic polypeptide cell or by an indirect influence on other islet cell secretions. In our previous studies [11], splanchnic neural stimulation during hyperglycemia with or without gastric inhibitory polypeptide resulted in augmented glucagon
Volume 155, January 1988
secretion, a response that parallels that of the pan9creatic polypeptide cell, whereas insulin and somatostatin secretion were inhibited [11,14]. Perfusion with a low physiologic concentration of insulin consistently resulted in an enhancement of immunoreactive pancreatic polypeptide secretion, either in the basal state or during concomitant perfusion with gastric inhibitory polypeptide, 16.7 mM glucose solution, or both, In the presence of 20 ~U/ ml insulin, insulin secretion of 2,000 ~U/ml and somatostatin secretion were significantly reduced, and glucagon secretion was strongly stimulated [10,14]. These findings confirm that the endocrine pancreas is especially sensitive to ambient levels of insulin, and suggest that paracrine mechanisms play a strong role in the regulation of islet cell secretion [27,28]. Although it is possible to ascribe the changes in pancreatic polypeptide secretion to a direct effect of infused insulin o n t h e pancreatic polypeptide cell, a recent morphologic study by Bonner-Weir and Orci [29] supports the concept of a discreet compartmentalization of the islet, with arterial inflow directed preferentially to the central/~-cell core followed by perfusion with the fl-cell products to the peripherally located a, 6, and pancreatic polypeptide cells. Our results seen in the isolated perfused human pancreas support such a theory. When the level of insulin secretion is decreased, as during splanchnic nerve stimulation or with low levels of inflow insulin, the pancreatic polypeptide cell responds by increasing its secretion of pancreatic polypeptide. This effect could represent a direct effect of insulin on the pancreatic polypeptide cell Or may be mediated by an indirect effect of insulin on ()ther islet cells, such as the somatostatin-secreting 5 cell, which may in turn regulate pancreatic polypeptide cell function. Further studies are required to clarify these mechanisms, but our data are nonetheless consistent with a centrifugal Pattern of blood flow within the islet and with the essentiality of the relationship of/3 cells to pancreatic polypeptide cell function. Summary The isolated perfused human pancreas was used as a model to assess factors mediating the pancreatic polypeptide cell response to glucose, insulin, gastric inhibitory polypeptide, and splanchnic nerve stimulation. Pancreases obtained from 18 cadaveric organ donors were isolated and perfused by way of the splenic artery utilizing a Krebs bicarbonate buffer in a single-pass perfusion system. Hormonal stimulation and inhibition of pancreatic polypeptide secretion were assessed, as was the influence of direct electrical stimulation of celiac neural fibers innervating the pancreas. In this in vitro human model, pancreatic polypeptide cell secretion was inhibited by hyperglycemia, although the presence of gastric inhibitory polypeptide augmented the
67
Brunicardi et al
pancreatic polypeptide cell response. Perfusion with low levels of insulin and splanchnic nerve stimulation augmented the response of the pancreatic polypeptide cell to hyperglycemia and gastric inhibitory polypeptide. Since the immunoreactive pancreatic polypeptide response was augmented when insulin and somatostatin release was inhibited by perfusion insulin or nerve stimulation, we conclude that the pancreatic polypeptide cell is regulated by the ambient degree of somatostatin release, insulin release, or both. These findings support a centrifugal pattern of intraislet blood flow. References 1. Kimmet JR, Pollock HG, Hazelwood RL. Isolation and characterization of chicken insulin. Endocrinol 1968; 83: 132330. 2. Lin TM, Chance RE. Spectrum gastrointestinal actions of a new bovine pancreas polypeptide. Gastroenterology 1972; 62: 852. 3. Hazelwood RL. Synthesis, storage, secretion, and significance of panci'eatlc polypeptide in vertebrates. In: Cooperstein SJ, Watkins D, eds. The islets of Langerhans. Biochemistry, physiology , and pathology; New York, Academic Press, 1981: 275-318. 4. Floyd JC Jr. Human pancreatic polypeptide. In: Buchanan KD, ed. Clinics in endocrinology and metabolism. Vol. 8. Philadelphia, WB Saunders, 1977: 379-94. 5. Stefan Y, Orci L, Malaisse-Legae F, Perrelet A, Patel Y, Unger RH. Quantitation of endocrine cell content in the pancreas of nondiabetic and diabetic humans. Diabetes 1982; 31! 694-700. 6. Gingerich RL. Survey of potential pancreatic polypeptide Secretagogues !abstr). Diabetes 1977; 26: 375. 7. Adrian TE, Bloom SR, Hermanson K, Iverson d. Pancreatic polypeptide, glucagon and insulin secretion from the isolated perfused canine pancreas. Diabetologia 1978; 14:413= 7. 8. Bloom SR, Adrian TE, Greenburg GR, et al. Effects of pancreatic POlypeptide infusion in man (abstr). Gastroenterology 1978; 74: 1012. 9. Sun YS, Brunicardi FC, Druck P, et al. Reversal of abnormal glucose metabolism in chronic pancreatitis by administration of pancreatic polypeptide. Am J Surg 1986; 151: 13040. 10. Brunicardi FC, Goulet RJ, Sun YS, Berlin SA, Elahi D, Andersen DK. Neural and hormonal regulation of insulin and glucagon secretion in the isolated perfused human pancreas. Surg Forum 1984; 35: 214-7. 11. Brunicardi FC, Sun YS, DrUck P, Goulet RJ, Elahi D, Andersen DK. Splanchnic neural regulation of insulin and glucagon secretion in the isolated perfused human pancreas. Am J Surg !987; 153: 34-40. 12. Kelly WD, Lillihei RC, Merkel FK, Idezuki Y, Goetz FC. AIIotransplantation of the pancreas and duodenum al0ng with the kidney in diabetic nephropathy. Surgery 1967; 61: 82737. 13. Gingerich RL, Lacy PE, Chance RE, Johnson MG. Regional pancreatic concentration and in vitro secretion of canine pancreatic polypeptide, insulin, and glucagon. Diabetes 1978; 27: 96-101. 14. Brunicardi FC, Druck P, Sun YS, Elahi D, Yamada T, Andersen DK: Neural and hormonal regulation of s0matostatin release in the isolated perfused human pancreas (abstr). Gastroenterology 1986; 90: 1360. 15. Gersell DJ, Gingerich RL, Greider MH. Regional distribution and concentration of pancreatic polypeptide in the human and canine pancreas. Diabetes 1979; 28:11-5.
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16. Weir GC, Samols E, Loo S, Patel YC, Gabbay KH. Somatostatin and pancreatic polypeptide secretion. Diabetes 1979; 28: 35-40. 17. Schusdziarra V, Stapelfe!dt W, Klier M, Mair V, Pfeiffer EF. Effect of physiological increments of blood glucose on plasma somatostatin and pancreatic polypeptide levels in dogs. Regul Pept 1981; 2:211-8. 18. Marco J, Hedo JA, Villanueva ME Control of pancreatic polypeptide secretion by glucose in man. J Clin Endocrinol Metabol 1978; 46: 140-5. 19. Villaneuva ML, Hedo JA, Castillo,Olivares J, Marco J. Effect of exogenous hyperglycemia on human pancreatic polypeptide secretion. Diabetologia 1978; 15: 278-9. 20. Prinz RA, El Sabbagh H, Adrian TE, et al. Neural regulation of pancreatic polypeptide release. Surgery 1983; 94:1011-7. 21. Adrian TE, Bloom SR, Besterman HS, Barnes AJ, Cooke TJ, Faber RG. Mechanism of pancreatic polypeptide release in man. Lancet 1977; 1: 161-3. 22. Floyd JC, Fajans SS, Pek S, Chance RE. A newly recognized pancreatic polypeptide: plasma levels in health and disease. Recent Prog Horm Res 1977; 33: 519-70. 23. Tsuda K, Seino Y, Mori K. Effect of truncal vagotomy on pancreatic polypeptide response after intravenous glucose administration. Regul Pept 1981; 1: 347-52. 24. Sire AA, Vinik AI, VanTonder SV. Pancreatic polypeptide responses to oral and intravenous glucose in man. Am J Gastroenterol 1979; 71: 183-5. 25. Lonovics J, Guzman S, Devitt P, et al. Release of pancreatic polypeptide in humans by infusion of cholecystokinin. Gastroenterology 1980; 79: 817:-22. 26. WoodsSC, Porte D Jr. Neural control of the endocrine pancreas. Physiol Rev 1974; 54: 596-619. 27. Elahi D, Nagulesparan M, Hershcopf R, et al. Feedback inhibition of insulin secretion by insulin. Relation to the hyperinsulinemia of obesity: N Engl J Med 1982; 306:1196-202. 28. Rappaport AM, Ohira S, Coddling JA, et al. Effects on insulin output and on pancreatic blood flow of exogenous insulin infusion into an in situ isolated portion of the pancreas. Endocrinology 1972; 91: 168-76. 29. Bonner-Weir S, Orci L. New perspectives on the microvasculature of the islets of Langerhans in the rat. Diabetes 1982; 31: 883-9.
Discussion
Aaron S. Fink (Los Angeles, CA): Dr. Brunicardi, what effect did splanchnic nerve stimulation alone, in the presence of a physiologic level of glucose, have on pancreatic polypeptide release? Furthermore, were you able to either enhance or block any of the effects of your tested secretogogues, by adding cholinergic blockers such as atropine? I-Iaile T. Debas (San Francisco, CA): Dr. Brunicardi, it is very imaginative to use organs from the cadaveric organ donors to perform appropriate studies in human subjects. I suspect it is the only way we are going to be able to confirm observations made in animal studies. At this level of nerve stimulation there are mixed nerves (cholinergic, adrenergic, and peptidegeric). Did you consider selective blocking to stimulate and define exact mechanisms? I am a bit confused by your results showing that hyperglycemia, when added to gastric inhibitory polypeptide stimulation, inhibits pancreatic polypeptide release. I believe your laboratory first described that gastric inhibitory polypeptide requires a certain level of hyperglycemia to release insulin. You showed insulin released as pancreatic polypeptide. Therefore, in the experiments in which you combined hyperglycemia with gastric inhibitory polypep-
The American Journal of Surgery
Regulation of Pancreatic Polypeptide Secretion
tide, you should see a release of insulin. So the results should be to augment, not inhibit. Finally, most of the pancreatic polypeptide cells are in the head and the uncinate process of the pancreas. Do you think these studies reflect the actual regulation of pancreatic polypeptide, which is mostly in the other part of the gland? James R. Upp, Jr. (Galveston, TX): Dr. Brunicardi, in your study basal pancreatic polypeptide levels in the older patients were elevated. Did you see increased stimulation of pancreatic polypeptide in those patients? Also, the response to gastric inhibitory polypeptide seems to be different in vivo than in your in vitro model. What do you think the different mechanisms are?
Gerald M. Larson (Louisville, KY): Dr. Brunicardi, you have clearly shown that nerve stimulation alters the release of several of these peptides, and we have also shown that surgical sympathectomy leads to changes in pancreatic polypeptide levels in the dog. What is the implication of this for the transplanted pancreas? In most transplants all nerves to the pancreas are cut. What significance does that have for the endocrine function of the pancreas down the road? Certainly insulin metabolism seems to be improved, but just what impact would you predict that denervation of the pancreas would have on hormone balance? F. Charles Brunicardi (closing): Dr. Fink, in additional studies we have shown that splanchnic nerve stimulation as well as perfusion with insulin alone during euglycemia both augment pancreatic polypeptide release. In an extensive study in this model in which we have looked at the neural regulation of all four islet hormones, cholinergic stimulation increased pancreatic polypeptide secretion, whereas a-adrenergic cell stimulation inhibited pancreatic polypeptide secretion and/~-cell stimulation had very little effect. Drs. Bonnet-Weir and Orci have shown that the arteriole penetrates the ~-cell mass first. Therefore, secretogogues reach the B cell first and its produces are carried peripherally to the outer cells, which are the a, ~, and pancreatic polypeptide cells. Therefore, Dr. Debas, our data are consistent with the hypothesis that insulin secretion actually inhibits pancreatic polypeptide secretion. When you administer gastric inhibitory polypeptide dur-
Volume 155, January 1988
ing hyperglycemia, you expect a marked increase of insulin release. Pancreatic polypeptide release should be inhibited compared with the effects seen during perfusion of gastric inhibitory polypeptide with euglycemia alone, and this is precisely what we saw. The response seen with somatostatin release paralleled that of insulin. Therefore, fl-cell secretion influences somatostatin release, both of which in turn influence the a and the pancreatic polypeptide cells. We were pleasantly surprised to find a pancreatic polypeptide response in this preparation, since we used only the body and tail of the pancreas. It is true that 90 to 95 percent of pancreatic polypeptide cell mass is located in the head of the pancreas, with 5 to 10 percent remaining in the tail. We found that in comparison to levels of the other islet hormones, the pancreatic polypeptide levels were relatively low. I can't tell you whether the tail of the pancreas differs from the head of the pancreas in terms of intraislet interactions. Dr. Upp, in vivo studies indicate that basal pancreatic polypeptide levels increase with age. When we compared the age of the donors with the basal pancreatic polypeptide response, we found a highly significant correlation. This seems to indicate that the increase in pancreatic polypeptide secretion one sees with aging is due to increased pancreatic polypeptide secretion and not decreased pancreatic polypeptide clearance. You asked about the effects of gastric inhibitory polypeptide in vivo versus in vitro. As we know from the studies from your own lab, there have been many possible enteric secretagogues that have been suggested for pancreatic polypeptide release, including cholecystokinin. In our model, the insulin and somatostatin responses to gastric inhibitory polypeptide were consistent with the findings of in vivo studies. However, for glucagon and pancreatic polypeptide, the stimulation by gastric inhibitory polypeptide was contradictory to in vivo results. The fact that our glands were denervated and that there was a lack of inflow hormones in the perfusate may have contributed to this finding. Dr. Larson, our findings may be relevant to the function of the transplanted pancreas. There are very few groups who are looking at all four islet hormones in the transplanted pancreas. We suspect that although the transplanted pancreas responds to stimuli, it lacks the finetuned neural regulation of the in situ pancreas.
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