Sara K. Swanston-Flatt

Specific binding of the C-peptide of proinsulin to cultured B-cells from a transplantable rat islet cell tumor

Specific binding of the C-peptide of proinsulin was evaluated using a transplantable NEDH rat islet cell tumour predominantly composed of insulin-secreting B-cells. Cultured tumour B-cells exhibited greater than 90% viability assessed by trypan blue exclusion, and retained the ability to form tumours with accompanying hypoglycaemia and hyperinsulinaemia after reimplantation. During binding experiments with synthetic rat C-peptide I and iodinated tyrosylated rat C-peptide I, turnout B-cells exhibited 54±6% specific binding. Displacement of tracer increased with increasing concentrations of unlabelled rat C-peptide I (0.25–1,000 ng/ml), and the specificity of binding was substantiated by reduced displacement with human C-peptide. Scatchard analysis of specific C-peptide binding revealed a curvilinear plot with upward concavity. The demonstration of specific C-peptide binding to insulin-secreting B-cells provides evidence for a physiological role of proinsulin C-peptide.

Effects of cytotoxic drugs and inhibitors of insulin secretion on a serially transplantable rat insulinoma and cultured rat insulinoma cells

The effects of cytotoxic drugs and inhibitors of insulin secretion were examined in vivo in rats with a radiation-induced transplantable insulinoma, and in vitro using cultured rat insulinoma cells and the derived RINm5F insulin-secreting cell line. Administration of diazoxide to insulinoma-bearing rats resulted in a transient decrease of plasma insulin with a temporary rise of glucose concentrations. Mannoheptulose and somatostatin failed to affect the marked hyperinsulinaemia and hypoglycaemia. Streptozotocin produced a rapid and sustained decrease of insulin concentrations in insulinoma-bearing rats, accompanied by a progressive elevation of plasma glucose. Administration of alloxan failed to affect circulating insulin or glucose concentrations. In vitro, streptozotocin and alloxan exerted approximately equipotent time-dependent and concentration-dependent cytotoxic effects on insulinoma cells and RINm5F cells as established by cell staining with trypan blue. The cytotoxic actions of both drugs were decreased by agents believed to scavenge free radicals or to act as inhibitors of poly(ADP-ribose) synthetase. The results suggest that the cytotoxic actions of streptozotocin and alloxan on rat insulinoma cells and RINm5F cells are mediated by the generation of hydroxyl free radicals and DNA strand breaks. The ineffectiveness of alloxan in insulinoma-bearing rats probably reflects the high rate of decomposition of the drug in vivo.

Metabolic effects and secretory properties of a radiation-induced transplantable rat insulinoma

The metabolic effects and secretory properties of a radiation-induced transplantable insulinoma were examined in 16-17 week old NEDH rats. Subcutaneous subscapular implantation of tumour fragments resulted in hyperphagia, increased body weight gain, marked hyperinsulinaemia and severe hypoglycaemia, with the resulting death of the recipient by 27 days. Ultimate tumour size was 2.1 +/- 0.4 g (mean +/- SEM). At 3 days after transplantation, plasma glucose and insulin responses to intraperitoneal glucose, insulin, arginine and adrenaline were similar to control rats. At 20 days, plasma glucose concentrations of insulinoma-bearing rats remained low throughout glucose tolerance tests, and insulin responsiveness to glucose stimulation was absent. 2-Deoxy-D-glucose produced only a small rise of glucose concentrations in tumour-bearing rats. Insulin sensitivity was not appreciably impaired at 20 days despite severe hyperinsulinaemia and hypoglycaemia. The ability of adrenaline and propranolol to suppress plasma insulin and raise plasma glucose concentrations was also retained. At 20 days, glucagon evoked a marked plasma insulin response with no change in plasma glucose concentrations. In contrast, arginine and glibenclamide failed to stimulate insulin above high basal concentrations.

Metabolic effects of radiation induced rat insulinoma at pancreatic, hepatic and subscapular transplantation sites

Metabolic effects of 3 different sites of transplantation of cultured tumour cells from a radiation induced insulinoma (28 X 10(6) viable cells per rat) were examined in 15-18 weeks old male NEDH rats. Subscapular implantation consistently produced a highly vascularised encapsulated tumour associated with hyperinsulinaemia, hyperphagia and hypoglycaemia by 21 days, which progressed to fatal neuroglycopaenic coma at 37 +/- 3 days (mean +/- SEM). Implantation of tumour cells into the hepatic portal vein resulted in a multilobular hepatic tumour in two out of nine rats, with hyperinsulinaemia and fatal hypoglycaemia by 49-54 days. Irregularities of glucose homeostasis were observed in a further three rats by 62 days. Intrapancreatic implantation consistently produced a similar tumour to that observed at the subscapular site. Implantation into the pancreas produced the most rapid onset of hyperinsulinaemia, hyperphagia and hypoglycaemia, with survival for only 28 +/- 3 days. The results demonstrate an important effect of transplantation site on the function and metabolic consequences of the NEDH rat insulinoma.

Defective Diurnal Changes of Food Intake, Plasma Glucose and Insulin in Rats with a Transplantable Islet Cell Tumour

Subcutaneous implantation of small fragments of a radiation-induced transplantable rat insulinoma into the subscapular region of 16- to 17-week-old male NEDH rats resulted, over a 22-day period, in the progressive development of marked hyperinsulinaemia and severe hypoglycaemia, despite a compensatory increase in food intake. Diurnal changes were examined at 3-hourly intervals for 24 h in control rats and tumour-bearing rats at 20-21 days after transplantation. The control animals exhibited distinct diurnal changes of food intake, glucose and insulin concentrations. Food intake was greatest between 17.00 and 23.00 h; plasma insulin was greatest between 20.00 and 23.00 h, and plasma glucose was raised at 20.00, 02.00 and 05.00 h, compared with the other times. In contrast, insulinoma-bearing rats displayed no diurnal changes other than a small decrease in food intake between 05.00 and 11.00 h. Plasma glucose and insulin concentrations were significantly different from control rats at all times, and food intake was significantly increased between 23.00 and 17.00 h. These observations demonstrate that the transplantable insulinoma not only causes hyperinsulinaemia and hypoglycaemia but results in hyperphagia and defective diurnal changes of food intake, plasma glucose and insulin concentrations. Interruption of nutrient intake by withdrawal of food for 6 h exacerbated the hypoglycaemia of insulinoma-bearing rats leading to coma.

Effects of amino acids, hormones and drugs on insulin release and 45Ca uptake by transplantable rat insulinoma cells maintained in tissue culture.

1. Acute effects of amino acids, hormones and drugs on transplantable rat insulinoma cells were examined after 2-3 days culture in RPMI-1640 (11.1 mM glucose) to eliminate necrotic cells and counter prior hypoglycaemia. 2. At 2.6 mM Ca2+, rat insulinoma cells (greater than 95% viability) released 48-97 ng insulin/10(6) cells during 60 min incubations with uptake of 1.0-1.8 nmol 45Ca/10(6) cells. 3. Insulin release and 45Ca uptake by rat insulinoma cells were not modified by arginine, leucine, 2-ketoisocaproate, tolbutamide, glibenclamide, somatostatin, adrenaline, noradrenaline, diazoxide or cyproheptadiene. 4. Responsiveness to acetylcholine (stimulation of insulin release and 45Ca uptake) and to GIP (stimulation of insulin release) was demonstrated. Thiol reagents (CMBS, CPDS and DTNB) and agents affecting microtubules-microfilaments (colchicine, vinblastine and cytochalasin B) enhanced insulin release. 5. The results suggest that rat insulinoma cells exhibit a generalized defect in the regulation of insulin release by nutrients, hormones and drugs which act in pancreatic B-cells by alteration of cellular Ca2+. Responsiveness to agents affecting insulin release through alternative mechanisms appears to be retained.

Reversal of diabetes by syngeneic transplantation of a radiation-induced rat insulinoma

The growth and metabolic effects of a radiation-induced rat insulinoma were examined after subcutaneous subscapular transplantation into normal and streptozotocin diabetic NEDH rats. Streptozotocin diabetic rats exhibited hyperglycaemia, hypoinsulinaemia, impaired glucose tolerance without an insulin response, polyuria, polydipsia, hyperphagia and weight loss. Transplantation of tumour fragments gradually improved the physical and metabolic state over the following 3 weeks. Coincident with a progressive rise in plasma insulin between 10 and 17 days, the diabetic rats gained weight and reduced their food intake. The rats remained hyperglycaemic during this time, but developed hypoglycaemia with marked hyperinsulinaemia by 24 days. Furthermore, plasma glucose and insulin concentrations were not increased by an intraperitoneal glucose challenge, indicating greatly accelerated glucose clearance. Both the streptozotocin-treated and normal insulinoma-bearing rats incurred a fatal hypoglycaemic coma by 28-33 days after transplantation. Final body weights, tumour weights and concentrations of glucose and insulin were similar in the two groups. This study demonstrates reversal of streptozotocin diabetes by insulinoma transplantation. The hyperglycaemia and the accompanying diabetic environment did not modify tumour growth and development.

Effects of cationic modification on 45Ca uptake and insulin release by transplantable rat insulinoma cells maintained in tissue culture.

1. Acute effects of cations on 45Ca uptake and insulin release by transplantable rat insulinoma cells were examined after 2-3 days culture in RPMI-1640 containing 11.1 mM glucose. 2. At 2.6 mM Ca2+, rat insulinoma cells (greater than 95% viability) released 78-158 ng insulin/10(6) cells during 60 min incubation with uptake at 2.19-3.24 nmol 45Ca/10(6) cells. 3. Addition of 2 mM La3+, Co2+, Mn2+, Zn2+ or Ba2+ did not affect 45Ca uptake. Insulin release was also unaffected by these cations with the exception of 87% inhibition in the presence of La3+ or Zn2+. 4. Omission of 5.9 mM K+, 1.2 mM Mg2+, 115 mM Na+ or H+ (pH 8.5) did not affect 45Ca uptake or insulin release, irrespective of osmotic compensation using choline chloride or sucrose. Rat insulinoma cells were similarly unresponsive to addition of 30.9 mM K+, 12 mM Mg2+ or H+ (pH 6.3). 5. Omission of 2.6 mM Ca2+ (with or without addition of 1 mM EGTA) or addition of 20.5 mM Ca2+ did not affect insulin release. 6. The results indicate that rat insulinoma cells are little affected by cationic modifications which have profound effects on Ca2+ handling and insulin release by pancreatic beta-cells. Dysregulation of insulin release by insulinoma cells is associated with marked irregularities in the control of transmembrane Ca2+ fluxes and sensitivity to extracellular Ca2+.