Intrapancreatic
Islet Transplantation Albert0
in Experimental
Diabetes
in the Rat
Hayek and Gillian M. Beattie
Pancreatic
islets have been transplanted successfully to a variety of organs except the pancreas. Since this organ is the most physiologic recipient for islets in rodents with chemically induced diabetes, we have placed neonatal islets intrapancreatically in highly inbred Lewis rats with streptozotocin (ST) diabetes. Transplantation of 2,000 neonatal islets into the pancreas of diabetic rats reversed diabetes within 2 weeks in all of the treated rats (seven of seven). The same success rate was obtained using the same number of islets placed under the kidney capsule in 11 recipients. Although cured of their diabetes, rats in both groups with inadequate islet-cell mass were not able to produce glucose tolerance tests equal to those observed in normal animals. Higher insulin levels were measured in the rats receiving islets under the kidney capsule, as compared with those placed intrapancreatically. In summary, syngeneic islet transplants to the pancreas of ST-diabetic rats are successful in reversing the diabetic state. This model may be useful to the study of islet function after islet transplants into their natural environment. Copyright 0 1992 by W.B. Saunders Company
HAVE PREVIOUSLY SHOWN that fetal and neonatal islets isolated and grown in culture are an excellent source of endocrine replacement tissue in rats with streptozotocin (ST)-induced diabetes.’ Data from those initial studies indicated that a greater number of perinatal islets as compared with adult islets is necessary to reverse experimental diabetes.‘,3 Subsequently, we demonstrated that strict metabolic control of diabetic rats led to a decrease in the number of neonatal islets required for successful transplantation.” However, normal glucose tolerance tests were only observed with the B-cell mass produced by transplanting at least 3,000 neonatal islets5 to the spleen and kidney. In the experiments described below, we investigated whether it was possible to place syngeneic islets into the pancreas of ST-diabetic rats and, if successful, whether this anatomically correct placement, even in the face of a diminished B-cell mass, would lead to glucose and insulin responses equal to those observed in normal rats and rats cured of diabetes by transplantation of islets under the kidney capsule.
w
MATERIALS
AND METHODS
I\nimals used in these experiments
were highly inbred Lewis rats, originally obtained from the National Institutes of Health Grantee Reimbursement Program through the Charles River Breeding Laboratories (Charles River, MA). Neonatal islets were isolated according to methods previously described.4,h Transplant recipients were male rats weighing 240 to 300 g, made diabetic by the intravenous (IV) administration of 65 mg/kg ST at least 2 weeks before transplantation. Only rats with nonfasting blood glucose levels higher than 350 mg/dL were used for these experiments. Following pentobarbital anesthesia (40 mg/kg, intraperitoneally [IP]). a midline or lateral abdominal incision was made and 2,000 neonatal islets were injected into the pancreas with a 25-guage butterfly infusion set (n = 7). For this procedure, islets were suspended in 200 uL Hanks solution containing blue agarose beads (BioRad Laboratories, Richmond, CA) to aid in the localization of transplanted islets within the pancreatic parenchyma. For comparison, a similar number of neonatal islets was transplanted under the kidney capsule as previously described5 (n = 11). Blood was withdrawn from the tail vein for weekly glucose measurements using a portable glucose meter (Lifescan, Mountain View, CA). For the glucose tolerance tests 1 month after the return to normoglycemia, fasted rats were bled for measurements of serum glucose and insulin at 0. 15. 30, and 60 minutes after an 1P glucose injection of 1.5 mgig body weight. During the test, the rats
Mefabolism,
Vol 41, No 12 (December), 1992: pp 1367-1369
were anesthetized by inhalation of metofane (Pitman-Moore, Mundelein, IL). Following the glucose tolerance tests, three of the animals that received the intrapancreatic islet transplant were killed. Visual inspection of their excised pancreas easily showed the blue beads. Portions of the pancreas where the beads were visualized were fixed in 10% neutral buffered Formalin, embedded in paraffin, and serially sectioned at 10 p,rn for histological analysis using hematoxylin and eosin staining. For the glucose tolerance tests, serum glucose was assayed using a glucose analyzer (Beckman Instruments, Fullerton, CA). Serum insulin was assayed using a solid-phase radioimmunoassay (DPC, Los Angeles, CA), as previously reported.’ Data were analyzed by Student’s t test for unpaired data using the Crunch statistical package (Jandel, San Francisco, CA). RESULTS
All animals receiving islets placed into the pancreatic parenchyma were normoglycemic within 2 weeks after the transplantation procedure. In rats with subcapsular islet transplants, normoglycemia was attained between 2 and 4 weeks after transplantation. The IP glucose tolerance tests (Fig 1) in animals that received transplanted islets showed that glucose and insulin responses following a glucose challenge were not equal to those observed in age-matched normal rats. Thus, 2,000 neonatal islets transplanted to either the kidney or pancreas did not produce normal insulin or glucose values compared with age-matched normal rats. Furthermore, it was apparent that the islets transplanted under the kidney capsule were more efficient than those transplanted in the pancreas, at both decreasing the peakglucose value (P < .02 at 30 and 60 minutes) and releasing insulin (P < .02 at 0 and 15 minutes). Histological examination of portions of the excised pancreas showed well-preserved islets placed near the agarose
From The Lucy Thome Whittier Children’s Cenrer, The Whittier Institute for Diabetes and Endocrinology, La Jollu, CA. Supported in purt by National Institute of Diabetes and Digestive and Kidney Diseases Grant No. DK39087 and grants from the Stem Foundation and the Deutz Fami&. Address reprint requests to Albert0 Hawk. MD, The Whittier Institute. 9894 Genesee Ave, La Jolla. CA 92037. Copyright 8 1992 by W.B. Saunders Cornpan\ 0026-0495/92/~112-01~~0.~.~0l0
1367
1368
HAYEK AND BEAT-TIE
Kidney 100 -
Pancreas
60 -
L”
.
;
1’5
3'0
6’0
I 15
I 30
I 60
300 -
200 -
100 .
I 0
Time
(min)
Fig 1. Glucose tolerance tests in normal (n = 8) rats and in rats receiving islet transplants under the kidney capsule (n = 11) or intrapancreatically (n = 7; mean f SE). *P c .02 for values in animals receiving islet transplants to the kidney compared with the pancreas.
beads (Fig 2) and surrounded by exocrine tissue, in much the same way native islets look in the normal pancreas. Positive insulin-secretory cells were identified solely in islets near agarose beads (not shown). The remaining transplanted animals maintained normoglycemia until they were killed more than 3 months after the transplant.
pancreatic enzymes are activated only in the intestinal lumen following contact with other brush-border enzymes.8 Because of the extensive experience reported with pancreas transplantation in humans9 the issue of islet placement becomes relevant in view of the reported metabolic effects of portal versus systemic venous drainage with whole-organ transplants. lo In these situations, individuals with pancreas allografts with systemic venous drainage exhibit basal and stimulated hyperinsulinemia due to the bypass of the first-pass hepatic insulin clearance. In this respect, data from the glucose tolerance tests become relevant (Fig 1). The higher insulin concentrations were measured in the normal rat, probably a reflection of a normal insulin output from an intact p-cell mass. In animals with decreased but comparable islet-cell mass, higher insulin levels were measured in rats receiving islets in the kidney, mimicking the situation observed in humans with systemic insulin drainage from the transplanted pancreas.‘* However, these higher insulin concentrations corresponded with glucose values closer to normal than those found in animals receiving intrapancreatic islet transplants. The data in this report indicate that intrapancreatic transplantation of 2,000 islets, representing approximately 50% of the total islet mass present in a normal rat,” is successful in reversing diabetes, but not sufficient to produce glucose or insulin responses similar to those observed in normal rats following a glucose challenge. An earlier report by Georgakakis12 showed partial improvement of diabetes following intrapancreatic transplantation of 200 to 400 islets. This information is in accordance with the results presented here, and emphasizes the importance of an adequate islet-cell mass and the purity of the islet preparation in islet transplantation. However, the number of transplanted islets by itself cannot completely account for a successful outcome; we have recently published data demonstrating that the same number of islets (2,000) injected intravenously and lodged in the lungs not only reversed chemical diabetes in rats, but also led to glucose tolerance tests indistinguishable from those performed in normal
DISCUSSION
This study demonstrates that it is feasible to transplant islets directly and successfully into the pancreatic parenchyma, in this way obtaining almost normal anatomical placement of the missing p-cell population in a diabetic animal. Although the most important reason for using the pancreas as the recipient of the islet transplant is to ensure insulin delivery directly into the liver through portal vein drainage, other possible advantages could be the influence of intestinal or exocrine paracrine factors in pancreatic islet hormone secretion. The possibility that injection of islets into the pancreas could lead to destruction of transplanted islets by activation of digestive enzymes did not occur, as shown by the normal function of the islets in reversing the diabetic process. This is probably due to the fact that
Fig 2. Microphotograph of a transplanted islet (arrow) lying between a blue agarose bead (arrowhead) and adjacent exocrine tissue. (Original magnification x200.)
INTRAPANCREATIC
1369
ISLET TRANSPLANTATION
animals.13 Perhaps islet engraftment also plays a critical role in the ultimate function of the graft, and this may be influenced by local factors within the recipient organ. In summary, we have presented data indicating that intrapancreatic syngeneic islet transplants are successful in
mental model of islet transplantation normal physiology.
reversing
the manuscript.
diabetes
in rats. This approach
offers an experi-
that closely parallels
ACKNOWLEDGMENT We are
grateful to Vicki Estey for assistance with preparation of
REFERENCES
1. Hayek A, Guardian C: Hormone release, islet yield and transplantation of fetal and neonatal rat dorsal and ventral pancreatic islets. Diabetes 35:1189-1190, 1986 2. Koncz L, Davidoff F, DeLellis RA, et al: Quantitative aspects of the metabolic response to pancreatic islet transplantation in rats with severe ketotic diabetes. Metabolism 25:147-156, 1976 3. Kemp CB, Knight MJ. &harp DW, et al: Transplantation of isolated pancreatic islets into the portal vein of diabetic rats. Nature 244:447-450, 1973 4. Hayek A, Lopez AD, Beattie GM: Decrease in the number of neonatal islets required for successful transplantation by strict metabolic control of diabetic rats. Transplantation 45:940-942. 1988 5. Hayek A, Lopez AD, Beattie GM: Factors influencing islet transplantation-Number. location, and metabolic control. Transplantation 49:224-225, 1990 6. Hellerstrom C, Lewis NJ, Borg H, et al: Method for largescale isolation of pancreatic islets by tissue-culture of fetal rat pancreas. Diabetes 28766-769. 1979
7. Beattie GM, Lappi DA, Baird A, et al: Functional impact of attachment and purification in the short term culture of human pancreatic islets. J Clin Endocrinol Metab 73:93-98, 1991 8. Leach SD, Modlin IM, Scheele GA, et al: Intracellular activation of digestive zymogens in rat pancreatic acini. J Clin Invest 87:362-366,199l 9. Robertson RP: Pancreas transplantation in humans with diabetes mellitus. Diabetes 40~1085-1089, 1991 10. Diem P, Abid M. Redmon JB, et al: Systemic venous drainage of pancreas allografts as independent cause of hyperinsulinemia in type I diabetic recipients. Diabetes 39:534-540. 1990 11. Jansson L, Hellerstrom C: A rapid method of visualizing the pancreatic islets for studies of islet capillary blood flow using nonradioactive microspheres. Acta Physiol Stand 113:371-374,198l 12. Georgakakis A: Experimental pancreatic islet transplantation. Ann R Coll Surg Engl59:231-235, 1977 13. Hayek A. Beattie GM: Reversal of experimental diabetes by injection of syngeneic islets into peripheral veins. Cell Transplantation 1:83-85, 1992
Islet Transplantation Albert0
in Experimental
Diabetes
in the Rat
Hayek and Gillian M. Beattie
Pancreatic
islets have been transplanted successfully to a variety of organs except the pancreas. Since this organ is the most physiologic recipient for islets in rodents with chemically induced diabetes, we have placed neonatal islets intrapancreatically in highly inbred Lewis rats with streptozotocin (ST) diabetes. Transplantation of 2,000 neonatal islets into the pancreas of diabetic rats reversed diabetes within 2 weeks in all of the treated rats (seven of seven). The same success rate was obtained using the same number of islets placed under the kidney capsule in 11 recipients. Although cured of their diabetes, rats in both groups with inadequate islet-cell mass were not able to produce glucose tolerance tests equal to those observed in normal animals. Higher insulin levels were measured in the rats receiving islets under the kidney capsule, as compared with those placed intrapancreatically. In summary, syngeneic islet transplants to the pancreas of ST-diabetic rats are successful in reversing the diabetic state. This model may be useful to the study of islet function after islet transplants into their natural environment. Copyright 0 1992 by W.B. Saunders Company
HAVE PREVIOUSLY SHOWN that fetal and neonatal islets isolated and grown in culture are an excellent source of endocrine replacement tissue in rats with streptozotocin (ST)-induced diabetes.’ Data from those initial studies indicated that a greater number of perinatal islets as compared with adult islets is necessary to reverse experimental diabetes.‘,3 Subsequently, we demonstrated that strict metabolic control of diabetic rats led to a decrease in the number of neonatal islets required for successful transplantation.” However, normal glucose tolerance tests were only observed with the B-cell mass produced by transplanting at least 3,000 neonatal islets5 to the spleen and kidney. In the experiments described below, we investigated whether it was possible to place syngeneic islets into the pancreas of ST-diabetic rats and, if successful, whether this anatomically correct placement, even in the face of a diminished B-cell mass, would lead to glucose and insulin responses equal to those observed in normal rats and rats cured of diabetes by transplantation of islets under the kidney capsule.
w
MATERIALS
AND METHODS
I\nimals used in these experiments
were highly inbred Lewis rats, originally obtained from the National Institutes of Health Grantee Reimbursement Program through the Charles River Breeding Laboratories (Charles River, MA). Neonatal islets were isolated according to methods previously described.4,h Transplant recipients were male rats weighing 240 to 300 g, made diabetic by the intravenous (IV) administration of 65 mg/kg ST at least 2 weeks before transplantation. Only rats with nonfasting blood glucose levels higher than 350 mg/dL were used for these experiments. Following pentobarbital anesthesia (40 mg/kg, intraperitoneally [IP]). a midline or lateral abdominal incision was made and 2,000 neonatal islets were injected into the pancreas with a 25-guage butterfly infusion set (n = 7). For this procedure, islets were suspended in 200 uL Hanks solution containing blue agarose beads (BioRad Laboratories, Richmond, CA) to aid in the localization of transplanted islets within the pancreatic parenchyma. For comparison, a similar number of neonatal islets was transplanted under the kidney capsule as previously described5 (n = 11). Blood was withdrawn from the tail vein for weekly glucose measurements using a portable glucose meter (Lifescan, Mountain View, CA). For the glucose tolerance tests 1 month after the return to normoglycemia, fasted rats were bled for measurements of serum glucose and insulin at 0. 15. 30, and 60 minutes after an 1P glucose injection of 1.5 mgig body weight. During the test, the rats
Mefabolism,
Vol 41, No 12 (December), 1992: pp 1367-1369
were anesthetized by inhalation of metofane (Pitman-Moore, Mundelein, IL). Following the glucose tolerance tests, three of the animals that received the intrapancreatic islet transplant were killed. Visual inspection of their excised pancreas easily showed the blue beads. Portions of the pancreas where the beads were visualized were fixed in 10% neutral buffered Formalin, embedded in paraffin, and serially sectioned at 10 p,rn for histological analysis using hematoxylin and eosin staining. For the glucose tolerance tests, serum glucose was assayed using a glucose analyzer (Beckman Instruments, Fullerton, CA). Serum insulin was assayed using a solid-phase radioimmunoassay (DPC, Los Angeles, CA), as previously reported.’ Data were analyzed by Student’s t test for unpaired data using the Crunch statistical package (Jandel, San Francisco, CA). RESULTS
All animals receiving islets placed into the pancreatic parenchyma were normoglycemic within 2 weeks after the transplantation procedure. In rats with subcapsular islet transplants, normoglycemia was attained between 2 and 4 weeks after transplantation. The IP glucose tolerance tests (Fig 1) in animals that received transplanted islets showed that glucose and insulin responses following a glucose challenge were not equal to those observed in age-matched normal rats. Thus, 2,000 neonatal islets transplanted to either the kidney or pancreas did not produce normal insulin or glucose values compared with age-matched normal rats. Furthermore, it was apparent that the islets transplanted under the kidney capsule were more efficient than those transplanted in the pancreas, at both decreasing the peakglucose value (P < .02 at 30 and 60 minutes) and releasing insulin (P < .02 at 0 and 15 minutes). Histological examination of portions of the excised pancreas showed well-preserved islets placed near the agarose
From The Lucy Thome Whittier Children’s Cenrer, The Whittier Institute for Diabetes and Endocrinology, La Jollu, CA. Supported in purt by National Institute of Diabetes and Digestive and Kidney Diseases Grant No. DK39087 and grants from the Stem Foundation and the Deutz Fami&. Address reprint requests to Albert0 Hawk. MD, The Whittier Institute. 9894 Genesee Ave, La Jolla. CA 92037. Copyright 8 1992 by W.B. Saunders Cornpan\ 0026-0495/92/~112-01~~0.~.~0l0
1367
1368
HAYEK AND BEAT-TIE
Kidney 100 -
Pancreas
60 -
L”
.
;
1’5
3'0
6’0
I 15
I 30
I 60
300 -
200 -
100 .
I 0
Time
(min)
Fig 1. Glucose tolerance tests in normal (n = 8) rats and in rats receiving islet transplants under the kidney capsule (n = 11) or intrapancreatically (n = 7; mean f SE). *P c .02 for values in animals receiving islet transplants to the kidney compared with the pancreas.
beads (Fig 2) and surrounded by exocrine tissue, in much the same way native islets look in the normal pancreas. Positive insulin-secretory cells were identified solely in islets near agarose beads (not shown). The remaining transplanted animals maintained normoglycemia until they were killed more than 3 months after the transplant.
pancreatic enzymes are activated only in the intestinal lumen following contact with other brush-border enzymes.8 Because of the extensive experience reported with pancreas transplantation in humans9 the issue of islet placement becomes relevant in view of the reported metabolic effects of portal versus systemic venous drainage with whole-organ transplants. lo In these situations, individuals with pancreas allografts with systemic venous drainage exhibit basal and stimulated hyperinsulinemia due to the bypass of the first-pass hepatic insulin clearance. In this respect, data from the glucose tolerance tests become relevant (Fig 1). The higher insulin concentrations were measured in the normal rat, probably a reflection of a normal insulin output from an intact p-cell mass. In animals with decreased but comparable islet-cell mass, higher insulin levels were measured in rats receiving islets in the kidney, mimicking the situation observed in humans with systemic insulin drainage from the transplanted pancreas.‘* However, these higher insulin concentrations corresponded with glucose values closer to normal than those found in animals receiving intrapancreatic islet transplants. The data in this report indicate that intrapancreatic transplantation of 2,000 islets, representing approximately 50% of the total islet mass present in a normal rat,” is successful in reversing diabetes, but not sufficient to produce glucose or insulin responses similar to those observed in normal rats following a glucose challenge. An earlier report by Georgakakis12 showed partial improvement of diabetes following intrapancreatic transplantation of 200 to 400 islets. This information is in accordance with the results presented here, and emphasizes the importance of an adequate islet-cell mass and the purity of the islet preparation in islet transplantation. However, the number of transplanted islets by itself cannot completely account for a successful outcome; we have recently published data demonstrating that the same number of islets (2,000) injected intravenously and lodged in the lungs not only reversed chemical diabetes in rats, but also led to glucose tolerance tests indistinguishable from those performed in normal
DISCUSSION
This study demonstrates that it is feasible to transplant islets directly and successfully into the pancreatic parenchyma, in this way obtaining almost normal anatomical placement of the missing p-cell population in a diabetic animal. Although the most important reason for using the pancreas as the recipient of the islet transplant is to ensure insulin delivery directly into the liver through portal vein drainage, other possible advantages could be the influence of intestinal or exocrine paracrine factors in pancreatic islet hormone secretion. The possibility that injection of islets into the pancreas could lead to destruction of transplanted islets by activation of digestive enzymes did not occur, as shown by the normal function of the islets in reversing the diabetic process. This is probably due to the fact that
Fig 2. Microphotograph of a transplanted islet (arrow) lying between a blue agarose bead (arrowhead) and adjacent exocrine tissue. (Original magnification x200.)
INTRAPANCREATIC
1369
ISLET TRANSPLANTATION
animals.13 Perhaps islet engraftment also plays a critical role in the ultimate function of the graft, and this may be influenced by local factors within the recipient organ. In summary, we have presented data indicating that intrapancreatic syngeneic islet transplants are successful in
mental model of islet transplantation normal physiology.
reversing
the manuscript.
diabetes
in rats. This approach
offers an experi-
that closely parallels
ACKNOWLEDGMENT We are
grateful to Vicki Estey for assistance with preparation of
REFERENCES
1. Hayek A, Guardian C: Hormone release, islet yield and transplantation of fetal and neonatal rat dorsal and ventral pancreatic islets. Diabetes 35:1189-1190, 1986 2. Koncz L, Davidoff F, DeLellis RA, et al: Quantitative aspects of the metabolic response to pancreatic islet transplantation in rats with severe ketotic diabetes. Metabolism 25:147-156, 1976 3. Kemp CB, Knight MJ. &harp DW, et al: Transplantation of isolated pancreatic islets into the portal vein of diabetic rats. Nature 244:447-450, 1973 4. Hayek A, Lopez AD, Beattie GM: Decrease in the number of neonatal islets required for successful transplantation by strict metabolic control of diabetic rats. Transplantation 45:940-942. 1988 5. Hayek A, Lopez AD, Beattie GM: Factors influencing islet transplantation-Number. location, and metabolic control. Transplantation 49:224-225, 1990 6. Hellerstrom C, Lewis NJ, Borg H, et al: Method for largescale isolation of pancreatic islets by tissue-culture of fetal rat pancreas. Diabetes 28766-769. 1979
7. Beattie GM, Lappi DA, Baird A, et al: Functional impact of attachment and purification in the short term culture of human pancreatic islets. J Clin Endocrinol Metab 73:93-98, 1991 8. Leach SD, Modlin IM, Scheele GA, et al: Intracellular activation of digestive zymogens in rat pancreatic acini. J Clin Invest 87:362-366,199l 9. Robertson RP: Pancreas transplantation in humans with diabetes mellitus. Diabetes 40~1085-1089, 1991 10. Diem P, Abid M. Redmon JB, et al: Systemic venous drainage of pancreas allografts as independent cause of hyperinsulinemia in type I diabetic recipients. Diabetes 39:534-540. 1990 11. Jansson L, Hellerstrom C: A rapid method of visualizing the pancreatic islets for studies of islet capillary blood flow using nonradioactive microspheres. Acta Physiol Stand 113:371-374,198l 12. Georgakakis A: Experimental pancreatic islet transplantation. Ann R Coll Surg Engl59:231-235, 1977 13. Hayek A. Beattie GM: Reversal of experimental diabetes by injection of syngeneic islets into peripheral veins. Cell Transplantation 1:83-85, 1992
