Short term culture with the caspases inhibitor z-VAD fmk reduces beta cell apoptosis in transplanted islets and improves the metabolic outcome of the graft

In the initial days after transplantation islets are particularly vulnerable and show increased apoptosis and necrosis. We have studied the effects of caspase inhibition on this early beta cell death in syngeneically transplanted islets. Streptozotocin-diabetic C57BL/6 mice were transplanted with 150 syngeneic islets, an insufficient mass to restore normoglycemia, preincubated with or without the pan-caspase inhibitor z-VAD. fmk 2 h before transplantation. Beta cell apoptosis was increased in control islets on day 3 after transplantation (0.28 ± 0.02%) compared with freshly isolated islets (0.08 ± 0.02%, p< 0.001), and was partially reduced in transplanted islets preincubated with z-VAD.fmk 200 μM (0.14 ± 0.02%, p = 0.003) or with z-VAD.fmk 500 μM (0.17 ± 0.01%, p = 0.012), but not with a lower z-VAD.fmk (100 μM) concentration. Diabetic mice transplanted with islets preincubated with z-VAD.fmk 500 μM showed an improved metabolic evolution compared with control and z-VAD.fmk 200 μM groups. The z-VAD.fmk 500 μM group showed an overall lower blood glucose after transplantation (p = 0.02), and at the end of the study blood glucose values were reduced compared with transplantation day (15.7 ± 3.6 vs. 32.5 ± 0.5 mmol/L, p = 0.001). In contrast, blood glucose was not significantly changed in control and z-VAD.fmk 200 μM groups. Four weeks after transplantation beta cell mass was higher in z-VAD.fmk 500 μM group (0.15 ± 0.02 mg) than in the control group (0.10 ± 0.02 mg) (p = 0.043). In summary, the treatment of freshly isolated islets with the caspase inhibitor z-VAD.fmk reduced the subsequent apoptosis of the islets once they were transplanted and improved the outcome of the graft

Mecanismes de lesió de la cèl·lula Beta en el trasplantament singènic d'illots de Langerhans

[cat] Una de les alternatives més atractives de futur pel tractament de la Diabetis Mellitus és el trasplantament d'illots pancreàtics. La insuficient quantitat de teixit insular és un dels principals obstacles pel trasplantament d'illots, a més el problema es veu agreujat per el gran nombre d'illots necessaris per realitzar un trasplantament amb èxit. S'ha suggerit que la presència d'una inflamació no-específica en el lloc d'implantació dels illots podria ser una de les causes de la pèrdua primerenca de l'empelt. L'objectiu general fou l'estudi dels fenòmens que tenen lloc en els primers dies després del trasplantament, i específicament els mecanismes de mort dels illots trasplantats, els fenòmens inflamatoris que es produeixen al lloc d'implantació dels illots i la possible contribució de l'estat metabòlic del receptor. Es van trasplantar animals singènics diabètics per injecció amb estreptozotocina amb una massa beta insuficient per restaurar la normoglicèmia. Uns animals es van mantenir hiperglicèmics al llarg de l'estudi i uns altres van ser tractats amb insulina i per tant van normalitzar els nivells de glicèmia. En els primers dies després del trasplantament més de la meitat de la massa beta trasplantada es va perdre malgrat les condicions òptimes del trasplantament degut a un augment de la mort cel·lular beta tant per processos d'apoptosi com de necrosi. A més es va detectar l'expressió del gen de la IL-1beta i iNOS en illots immediatament després de l'aïllament i l'augment de l'expressió d'aquests gens en empelts inicialment després del trasplantament. L'augment en l'expressió dels gens de la IL-1beta i iNOS es produí tant a animals diabètics hiperglicèmics com a animals diabètics normoglicèmics, però els animals hiperglicèmics presentaven una menor expressió dels gens de la IL-1beta i iNOS que els normoglicèmics, suggerint la possible existència d'una limitació en l'augment d'expressió d'aquests gens en els empelts exposats a hiperglicèmia. Per tant en els primers dies després del trasplantament hem demostrat la presència d'una important pèrdua de massa beta que podria contribuir a que sigui necessària una major quantitat d'illots a trasplantar. La detecció de l'expressió dels gens de la IL-1beta i iNOS en els empelts en aquests primers dies després del trasplantament indicaria la presència d'un procés inflamatori en el lloc d'implantació dels illots que podria jugar un paper en l'augment de l'apoptosi i necrosi detectada. Una de les alternatives més atractives de futur pel tractament de la Diabetis Mellitus és el trasplantament d'illots pancreàtics. La insuficient quantitat de teixit insular és un dels principals obstacles pel trasplantament d'illots, a més el problema es veu agreujat per el gran nombre d'illots necessaris per realitzar un trasplantament amb èxit. S'ha suggerit que la presència d'una inflamació no-específica en el lloc d'implantació dels illots podria ser una de les causes de la pèrdua primerenca de l'empelt. L'objectiu general fou l'estudi dels fenòmens que tenen lloc en els primers dies després del trasplantament, i específicament els mecanismes de mort dels illots trasplantats, els fenòmens inflamatoris que es produeixen al lloc d'implantació dels illots i la possible contribució de l'estat metabòlic del receptor. Es van trasplantar animals singènics diabètics per injecció amb estreptozotocina amb una massa beta insuficient per restaurar la normoglicèmia. Uns animals es van mantenir hiperglicèmics al llarg de l'estudi i uns altres van ser tractats amb insulina i per tant van normalitzar els nivells de glicèmia. En els primers dies després del trasplantament més de la meitat de la massa beta trasplantada es va perdre malgrat les condicions òptimes del trasplantament degut a un augment de la mort cel·lular beta tant per processos d'apoptosi com de necrosi. A més es va detectar l'expressió del gen de la IL-1beta i iNOS en illots immediatament després de l'aïllament i l'augment de l'expressió d'aquests gens en empelts inicialment després del trasplantament. L'augment en l'expressió dels gens de la IL-1beta i iNOS es produí tant a animals diabètics hiperglicèmics com a animals diabètics normoglicèmics, però els animals hiperglicèmics presentaven una menor expressió dels gens de la IL-1beta i iNOS que els normoglicèmics, suggerint la possible existència d'una limitació en l'augment d'expressió d'aquests gens en els empelts exposats a hiperglicèmia. Per tant en els primers dies després del trasplantament hem demostrat la presència d'una important pèrdua de massa beta que podria contribuir a que sigui necessària una major quantitat d'illots a trasplantar. La detecció de l'expressió dels gens de la IL-1beta i iNOS en els empelts en aquests primers dies després del trasplantament indicaria la presència d'un procés inflamatori en el lloc d'implantació dels illots que podria jugar un paper en l'augment de l'apoptosi i necrosi detectada

Linear correlation between beta cell mass and body wight throughout the lifespan in Lewis rats: role of beta cell hyperplasia and hypertrophy

We determined the beta-cell replicative rate, beta-cell apoptosis, cross-sectional beta-cell area, and pancreatic beta-cell mass throughout the entire postweaning lifespan (months 1, 3, 7, 10, 15, and 20) of Lewis rats. Beta-cell replication was progressively reduced in the initial months of life but remained stable after month 7 (month 1, 0.99 +/- 0.10%; month 3, 0.24 +/- 0.04%; month 7, 0.12 +/- 0.02%; month 10, 0.14 +/- 0.02%; month 15, 0.10 +/- 0.03%; month 20, 0.13 +/- 0.03%; analysis of variance [ANOVA], P < 0.001). Beta-cell apoptosis was low and did not change significantly from month 1 to 20 of life. Cross-sectional area of individual beta-cells increased progressively in the initial months, remained stable from month 7 to 15, and increased again on month 20. The estimated number of beta-cells per pancreas, calculated as the ratio of total beta-cell mass to individual beta-cell mass, tripled from month 1 to 7 but did not change significantly thereafter. Beta-cell mass increased approximately 8 times from month 1 to 20 (month 1, 2.04 +/- 0.28 mg; month 20, 15.5 +/- 2.32 mg; ANOVA, P < 0.001) and showed a strong and significant linear correlation with body weight (r = 0.98, P < 0.001). In summary, we have shown that beta-cell replication was maintained throughout the lifespan in normal rats, clearly establishing that the beta-cell birth rate does not fall to 0, even in very old rats. Beta-cell mass increased throughout the lifespan, closely matching the increment in total body weight at any time point. This increment was selective for beta-cells, since the growth of the endocrine non-beta-cell mass was limited to the initial months of life. Both beta-cell hypertrophy and hyperplasia contributed to increased beta-cell mass in young animals, but only beta-cell hypertrophy was responsible for the increased beta-cell mass found in old animals. This study provides a global perspective for understanding the dynamics of beta-cell mass in young, adult, and aged animals

B-cell death and mass in syngeneically transplanted islets exposed to short and long-term hyperglycemia

We studied the effects of hyperglycemia on beta-cell death and mass in syngeneically transplanted islets. Six groups of STZ-induced diabetic C57BL/6 mice were transplanted with 100 syngeneic islets, an insufficient beta-cell mass to restore normoglycemia. Groups 1, 2, and 3 remained hyperglycemic throughout the study. Groups 4, 5, and 6 were treated with insulin from day 7 before transplantation to day 10 after transplantation. After insulin discontinuation, group 6 mice achieved definitive normoglycemia. Grafts were harvested at 3 (groups 1 and 4), 10 (groups 2 and 5), and 30 (groups 3 and 6) days after transplantation. On day 3, the initially transplanted beta-cell mass (0.13 +/- 0.01 mg) was dramatically and similarly reduced in the hyperglycemic and insulin-treated groups (group 1: 0.048 +/- 0.002 mg; group 4: 0.046 +/- 0.007 mg; P < 0.001). Extensive islet necrosis (group 1: 30.7%; group 4: 26.8%) and increased beta-cell apoptosis (group 1: 0.30 +/- 0.05%; group 4: 0.42 +/- 0.07%) were found. On day 10, apoptosis remained increased in both hyperglycemic and insulin-treated mice (group 2: 0.44 +/- 0.09%; group 5: 0.48 +/- 0.08%) compared with normal pancreas (0.04 +/- 0.03%; P < 0.001). In contrast, on day 30, beta-cell apoptosis was increased in grafts exposed to sustained hyperglycemia (group 3: 0.37 +/- 0.03%) but not in normoglycemic mice (group 6: 0.12 +/- 0.02%); beta-cell mass was selectively reduced in islets exposed to hyperglycemia (group 3: 0.046 +/- 0.02 mg; group 6: 0.102 +/- 0.009 mg; P < 0.01). In summary, even in optimal conditions, approximately 60% of transplanted islet tissue was lost 3 days after syngeneic transplantation, and both apoptosis and necrosis contributed to beta-cell death. Increased apoptosis and reduced beta-cell mass were also found in islets exposed to chronic hyperglycemia, suggesting that sustained hyperglycemia increased apoptosis in transplanted beta-cells

B-cell death and mass in syngeneically transplanted islets exposed to short and long-term hyperglycemia

We studied the effects of hyperglycemia on beta-cell death and mass in syngeneically transplanted islets. Six groups of STZ-induced diabetic C57BL/6 mice were transplanted with 100 syngeneic islets, an insufficient beta-cell mass to restore normoglycemia. Groups 1, 2, and 3 remained hyperglycemic throughout the study. Groups 4, 5, and 6 were treated with insulin from day 7 before transplantation to day 10 after transplantation. After insulin discontinuation, group 6 mice achieved definitive normoglycemia. Grafts were harvested at 3 (groups 1 and 4), 10 (groups 2 and 5), and 30 (groups 3 and 6) days after transplantation. On day 3, the initially transplanted beta-cell mass (0.13 +/- 0.01 mg) was dramatically and similarly reduced in the hyperglycemic and insulin-treated groups (group 1: 0.048 +/- 0.002 mg; group 4: 0.046 +/- 0.007 mg; P < 0.001). Extensive islet necrosis (group 1: 30.7%; group 4: 26.8%) and increased beta-cell apoptosis (group 1: 0.30 +/- 0.05%; group 4: 0.42 +/- 0.07%) were found. On day 10, apoptosis remained increased in both hyperglycemic and insulin-treated mice (group 2: 0.44 +/- 0.09%; group 5: 0.48 +/- 0.08%) compared with normal pancreas (0.04 +/- 0.03%; P < 0.001). In contrast, on day 30, beta-cell apoptosis was increased in grafts exposed to sustained hyperglycemia (group 3: 0.37 +/- 0.03%) but not in normoglycemic mice (group 6: 0.12 +/- 0.02%); beta-cell mass was selectively reduced in islets exposed to hyperglycemia (group 3: 0.046 +/- 0.02 mg; group 6: 0.102 +/- 0.009 mg; P < 0.01). In summary, even in optimal conditions, approximately 60% of transplanted islet tissue was lost 3 days after syngeneic transplantation, and both apoptosis and necrosis contributed to beta-cell death. Increased apoptosis and reduced beta-cell mass were also found in islets exposed to chronic hyperglycemia, suggesting that sustained hyperglycemia increased apoptosis in transplanted beta-cells

Short term culture with the caspases inhibitor z-VAD fmk reduces beta cell apoptosis in transplanted islets and improves the metabolic outcome of the graft

In the initial days after transplantation islets are particularly vulnerable and show increased apoptosis and necrosis. We have studied the effects of caspase inhibition on this early beta cell death in syngeneically transplanted islets. Streptozotocin-diabetic C57BL/6 mice were transplanted with 150 syngeneic islets, an insufficient mass to restore normoglycemia, preincubated with or without the pan-caspase inhibitor z-VAD. fmk 2 h before transplantation. Beta cell apoptosis was increased in control islets on day 3 after transplantation (0.28 ± 0.02%) compared with freshly isolated islets (0.08 ± 0.02%, p< 0.001), and was partially reduced in transplanted islets preincubated with z-VAD.fmk 200 μM (0.14 ± 0.02%, p = 0.003) or with z-VAD.fmk 500 μM (0.17 ± 0.01%, p = 0.012), but not with a lower z-VAD.fmk (100 μM) concentration. Diabetic mice transplanted with islets preincubated with z-VAD.fmk 500 μM showed an improved metabolic evolution compared with control and z-VAD.fmk 200 μM groups. The z-VAD.fmk 500 μM group showed an overall lower blood glucose after transplantation (p = 0.02), and at the end of the study blood glucose values were reduced compared with transplantation day (15.7 ± 3.6 vs. 32.5 ± 0.5 mmol/L, p = 0.001). In contrast, blood glucose was not significantly changed in control and z-VAD.fmk 200 μM groups. Four weeks after transplantation beta cell mass was higher in z-VAD.fmk 500 μM group (0.15 ± 0.02 mg) than in the control group (0.10 ± 0.02 mg) (p = 0.043). In summary, the treatment of freshly isolated islets with the caspase inhibitor z-VAD.fmk reduced the subsequent apoptosis of the islets once they were transplanted and improved the outcome of the graft

Optimal insulin tratment in experimental islet transplantation

Insulin-induced normoglycemia has shown to have a beneficial effect on the outcome of pancreatic islets transplanted to diabetic recipients. The aim of the study was to identify the insulin treatment that can maximize its beneficial effect on islet transplants. Six groups of streptozotocin diabetic C57Bl/6 mice were transplanted (Tx) with 100 syngeneic islets, an insufficient beta cell mass to restore normoglycemia, and were treated with insulin as follows: group 1 (n = 9): from day 10 before Tx to day 14 after Tx; group 2 (n = 11): from day 6 before Tx to Tx day; group 3 (n = 11): from Tx day to day 6 after Tx; group 4 (n = 7): from Tx day to day 14 after Tx; group 5 (n = 8): from day 10 to day 24 after Tx; group 6 (n = 18): Tx mice were not treated with insulin. Sixty days after Tx, normoglycemia was achieved in 100% of mice in groups 1, 4, and 5, in 73% of mice in group 2, and in only 45% and 33% of mice in groups 3 and 6, respectively (p < 0.01). Intraperitoneal glucose tolerance, determined only in normoglycemic mice, was similar in groups 1, 2, 4, and normal controls. In contrast, normoglycemic mice from groups 3, 5, and 6, exposed to more severe and prolonged hyperglycemia after Tx, showed higher glucose values after glucose injection, suggesting that hyperglycemia had a long-lasting deleterious effect on transplanted beta cell function. The initially transplanted beta cell mass was maintained in the grafts of normoglycemic mice, but was severely reduced in hyperglycemic mice. Transplanted beta cell mass was similar in normoglycemic groups with normal or impaired glucose tolerance, indicating that impaired glucose tolerance was not due to reduced beta cell mass. In summary, the beneficial effect of insulin-induced normoglycemia on transplanted islets was maximal when insulin treatment was maintained the initial 14 days after transplantation. Exposure to sustained hyperglycemia initially after transplantation had a long-lasting deleterious effect on transplanted islets

Optimal insulin tratment in experimental islet transplantation

Insulin-induced normoglycemia has shown to have a beneficial effect on the outcome of pancreatic islets transplanted to diabetic recipients. The aim of the study was to identify the insulin treatment that can maximize its beneficial effect on islet transplants. Six groups of streptozotocin diabetic C57Bl/6 mice were transplanted (Tx) with 100 syngeneic islets, an insufficient beta cell mass to restore normoglycemia, and were treated with insulin as follows: group 1 (n = 9): from day 10 before Tx to day 14 after Tx; group 2 (n = 11): from day 6 before Tx to Tx day; group 3 (n = 11): from Tx day to day 6 after Tx; group 4 (n = 7): from Tx day to day 14 after Tx; group 5 (n = 8): from day 10 to day 24 after Tx; group 6 (n = 18): Tx mice were not treated with insulin. Sixty days after Tx, normoglycemia was achieved in 100% of mice in groups 1, 4, and 5, in 73% of mice in group 2, and in only 45% and 33% of mice in groups 3 and 6, respectively (p < 0.01). Intraperitoneal glucose tolerance, determined only in normoglycemic mice, was similar in groups 1, 2, 4, and normal controls. In contrast, normoglycemic mice from groups 3, 5, and 6, exposed to more severe and prolonged hyperglycemia after Tx, showed higher glucose values after glucose injection, suggesting that hyperglycemia had a long-lasting deleterious effect on transplanted beta cell function. The initially transplanted beta cell mass was maintained in the grafts of normoglycemic mice, but was severely reduced in hyperglycemic mice. Transplanted beta cell mass was similar in normoglycemic groups with normal or impaired glucose tolerance, indicating that impaired glucose tolerance was not due to reduced beta cell mass. In summary, the beneficial effect of insulin-induced normoglycemia on transplanted islets was maximal when insulin treatment was maintained the initial 14 days after transplantation. Exposure to sustained hyperglycemia initially after transplantation had a long-lasting deleterious effect on transplanted islets