It has been shown that peripheral glucagon secreting cells (A-cells) are lost during most of the isolation procedures employed for pig islets. Loss of A-cells decreases intra-islet glucagon levels and cAMP levels in B-cells and might reduce glucose-induced insulin release. This study was designed to test this hypothesis, by evaluating the effects of culture of porcine islets with exogenous glucagon on insulin secretion and on insulin and cAMP content in islets. Islets were isolated from adult 2-year old Large White pigs using an automated method. The number of A-cells was calculated by immunostaining for glucagon in islets before and after isolation and a significant decrease in A-cells was observed. After an overnight culture, islets were cultured for 48 h in a standard medium (CMRL 1066, 10% foetal calf serum, 1% antibiotics, 1% glutamine) alone or in the presence of glucagon at two different concentrations (1.0 and 10.0 mu M); exposure to glucagon was either continuous or alternated with periods of incubation in CMRL 1066 alone. After the 48-h culture in standard medium, the islet glucagon response to arginine was almost negligible and significantly lower than that observed in human islets. After culture, islet insulin response to glucose, and islet insulin and cAMP content were evaluated; continuous exposure to glucagon did not produce any significant effect on either insulin secretion or insulin and cAMP content; in contrast, discontinuous exposure to glucagon induced a significant improvement in insulin release, proportional to glucagon concentrations (integrated insulin release: -13.8 +/- 20.12 pg/islet/20 min in control islets, 111.0 +/- 50.73 and 144.7 +/- 47.54 pg/islet/20 min in islets exposed to 1.0 and 10.0 mu M glucagon respectively; n=10, P=0.01). Intracellular insulin and cAMP content of islets cultured in different culture media were not different. In conclusion, discontinuous exposure of isolated pig islets to exogenous glucagon induced a significant increase in glucose-induced insulin release which was not associated with an increase in cAMP content. The fact that even in the presence of glucagon the secretory activity of pig islets was lower than the reported activity of human or rat islets suggests that glucagon is only one of the factors involved in the poor insulin responsiveness of pig islets.

GLUCAGON IMPROVES INSULIN-SECRETION FROM PIG ISLETS IN-VITRO

PIEMONTI , LORENZO;
1995

Abstract

It has been shown that peripheral glucagon secreting cells (A-cells) are lost during most of the isolation procedures employed for pig islets. Loss of A-cells decreases intra-islet glucagon levels and cAMP levels in B-cells and might reduce glucose-induced insulin release. This study was designed to test this hypothesis, by evaluating the effects of culture of porcine islets with exogenous glucagon on insulin secretion and on insulin and cAMP content in islets. Islets were isolated from adult 2-year old Large White pigs using an automated method. The number of A-cells was calculated by immunostaining for glucagon in islets before and after isolation and a significant decrease in A-cells was observed. After an overnight culture, islets were cultured for 48 h in a standard medium (CMRL 1066, 10% foetal calf serum, 1% antibiotics, 1% glutamine) alone or in the presence of glucagon at two different concentrations (1.0 and 10.0 mu M); exposure to glucagon was either continuous or alternated with periods of incubation in CMRL 1066 alone. After the 48-h culture in standard medium, the islet glucagon response to arginine was almost negligible and significantly lower than that observed in human islets. After culture, islet insulin response to glucose, and islet insulin and cAMP content were evaluated; continuous exposure to glucagon did not produce any significant effect on either insulin secretion or insulin and cAMP content; in contrast, discontinuous exposure to glucagon induced a significant improvement in insulin release, proportional to glucagon concentrations (integrated insulin release: -13.8 +/- 20.12 pg/islet/20 min in control islets, 111.0 +/- 50.73 and 144.7 +/- 47.54 pg/islet/20 min in islets exposed to 1.0 and 10.0 mu M glucagon respectively; n=10, P=0.01). Intracellular insulin and cAMP content of islets cultured in different culture media were not different. In conclusion, discontinuous exposure of isolated pig islets to exogenous glucagon induced a significant increase in glucose-induced insulin release which was not associated with an increase in cAMP content. The fact that even in the presence of glucagon the secretory activity of pig islets was lower than the reported activity of human or rat islets suggests that glucagon is only one of the factors involved in the poor insulin responsiveness of pig islets.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/20.500.11768/1775
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