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Effect of gliclazide on islet transplants
Effect of Gliclazide on Islet Transplants J.-H. Juang, C.-H. Kuo, and B.R.-S. Hsu
I
SLET TRANSPLANTATION has produced insulin-independence in humans with insulin-dependent diabetes mellitus.1 However, a large number of islets are needed to cure the diabetes.2 Therefore, it is important to look for ways to improve the outcome of transplantation using fewer islets. Strict metabolic control of diabetic animals, either by transplantation of additional islets or by insulin treatment, can lessen the number of islets required to reverse diabetes.3– 8 Furthermore, control of the diabetic state may improve the growth and function of the islet graft.7–9 Sulfonylureas have been widely used in the treatment of type 2 diabetes mellitus. They can stimulate insulin secretion from native pancreatic beta cells. However, little is known about their effects on transplanted islets. In this study, we investigated the influence of gliclazide, a second generation sulfonylurea, on the outcome of islet transplantation. MATERIALS AND METHODS Animals Male inbred C57BL/6 mice, aged 8 to 12 weeks, were used as donors and recipients for transplantation. The recipients were made diabetic by a single intraperitoneal (IP) injection of streptozocin (Sigma Immunochemicals, St Louis, Mo, 200 mg/kg body weight, freshly dissolved in citrate buffer, pH 4.5).
Islet Isolation and Transplantation The method has been previously described.9 –15 Briefly, under anesthesia with sodium amobarbital, pancreata were distended with 2.5 mL of RPMI medium 1640 (GIBCO BRL, Grand Island, NY) containing 1.5 mg/mL of collagenase (collagenase from Clostridium histolyticum, type XI, Sigma Immunochemicals), excised, and incubated in a water bath at 37°C. Islets separated by density gradient (Histopaque-1077; Sigma Immunochemicals) were handpicked under a dissecting microscope to select clusters with a diameter between 75 and 250 m. They were aliquotted into groups of 150 islets for transplantation under the left kidney capsule of the recipients on the same day as the isolation. Blood glucose levels and body weights were measured twice a week during the first 2 weeks and then once weekly.
Gliclazide Treatment Two weeks after transplantation, 8 recipients were injected with glicalzide (Diamicron, Les Laboratoires Servier, France) 10 mg/kg IP once daily for 2 weeks. Seventeen recipients who did not receive gliclazide treatment were used as controls.
IP Glucose Tolerance Test After an overnight fast, glucose was injected IP as a 5% glucose solution (1.5 g/kg). At 2 and 4 weeks after transplantation blood glucose levels were measured at 0, 30, 60, 90, and 120 minutes thereafter by tail snipping.9 –16 The area under the curve (AUC) was calculated as the sum of blood glucose values during the test.15
Insulin Content of the Graft The graft-bearing kidney was removed and homogenized in acid ethanol. After extraction overnight at 4°C, they were centrifuged (2,400 rpm/30 min) and the supernatant was stored at ⫺20°C. The pellet was again homogenized in acid ethanol and insulin was extracted overnight. After centrifugation this second supernatant was added to the first extraction sample. Insulin was measured by radioimmunoassay.9 –15
Statistical Analysis Results expressed as the mean and standard error of the mean (M ⫾ SEM) were compared using unpaired Student t test. P ⬍ .05 was significant.
RESULTS
After transplantation, the blood glucose value slightly decreased and body weight slightly increased in both gliclazide-treated and control groups (Fig 1). At 4 weeks after transplantation both groups remained hyperglycemic (Fig 1) with comparable glucose tolerance values (AUC: 1261 ⫾ 130 vs 1274 ⫾ 104 mg/dL, P ⬎ .5) (Fig 2) and insulin content of the grafts (1.63 ⫾ 0.59 vs 1.02 ⫾ 0.41 g, P ⫽ .5). DISCUSSION
Gliclazide is one of the sulfonylureas that lower blood glucose primarily by stimulating the second phase of insulin secretion. It binds to sulfonylurea receptors on the pancreatic beta-cell membrane, closing ATP-sensitive potassium From the Division of Endocrinology and Metabolism, Department of Internal Medicine, Chang Gung Memorial Hospital and Chang Gung University Medical College, Taoyuan, Taiwan. Supported by grants from the Chang Gung Memorial Hospital (CMRP 1018) and the National Science Council (NSC89-2314B-182A-202) of Taiwan. Address reprint requests to Dr Jyuhn-Huarng Juang, Division of Endocrinology and Metabolism, Chang Gung Memorial Hospital, 5 Fu-Shin St, Kweishan, Taoyuan, Taiwan.
0041-1345/02/$–see front matter PII S0041-1345(02)03379-1
© 2002 by Elsevier Science Inc. 360 Park Avenue South, New York, NY 10010-1710
2696
Transplantation Proceedings, 34, 2696 –2697 (2002)
EFFECT OF GLICLAZIDE ON ISLET TRANSPLANTS
2697
Fig 2. Blood glucose responses to IP glucose challenge in the recipients treated with (circle) or without (triangle) gliclazide at 4 weeks after transplantation. Values are M ⫾ SEM.
ably, membrane receptors, including sulfonylurea receptors, are destroyed first. In addition, when islets are transplanted into diabetic recipients, hyperglycemia may cause emptying of insulin granules.11,12 Thus, the transplanted islets may be unable to respond to gliclazide. REFERENCES
Fig 1. Evolution of blood glucose (upper) and body weight (lower) of the recipients treated with (circle) or without (triangle) gliclazide at 2 to 4 weeks after transplanting 150 islets under the left kidney capsule. Values are M ⫾ SEM.
channels and causing depolarization of the membrane; this allows calcium ions to enter the beta cell, triggering insulin release.17 In the present study, we tried to increase insulin secretion from transplanted islets by giving gliclazide to diabetic recipients between 2 to 4 weeks after transplantation with 150 islets. The reason why we started gliclazide treatment at 2 weeks was to avoid beta-cell exhaustion caused by overstimulation. At 4 weeks, we found that gliclazide-treated recipient blood glucose, body weight, glucose tolerance, and graft insulin content were not significantly different from those of controls. Our results suggest gliclazide has different effects on endogenous and transplanted islets. During the isolation and purification procedure, isolated islets may be damaged by digestion enzymes and by the density gradient. Presum-
1. Lacy PE: Diabetes Rev 1:76, 1993 2. International Islet Transplant Registry: Newsletter 5:21, 1995 3. Hayek A, Lopez AD, Beattie GM: Transplantation 45:940, 1988 4. Ar’Rajab A, Ahren B: Pancreas 7:435, 1992 5. Keymeulen B, Vetri M, Gorus F, et al: Transplantation 56:60, 1993 6. Ohzato H, Porter J, Monaco AP, et al: Transplantation 56:270, 1993 7. Brown J, Heininger D, Kuret J, et al: Diabetes 30:9, 1981 8. Juang J-H, Kuo C-H, Huang H-S: Transplant Proc 30:576, 1998 9. Juang J-H, Bonner-Weir S, Wu Y-J, et al: Diabetes 43:1334, 1994 10. Juang J-H, Kuo C-H, Huang H-S: Transplant Proc 29:2026, 1997 11. Juang J-H, Hsu BR-S, Kuo C-H, et al: Transplant Proc 30:565, 1998 12. Juang J-H, Hsu BR-S, Kuo C-H, et al: Transplant Proc 30:576, 1998 13. Juang J-H, Kuo C-H, Hsu BR-S: Transplant Proc 32:1073, 2000 14. Juang J-H, Kuo C-H, Hsu BR-S: Transplant Proc 32:1076, 2000 15. Juang J-H, Hsu BR-S, Kuo C-H, et al: Cell Transplant 10:277, 2001 16. Juang J-H, Bonner-Weir S, Ogawa Y, et al: Transplantation 61:1557, 1996 17. Pickup J, Williams G (eds): Textbook of Diabetes 2nd ed. Blackwell Science; 1997, p 38.1
I
SLET TRANSPLANTATION has produced insulin-independence in humans with insulin-dependent diabetes mellitus.1 However, a large number of islets are needed to cure the diabetes.2 Therefore, it is important to look for ways to improve the outcome of transplantation using fewer islets. Strict metabolic control of diabetic animals, either by transplantation of additional islets or by insulin treatment, can lessen the number of islets required to reverse diabetes.3– 8 Furthermore, control of the diabetic state may improve the growth and function of the islet graft.7–9 Sulfonylureas have been widely used in the treatment of type 2 diabetes mellitus. They can stimulate insulin secretion from native pancreatic beta cells. However, little is known about their effects on transplanted islets. In this study, we investigated the influence of gliclazide, a second generation sulfonylurea, on the outcome of islet transplantation. MATERIALS AND METHODS Animals Male inbred C57BL/6 mice, aged 8 to 12 weeks, were used as donors and recipients for transplantation. The recipients were made diabetic by a single intraperitoneal (IP) injection of streptozocin (Sigma Immunochemicals, St Louis, Mo, 200 mg/kg body weight, freshly dissolved in citrate buffer, pH 4.5).
Islet Isolation and Transplantation The method has been previously described.9 –15 Briefly, under anesthesia with sodium amobarbital, pancreata were distended with 2.5 mL of RPMI medium 1640 (GIBCO BRL, Grand Island, NY) containing 1.5 mg/mL of collagenase (collagenase from Clostridium histolyticum, type XI, Sigma Immunochemicals), excised, and incubated in a water bath at 37°C. Islets separated by density gradient (Histopaque-1077; Sigma Immunochemicals) were handpicked under a dissecting microscope to select clusters with a diameter between 75 and 250 m. They were aliquotted into groups of 150 islets for transplantation under the left kidney capsule of the recipients on the same day as the isolation. Blood glucose levels and body weights were measured twice a week during the first 2 weeks and then once weekly.
Gliclazide Treatment Two weeks after transplantation, 8 recipients were injected with glicalzide (Diamicron, Les Laboratoires Servier, France) 10 mg/kg IP once daily for 2 weeks. Seventeen recipients who did not receive gliclazide treatment were used as controls.
IP Glucose Tolerance Test After an overnight fast, glucose was injected IP as a 5% glucose solution (1.5 g/kg). At 2 and 4 weeks after transplantation blood glucose levels were measured at 0, 30, 60, 90, and 120 minutes thereafter by tail snipping.9 –16 The area under the curve (AUC) was calculated as the sum of blood glucose values during the test.15
Insulin Content of the Graft The graft-bearing kidney was removed and homogenized in acid ethanol. After extraction overnight at 4°C, they were centrifuged (2,400 rpm/30 min) and the supernatant was stored at ⫺20°C. The pellet was again homogenized in acid ethanol and insulin was extracted overnight. After centrifugation this second supernatant was added to the first extraction sample. Insulin was measured by radioimmunoassay.9 –15
Statistical Analysis Results expressed as the mean and standard error of the mean (M ⫾ SEM) were compared using unpaired Student t test. P ⬍ .05 was significant.
RESULTS
After transplantation, the blood glucose value slightly decreased and body weight slightly increased in both gliclazide-treated and control groups (Fig 1). At 4 weeks after transplantation both groups remained hyperglycemic (Fig 1) with comparable glucose tolerance values (AUC: 1261 ⫾ 130 vs 1274 ⫾ 104 mg/dL, P ⬎ .5) (Fig 2) and insulin content of the grafts (1.63 ⫾ 0.59 vs 1.02 ⫾ 0.41 g, P ⫽ .5). DISCUSSION
Gliclazide is one of the sulfonylureas that lower blood glucose primarily by stimulating the second phase of insulin secretion. It binds to sulfonylurea receptors on the pancreatic beta-cell membrane, closing ATP-sensitive potassium From the Division of Endocrinology and Metabolism, Department of Internal Medicine, Chang Gung Memorial Hospital and Chang Gung University Medical College, Taoyuan, Taiwan. Supported by grants from the Chang Gung Memorial Hospital (CMRP 1018) and the National Science Council (NSC89-2314B-182A-202) of Taiwan. Address reprint requests to Dr Jyuhn-Huarng Juang, Division of Endocrinology and Metabolism, Chang Gung Memorial Hospital, 5 Fu-Shin St, Kweishan, Taoyuan, Taiwan.
0041-1345/02/$–see front matter PII S0041-1345(02)03379-1
© 2002 by Elsevier Science Inc. 360 Park Avenue South, New York, NY 10010-1710
2696
Transplantation Proceedings, 34, 2696 –2697 (2002)
EFFECT OF GLICLAZIDE ON ISLET TRANSPLANTS
2697
Fig 2. Blood glucose responses to IP glucose challenge in the recipients treated with (circle) or without (triangle) gliclazide at 4 weeks after transplantation. Values are M ⫾ SEM.
ably, membrane receptors, including sulfonylurea receptors, are destroyed first. In addition, when islets are transplanted into diabetic recipients, hyperglycemia may cause emptying of insulin granules.11,12 Thus, the transplanted islets may be unable to respond to gliclazide. REFERENCES
Fig 1. Evolution of blood glucose (upper) and body weight (lower) of the recipients treated with (circle) or without (triangle) gliclazide at 2 to 4 weeks after transplanting 150 islets under the left kidney capsule. Values are M ⫾ SEM.
channels and causing depolarization of the membrane; this allows calcium ions to enter the beta cell, triggering insulin release.17 In the present study, we tried to increase insulin secretion from transplanted islets by giving gliclazide to diabetic recipients between 2 to 4 weeks after transplantation with 150 islets. The reason why we started gliclazide treatment at 2 weeks was to avoid beta-cell exhaustion caused by overstimulation. At 4 weeks, we found that gliclazide-treated recipient blood glucose, body weight, glucose tolerance, and graft insulin content were not significantly different from those of controls. Our results suggest gliclazide has different effects on endogenous and transplanted islets. During the isolation and purification procedure, isolated islets may be damaged by digestion enzymes and by the density gradient. Presum-
1. Lacy PE: Diabetes Rev 1:76, 1993 2. International Islet Transplant Registry: Newsletter 5:21, 1995 3. Hayek A, Lopez AD, Beattie GM: Transplantation 45:940, 1988 4. Ar’Rajab A, Ahren B: Pancreas 7:435, 1992 5. Keymeulen B, Vetri M, Gorus F, et al: Transplantation 56:60, 1993 6. Ohzato H, Porter J, Monaco AP, et al: Transplantation 56:270, 1993 7. Brown J, Heininger D, Kuret J, et al: Diabetes 30:9, 1981 8. Juang J-H, Kuo C-H, Huang H-S: Transplant Proc 30:576, 1998 9. Juang J-H, Bonner-Weir S, Wu Y-J, et al: Diabetes 43:1334, 1994 10. Juang J-H, Kuo C-H, Huang H-S: Transplant Proc 29:2026, 1997 11. Juang J-H, Hsu BR-S, Kuo C-H, et al: Transplant Proc 30:565, 1998 12. Juang J-H, Hsu BR-S, Kuo C-H, et al: Transplant Proc 30:576, 1998 13. Juang J-H, Kuo C-H, Hsu BR-S: Transplant Proc 32:1073, 2000 14. Juang J-H, Kuo C-H, Hsu BR-S: Transplant Proc 32:1076, 2000 15. Juang J-H, Hsu BR-S, Kuo C-H, et al: Cell Transplant 10:277, 2001 16. Juang J-H, Bonner-Weir S, Ogawa Y, et al: Transplantation 61:1557, 1996 17. Pickup J, Williams G (eds): Textbook of Diabetes 2nd ed. Blackwell Science; 1997, p 38.1