PARADOXICAL RELEASE OF INSULIN BY ADULT PIG ISLETS IN VITRO

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Abstract
In this study, in vitro responsiveness to glucose of fresh and cultured islets from adult pigs was tested under both static (incubation) and dynamic (perifusion) conditions. Islets were isolated by an automated method from pancreases of 24-month-old animals and cultured overnight in CMRL 1066 and 10% FCS plus antibiotics; islets, perifused immediately after the overnight culture, showed a paradoxical decrease in insulin release when exposed to an acute glucose stimulus (16.7 mmol/L), and a normal response to acute glucose when isobutylmethylxanthine (IBMX) was added to the perifusing buffer. In addition, an acute reduction of glucose concentration in the perifusate elicited a paradoxical insulin release. At the microscope, islets appeared loose and irregularly shaped after the overnight culture; immunohistochemistry showed loss of peripheral A and other mantle cells. After the overnight culture, islets were divided into 5 groups and were cultured for a further 48 hr in different tissue culture media: CMRL 1066; RPMI1640 (without glucose); RPMI 1640 (plus 11.1 mmol/L glucose); Ham's F12; and medium 199 (all media were supplemented with 10% FCS and antibiotics). During this period, insulin release was 11.4± 1.1 pg/islet/min in islets cultured in CMRL 1066, 16.2±2.4 in islets cultured in RPMI 1640 (11.1 mmol/L glucose), 1.8±0.2 (P After the 48-hr culture in different media, the islets' responsiveness to an acute glucose stimulus (16.7 mmol/L, static incubation) was evaluated: islets cultured in CMRL 1066 and in RPMI 1640 (with and without glucose) showed no insulin response to the acute glucose stimulus; in contrast, insulin release rose from 0.42± 0.06 to 0.60±0.12 pg/islet/min (NS) in islets cultured in Ham's F12, and from 0.24±0.06 to 0.48±0.06 pg/islet/min (P After the 48-hr culture, islet insulin content was similar in the 5 different media, being 4.7±0.5, 4.4±1.3, 3.1±0.3, 3.4±0.4, and 3.7±0.3 ng/islet, respectively. These data indicate that: (1) insulin release from freshly isolated pig islets is poorly glucose regulated, probably because of islet damage subsequent to the isolation procedure; (2) glucose regulation of insulin release improves after an additional 48 hr of culture; and (3) a significant increase in the glucose-induced insulin release is achievable only after the culture in medium 199.

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