<b>ACUTE AND LONG-TERM EFFECTS OF GLUCOSE ON THE FUNCTION OF TRANSPLANTABLE RAT INSULINOMA CELLS MAINTAINED IN TISSUE </b><b>CULTURE </b>

Abstract

Acute and long-term effects of glucose on B-cells from an X-ray induced transplantable rat insulinoma were examined in vitro. Fresh tumour cells failed to respond acutely to glucose or glucose plus theophylline with increased "Ca uptake and insulin release. During culture in RPMI-1640, blood cells and necrotic tumour cells rapidly disappeared. Cultured secretory cells did not proliferate but exhibited >95% viability and consumed substantial amounts of glucose. Glucose (1.4-22.2 mM) did not affect insulin content or insulin release in 7-day cultures, but both parameters decreased by 51% and 80% compared with 2-day cultures. Insulin output represented 1% of the fall in insulin content suggesting substantial cellular insulin degradation. Acute exposure of 2-day cultured cells to 16.7 mM glucose did not affect insulin release (64-88 ng/l06ce1ls/h) or "Ca uptake (1.4-2.0 nmoll 106 cells/h). However, combination with 5 mM theophylline evoked la 22-30% stimulatory insulin response. Cells cultured for 7 days also failed to respond acutely to glucose with increased "'5Ca uptake, but a 22-34% stimulation of insulin secretion was evident after maintenance at 5.6-22.2 but not 1.4 mM glucose. Addition of theophylline potentiated the insulin response irrespective of a change in "Ca uptake.Considerable interest has been generated by the availability of serially transplantable insulinomas in small laboratory animals. These have been produced in the rat by X-ray irradiation or streptozotocin-nicotinamide injection (3, 4), in the hamster by BK virus innoculation (15), and in the mouse by transfer into fertilised eggs of recombinant genes (12). Research conducted using these insulinomas has focused on two questions, namely what can these tumours tell us about human insulinomas, and secondly what might be learnt about pancreatic B-cell function by exploiting the availability of large amounts of insulinoma tissue. Despite these different approaches, interests have converged on the responsiveness of these insulinomas to glucose. Thus, glucose is not only recognised as the major physiological regulator of insulin secretion (27), but an inappropriately high rate of insulin secretion in the face of hypoglycaemia is also recognised as the cardinal feature of insulinoma (19). A The transplantable radiation-induced New England Deaconess Hospital (NEDH) rat insulinoma has a rapid growth rate and is composed of well granulated B-cells of high insulin content with only small amounts of other islet hormones and regulatory peptides (5, 22). These features together with the rapid formation of large vascularised tumours associated with hyperinsulinaemia and hypoglycaemiahave made the NEDH rat insulinoma a most attractive model for research. However, there is considerable controversy in the literature concerning the in viva and in vitro § 2 E 2

Measurements of membrane potential, transmembrane 45Ca fluxes, cytoplasmic free Ca2+ concentration and insulin release by transplantable rat insulinoma cells maintained in tissue culture

“…In accordance with previous studies with the Surrey insulinoma subline (Flatt et al, 1987b;Swanston-Flatt & Flatt, 1987), short-term cultured rat insulinoma cells did not respond to glucose with (2) 292+174 (2) +151+97 ( increased insulin release. This unresponsiveness may partly reflect defective glucose recognition since tumour cells exhibit a marked depletion of glucokinase with a corresponding increase in high affinity hexokinase (Lenzen et al, 1987 [Ca2+] were small compared with those evoked in pancreatic fl-cells .…”

“…Isolation and culture of tumour cells Tumours were excised from the subscapular site of hypoglycaemic insulinoma-bearing rats, and used to prepare tumour cell suspensions as previously described (Flatt et al, 1987d;Swanston-Flatt & Flatt, 1987). Isolated cells were cultured for 2-3 days at 37°C in a humidified atmosphere of 5% CO2 in air.…”