Differential effects of age versus gylcemic stimulation on the maturation of insulin stimulus-secretion coupling during culture of fetal rat islets

We have cultured islets from 21.5-day-old fetal rats for 1-7 days in RMPI 1640/10% fetal calf serum containing 2.8 or 1.1 mM glucose to evaluate the differential effects of age vis-a-vis glycemic stimulation on the maturation of selected components of stimulus-secretion coupling. After 1 day of culture in either media, acute stimulation with 3.0 mg/ml glucose during basal perifusion with 0.5 mg/ml glucose elicited only a small first phase of stimulated insulin secretion and no second phase. The acute exposure to 3.0 mg/ml glucose produced no change in the prevailing high rates of oxygen consumption (Q(O₂) and caused only minor increments in phosphate efflux (i.e., peak values of phosphate flush of 126 ± 16% of baseline for islets that had been cultured in 11.1 mM glucose and 162 ± 30% for islets cultured in 2.8 mM glucose). After 7 days of culture in 11.1 mM glucose, acute stimulation with 3.0 mg/ml glucose increased Q(O₂) (as in adult islets) and effected acute increases in the AT³²P and GT³²P content of prelabeled islets. The 3.0 mg/ml glucose also triggered phosphate flush to 599 ± 45% of baseline and elicited first as well as early second phases of stimulated insulin secretion that replicated the performance of adult islets. By contrast, after 7 days of culture in 2.8 mM glucose, similar acute exposures of fetal islets to 3.0 mg/ml glucose effected only a small first phase and a negligible second phase of stimulated insulin secretion despite the occurrence of the same increments in Q(O₂) as after culture in 11.1 mM glucose, highly significant increases in AT³²P and GT³²P, and phosphate flushes that peaked at 306 ± 16% of basal values. Thus, the ontogeny of individual components of stimulus-secretion coupling may occur in asynchronous fashion and varyingly require glycemic stimulation in addition to aging per se. The capacities to augment efflux of orthophosphate, enhance respiration, and heighten nucleotide turnover in response to acute stimulation with glucose seem to mature in large measure in time-dependent fashion, whereas some chronic exposure to ambient glucose in excess of basal levels may be required to establish and/or maintain the other coupled components that underlie biphasic stimulated insulin release. However, we did not achieve full exocytotic maturation even after 7 days of culture with 11.1 mM glucose. When stimulatory perifusion of such islets was prolonged beyond 30 min, the adult-like second phase of stimulated insulin release began to diminish. The fall-off was not due to limitations in preformed islet stores of immunoreactive insulin and it was not prevented by supplementing the perifusates with secretory-enhancing amounts of theophylline. Our experiences indicate that controlled tissue culture of fetal islets may provide a useful approach for unmasking the relative interdependence and independence of individual steps in insulin stimulus-secretion coupling. They also underscore the multiplicity of potential sites through which arrest of functional maturation could result in faulty insulin release.