Characterization of a novel glucose-responsive insulin-secreting cell line, BRIN-BD11, produced by electrofusion.

A novel insulin-secreting cell line (BRIN-BD11) was established after electrofusion of RINm5F cells with New England Deaconess Hospital rat pancreatic islet cells. Wells of cell fusion mixture with insulin output 5-10 times greater than parent RINm5F cells were subcultured with eventual establishment of clones, including BRIN-BD11. Morphological studies established that these cells grow as monolayers with epithelioid characteristics, maintaining stability in tissue culture for > 50 passages. Culture of these cells for 24 h at 5.6-33.3 mmol/l glucose revealed a 1.8- to 2.0-fold increase of insulin output compared with 1.4 mmol/l glucose. Dynamic insulin release was recorded in response to 16.7 mmol/l glucose, resulting in a rapid threefold insulin secretory peak followed by a sustained output slightly above basal. In acute 20-min tests, 4.2-16.7 mmol/l glucose evoked a stepwise two- to three-fold stimulation of insulin release. 3-Isobutyl-1-methylxanthine (1 mmol/l) served to increase basal and glucose-stimulated insulin release, shifting the threshold from 4.4 to 1.1 mmol/l glucose. Stimulation of insulin secretion with 16.7 mmol/l glucose was abolished by mannoheptulose or diazoxide (15 or 0.5 mmol/l). In contrast, glyceraldehyde (10 mmol/l) and 25 mmol/l K+ evoked 1.7- to 9.0-fold insulin responses. L-Alanine (10 mmol/l) evoked a twofold secretory response, which was potentiated 1.4-fold by increasing the Ca2+ concentration from 1.28 to 7.68 mmol/l. Forskolin (25 mumol/l) and phorbol 12-myristate 13-acetate (10 nmol/l) both increased insulin secretion in the presence of L-alanine (1.4- and 1.8-fold, respectively). Western blotting confirmed that BRIN-BD11 cells expressed the GLUT2 glucose transporter. This, coupled with a high glucokinase/hexokinase ratio in the cells, confirms an intact glucose sensing mechanism. High-performance liquid chromatography analysis demonstrated that insulin was the major product secreted under stimulatory conditions. Collectively, these data indicate that the BRIN-BD11 cell line represents an important stable glucose-responsive insulin-secreting beta-cell line for future studies.

Characteristics of BRIN-BG5 and BRIN-BG7, two novel glucose-responsive insulin-secreting cell lines produced by electrofusion.

Two hybrid insulin-secreting cell lines (BRIN-BG5 and BRIN-BG7) were established by the novel approach of electrofusing RINm5F cells with New England Deaconess Hospital rat pancreatic islet cells. Cells were selected from the fusion mixture on the basis of insulin output. Wells showing five to ten times greater insulin output than parental RINm5F cells were selected, subcultured and cloned. Clonal BRIN-BG5 and BRIN-G7 cells grow as monolayers with epithelial morphology. The differences in doubling time of 28 and 20 h respectively were associated with morphological differences; the growth pattern and insulin content of each cell line remaining stable for over 50 passages. In acute 20-min tests, both cell lines showed peak secretory responses (1.9- and 1.8-fold respectively) to 8.4 mmol/l glucose. Membrane depolarization with 25 mmol/l K+ evoked 3.7- and 3.9-fold increases in insulin output. L-Alanine (10 mmol/l) also served to promote 2.4- and 1.6-fold increases in insulin release respectively. Increasing the Ca2+ concentration from 1.28 to 7.68 mmol/l potentiated this effect by 1.8- and 1.5-fold. Incubation with forskolin (25 mumol/l) or phorbol-12-myristate 13-acetate (10 nmol/l), in the presence of L-alanine, similarly enhanced the secretory effect on BRIN-BG5 and BRIN-BG7 cells by 1.3- to 2.1-fold and 1.2- to 1.5-fold respectively. The presence of a functional glucose-sensing mechanism in both cell lines was confirmed by the demonstration of the glucose transporter GLUT-2 and measurement of glucokinase activity. These functional properties suggest that insulin-secreting BRIN-BG5 and BRIN-BG7 cells represent two useful glucose-responsive cell lines for future studies of the function of the pancreatic B-cell.

Substance P, neurokinin A and calcitonin gene-related peptide during development of the rat gastrointestinal tract.

Substance P, neurokinin A and calcitonin gene-related peptide (CGRP) were determined in the stomach and small intestine of rats during late foetal development and up to 35 days postnatal life. Concentrations of substance P in stomach and intestine increased from 14 gestational days to 3 days postpartum, and declined thereafter. Concentrations of neurokinin A in stomach declined from 14 days gestation over the period 3-35 postnatal days. In the intestine, concentrations of neurokinin A increased steadily from 14 days gestation to 21-35 postnatal days. Concentrations of CGRP in stomach and intestine declined from 14 days gestation to 7 postnatal days. Thereafter, concentrations of CGRP increased in both stomach and intestine. Total contents of each of the three peptides increased progressively with gestational and postnatal age in parallel with increasing stomach and intestinal weights. The results demonstrate different patterns of change in the concentrations of substance P, neurokinin A and CGRP during the dynamic phases of growth and maturation of the gastrointestinal tract in the foetal and postnatal rat.

Traditional plant treatments for diabetes. Studies in normal and streptozotocin diabetic mice.

The effects on glucose homeostasis of eleven plants used as traditional treatments for diabetes mellitus were evaluated in normal and streptozotocin diabetic mice. Dried leaves of agrimony (Agrimonia eupatoria), alfalfa (Medicago sativa), blackberry (Rubus fructicosus), celandine (Chelidonium majus), eucalyptus (Eucalyptus globulus), lady's mantle (Alchemilla vulgaris), and lily of the valley (Convallaria majalis); seeds of coriander (Coriandrum sativum); dried berries of juniper (Juniperus communis); bulbs of garlic (Allium sativum) and roots of liquorice (Glycyrhizza glabra) were studied. Each plant material was supplied in the diet (6.25% by weight) and some plants were additionally supplied as decoctions or infusions (1 g/400 ml) in place of drinking water to coincide with the traditional method of preparation. Food and fluid intake, body weight gain, plasma glucose and insulin concentrations in normal mice were not altered by 12 days of treatment with any of the plants. After administration of streptozotocin (200 mg/kg i.p.) on day 12 the development of hyperphagia, polydipsia, body weight loss, hyperglycaemia and hypoinsulinaemia were not affected by blackberry, celandine, lady's mantle or lily of the valley. Garlic and liquorice reduced the hyperphagia and polydipsia but did not significantly alter the hyperglycaemia or hypoinsulinaemia. Treatment with agrimony, alfalfa, coriander, eucalyptus and juniper reduced the level of hyperglycaemia during the development of streptozotocin diabetes. This was associated with reduced polydipsia (except coriander) and a reduced rate of body weight loss (except agrimony). Alfalfa initially countered the hypoinsulinaemic effect of streptozotocin, but the other treatments did not affect the fall in plasma insulin. The results suggest that certain traditional plant treatments for diabetes, namely agrimony, alfalfa, coriander, eucalyptus and juniper, can retard the development of streptozotocin diabetes in mice.

Effects of diets rich in sucrose, coconut fat and safflowerseed oil on the development of the obese hyperglycaemic (ob/ob) syndrome in mice.

The effect of dietary sucrose and fat in the form of coconut fat (rich in saturated fatty acids) or safflowerseed oil (rich in polyunsaturated fatty acids) was examined on the development of obesity and impaired glucose homeostasis in ob/ob mice. Isoenergetic high sucrose or high fat diets were fed to ob/ob mice from 3-11 weeks of age. Energy intake of mice fed diets rich in fat were similar, and exceeded that attained with the sucrose diet. Body weight gain was greatest in the sucrose-fed mice and least in those fed safflowerseed oil. With the exception of insulin sensitivity which was enhanced with safflowerseed oil, plasma concentrations of glucose and gastric inhibitory polypeptide (GIP), glucose tolerance, intestinal GIP content and the GIP response to oral fat were similar. However, mice fed the high sucrose diet exhibited markedly elevated plasma insulin concentrations and an enhanced pancreatic insulin content. Since the hyperinsulinaemic action of sucrose cannot be attributed to elevated GIP or glucose concentrations, the involvement of other insulin-releasing hormones released from the intestine by sucrose is suggested. The results indicate that the relative amounts of carbohydrate and fat in the diet have an important modulating effect on the development of the ob/ob syndrome. The type of fatty acids in the diet does not appear to be a particularly important determinant for expression of the ob gene.

Studies on the chemical and immunological destruction of insulin-secreting islet cell tumours.

Chemical and immunological destruction of insulin-secreting islet cell tumours were evaluated in vivo and in vitro using the transplantable radiation-induced NEDH rat insulinoma and the derived clonal RINm5F cell line. Administration of a large amount of polyclonal insulin antibody did not affect the development of hypoglycaemia, tumour weights or the survival of insulinoma-bearing rats. Streptozotocin (100 mg/kg body weight) evoked a rapid and sustained decrease of insulin concentrations, accompanied by tumour regression and elevation of plasma glucose. Administration of alloxan (200 mg/kg) was without effect. In vitro, streptozotocin and alloxan exerted approximately equipotent time-dependent and concentration-dependent cytotoxic effects on cultured insulinoma 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. Exposure of clonal RINm5F cells to the nitrosocompounds, N-methyl-N'-nitro-N-nitrosoguanidine and N-nitroso-N-methylurea, resulted in a particularly marked reduction in cell viability compared with streptozotocin.

Failure of glucose tolerance factor-containing Brewer's yeast to ameliorate spontaneous diabetes in C57BL/KsJ DB/DB mice.

Glucose tolerance factor (GTF), the biologically active form of Cr3+, has been ascribed a role in the potentiation of insulin action and glucose homeostasis. The present study investigated the effects of dietary supplementation with GTF-rich brewer's yeast (Saccharomyces cerevisiae) or GTF-deprived Torula yeast (Torulaspora delbrueckii) in genetically diabetic C57BL/KsJ db/db mice. At 15 weeks of age, db/db mice exhibited increased body weight, hyperphagia, hyperglycaemia, hyperinsulinaemia and increased glycosylated haemoglobins compared with control (+/+) mice. During 56 days consumption of diets supplemented with 50g brewer's yeast or Torula yeast per kg, body weights of both groups of db/db mice decreased by 35%, in association with 1.2-1.7 fold increases of food intake, plasma glucose, glycosylated haemoglobins and an 83% decrease of plasma insulin. With the exception of slightly decreased weight loss, addition of brewer's yeast as opposed to Torula yeast did not affect tissue or plasma chromium nor ameliorate any of the parameters monitored including glucose tolerance and insulin sensitivity at 52-54 days. These findings do not support the contention that the glucose intolerance of genetically diabetic C57BL/KsJ db/db mice partly reflects GTF deficiency.

Effects of dietary inorganic trivalent chromium (Cr3+) on the development of glucose homeostasis in rats.

Inorganic chromium (Cr3+) is classified as an essential trace element on the basis of a small dietary requirement for the maintenance of efficient glucose homeostasis. The present study has carefully scrutinised this claim by examining food intake, body weight gain, glycosylated haemoglobins, plasma glucose and insulin concentrations, glucose tolerance and insulin sensitivity in two groups of Wistar rats fed either a Cr3+-deficient (0.03 mg/kg) or Cr3+-containing (1 mg/kg) diet for 32 days from weaning at 3 weeks. At 53 days of age, control rats weighed 183 +/- 7 g (body weight gain 155 +/- 6 g, mean +/- SEM) and consumed 17 +/- 1 g diet/rat/day. Glycosylated haemoglobins, plasma glucose and plasma insulin were 2.5 +/- 1%, 6.4 +/- 0.3 mmol/l and 1.2 +/- 0.3 ng/ml, respectively. Compared with control rats, the rats fed the Cr3+-deficient diet consumed the same total amount of diet and exhibited up to a maximum 40% decrease in the chromium content of selected tissues at 53 days of age. At no time during the study did food intake, body weight, glycosylated haemoglobins or plasma concentrations of glucose and insulin differ between the two groups of rats. Furthermore, body weight gain and both glucose tolerance and insulin sensitivity were identical at 50-53 days of age. These results cast doubt on the significance of dietary trivalent chromium for the maintenance of glucose homeostasis in healthy animals.

Glycaemic effects of traditional European plant treatments for diabetes. Studies in normal and streptozotocin diabetic mice.

Twelve plants used for the traditional treatment of diabetes mellitus in northern Europe were studied using normal and streptozotocin diabetic mice to evaluate effects on glucose homeostasis. The plants were administered in the diet (6.25% by weight) and/or as decoctions or infusions in place of drinking water, to coincide with the traditional method of preparation. Treatment for 28 days with preparations of burdock (Arctium lappa), cashew (Anacardium occidentale), dandelion (Taraxacum officinale), elder (Sambucus nigra), fenugreek (Trigonella foenum-graecum), guayusa (Ilex guayusa), hop (Humulus lupulus), nettle (Urtica dioica), cultivated mushroom (Agaricus bisporus), periwinkle (Catharanthus roseus), sage (Salvia officinale), and wild carrot (Daucus carrota) did not affect the parameters of glucose homeostasis examined in normal mice (basal plasma glucose and insulin, glucose tolerance, insulin-induced hypoglycaemia and glycated haemoglobin). After administration of streptozotocin (200 mg/kg) burdock and nettle aggravated the diabetic condition, while cashew, dandelion, elder, fenugreek, hop, periwinkle, sage and wild carrot did not significantly affect the parameters of glucose homeostasis studied (basal glucose and insulin, insulin-induced hypoglycaemia, glycated haemoglobin and pancreatic insulin concentration). Guayusa and mushroom retarded the development of hyperglycaemia in streptozotocin diabetes and reduced the hyperphagia, polydipsia, body weight loss, and glycated haemoglobin. Mushroom also countered the initial reduction in plasma insulin and the reduction in pancreatic insulin concentration, and improved the hypoglycaemic effect of exogenous insulin. These studies suggest the presence of potentially useful antidiabetic agents in guayusa and mushroom.

Evaluation of traditional plant treatments for diabetes: studies in streptozotocin diabetic mice.

Seven plants and a herbal mixture used for traditional treatment of diabetes were studied in streptozotocin diabetic mice. The treatments were supplied as 6.25% by weight of the diet for 9 days. Consumption of diets containing bearberry (Arctostaphylos uva-ursi), golden seal (Hydrastis canadensis), mistletoe (Viscum album) and tarragon (Artemisia dracunculus) significantly reduced the hyperphagia and polydipsia associated with streptozotocin diabetes, but bayberry (Cinnamomum tamala), meadowsweet (Filipendula ulmaria), senna (Cassia occidentalis) and the herbal mixture did not alter these parameters. Bearberry, mistletoe and tarragon retarded the body weight loss but none of the eight treatments significantly altered plasma glucose or insulin concentrations. These studies suggest that bearberry, golden seal, mistletoe and tarragon may counter some of the symptoms of streptozotocin diabetes without, however, affecting glycemic control.

Tumour formation and insulin secretion by clonal RINm5F cells following repeated subcutaneous transplantation in NEDH rats.

The effects of repeated s.c. transplantation of clonal insulin-secreting RINm5F cells in NEDH rats on tumour morphology, insulin-glucose homeostasis and the function of RINm5F cells re-established in culture were examined after maintenance in vivo for periods of up to 308 days. Transplantation of RINm5F cells for ten consecutive passages consistently produced a single encapsulated vascularized tumour associated with the development in recipient rats of hyperinsulinaemia, hypoglycaemia and eventual neuroglycopenic coma. At the tenth passage, tumour-bearing rats exhibited a markedly enhanced rate of insulin-stimulated glucose uptake by 19 days, with evidence of a large and variable insulin response to i.p. glucose. Survival was 22 +/- 2 days, and resected RINm5F cell tumours exhibited weak immunostaining for insulin in scattered cells, with strands of fibrous tissue separating clusters of tumour cells many of which had distinct polarity. There was no detectable immunostaining for glucagon, somatostatin or pancreatic polypeptide. The insulin content and insulin secretory output of RINm5F cells re-established in culture after 20, 146, 259 or 308 days propagation in vivo were generally enhanced compared with non-passaged RINm5F cells. The magnitude of the effect was not appreciably affected by the duration of maintenance in vivo, but it was critically dependent upon the subsequent period of culture in vitro. Thus, whereas 2-day cultured RINm5F cells from the eighth tumour passage exhibited a greater than 100-fold increment of insulin content and release, with enhanced secretory responsiveness to leucine, arginine, theophylline, K+ (25 mmol/l) or Ca2+ (7.6 mmol/l), RINm5F cells cultured for a further 19 days had almost completely lost the attributes resulting from 259 days of maintenance in vivo. The results indicate substantial enhancement of the functional capabilities of RINm5F cells in vivo, and suggest that the resulting tumours afford a novel model in NEDH rats of responsive trabecular-type insulinomas.

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

Regulation of insulin release, membrane potential, transmembrane 45Ca fluxes and cytoplasmic free Ca2+ concentration, [Ca2+]i, was examined using suspensions of transplantable NEDH rat insulinoma cells previously cultured for 2-3 days to eliminate necrotic tumour cells and counter prior hypoglycaemia. Insulinoma cells displayed a resting [Ca2+]i of 94 +/- 8 nM (n = 17) and released 104 +/- 15 ng insulin 10(-6) cells (n = 7) during 60 min incubations with uptake of 2.7 +/- 0.2 nmol 45Ca 10(-6) cells (n = 7). High concentrations of glucose did not affect membrane potential, transmembrane 45Ca fluxes, [Ca2+]i or insulin release by insulinoma cells. K+ at 25 mM depolarised the plasma membrane, induced a small increase in 45Ca efflux and increased [Ca2+]i by 65%. This modest action was not associated with demonstrable effects on 45Ca uptake and insulin release. The effect of 25 mMK+ on [Ca2+]i was counteracted by D-600, but this blocker of voltage-activated Ca2+ channels and verapamil lacked effects on transmembrane 45Ca fluxes and insulin release. The Ca2+-calmodulin antagonist, trifluoroperazine, was also without effect on 45Ca fluxes and insulin release. Ca2+ ionophore ionomycin increased [Ca2+]i, whereas A23187 and X537A did not affect transmembrane 45Ca fluxes. Moreover, insulin release was independent of extracellular Ca2+ over the range 0-20.4 mM despite marked affects on transmembrane 45Ca fluxes and a greater than 4-fold change of [Ca2+]i. Dibutyryl cyclic AMP increased insulin release by 55% without affecting transmembrane 45Ca fluxes or [Ca2+]i. The phosphodiesterase inhibitor, theophylline, also enhanced insulin release by 10-36% with no change of 45Ca uptake. The effectiveness of theophylline was independent of extracellular Ca2+ over the range 0-10.2 mM. These results indicate that inappropriate Ca2+ regulation is a key pathogenic feature underlying the inappropriate insulin secretion of rat insulinoma cells.

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+.

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.

Defective regulation of insulin release and transmembrane Ca2+ fluxes by human islet cell tumours.

Regulation of insulin release and transmembrane Ca2+ fluxes was examined using pieces of 3 benign medullary-type insulinomas removed from the pancreas of female patients at surgery. Immunocytochemical staining confirmed the presence of insulin-containing cells with no demonstrable glucagon, somatostatin or pancreatic polypeptide. After 3 days of culture in RPMI-1640, tumour pieces released 11-158 mg insulin kg-1 dry wt during acute 60 min incubations with the concomitant uptake of 2-47 mmol 45Ca kg-1 into the intracellular lanthanum-nondisplaceable pool. At 2.56 mM Ca2+, glucose alone or in combination with glyceraldehyde, mannoheptulose or diazoxide did not modify insulin release or 45Ca uptake. Theophylline significantly increased insulin release from 2 tumours with a small stimulatory effect on the third. A depolarising concentration of K+ enhanced insulin release from one tumour but this was not associated with an increase of 45Ca uptake. Calcium antagonists, (verapamil, D-600 and trifluoroperazine) and calcium ionophores (A23187 and Br-X537A) failed to modify insulin release or 45Ca uptake by each of the two tumours tested. Evaluation of 45Ca efflux from one tumour confirmed the unresponsiveness to glucose, K+, verapamil and A23187. Prolonged culture of 2 tumours for up to 16 days was associated with the gradual decline of insulin release to a steady output of 2-15 ng 24 h-1. Addition of verapamil to the cultures inhibited insulin output from one tumour, but mannoheptulose or diazoxide were without effect. The results indicate that inappropriate insulin release from these 3 benign medullary-type insulinomas is associated with disturbances in the regulation of transmembrane Ca2+ fluxes.

Insulin secretion in vivo and in vitro from transplantable NEDH rat insulinoma and derived clonal RINm5F cell line.

The function of clonal insulin-secreting RINm5F cells was compared with parent tumoural B-cells from radiation-induced NEDH rat insulinoma and a RINm5Fr cell line established following transplantation of RINm5F cells in NEDH rat. After 3 days culture, tumoural B-cells contained 156 micrograms insulin/10(6) cells and released 57-82 ng insulin/10(6) cells/h during acute incubations at 2.6 mM Ca2+. RINm5F cells contained 0.56 ng insulin/10(6) cells and released 62-181 pg insulin/10(6) cells/h. Unlike tumoural B-cells, secretion was stimulated 1.7-2.4-fold by 5 mM theophylline, 1 microM glucagon, 25 mM K+, or 7.6 mM Ca2+. Subscapular transplantation of cultured tumoural B-cells or RINm5F cells (2.8 X 10(7) cells/rat) resulted in an encapsulated tumour associated with progressive hyperinsulinaemia, hypoglycaemia and death by 28-46 days and 39-44 days respectively. A RINm5Fr cell line was established in culture from a 19 g tumour 20 days after transplantation. RINm5Fr cells contained 2.69 ng insulin/10(6) cells and released 385-1,017 pg insulin/10(6) cells/h (p less than 0.001 compared with RINm5F cells). Secretion was not augmented by glucose, but at 16.7 mM glucose it was stimulated 1.5-fold by 5 mM theophylline, 1.6-fold by 1 microM glucagon and inhibited 0.6-fold by somatostatin. At 5.6 mM glucose, secretion was stimulated 1.6-fold by 25 mM K+, 2.5-fold by 7.8 mM Ca2+, 2.1-fold by 20 microM A23187, 1.5-fold by 20 mM leucine and 1.4-fold by 100 microM tolbutamide. These data indicate fundamental differences between rat insulinoma cells and the derived RIN cell lines. Transplantation is a useful means to enhance the function of RINm5F cells.