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Rat insulinoma-derived INS-1 cells constitute a widely used beta-cell surrogate. However, due to their nonclonal nature, INS-1 cells are heterogeneous and are not stable over extended culture periods. We have isolated clonal INS-1E cells from parental INS-1 based on both their insulin content and their secretory responses to glucose. Here we describe the stable differentiated INS-1E beta-cell phenotype over 116 passages (no. 27-142) representing a 2.2-yr continuous follow-up. INS-1E cells can be safely cultured and used within passages 40-100 with average insulin contents of 2.30 +/- 0.11 microg/million cells. Glucose-induced insulin secretion was dose-related and similar to rat islet responses. Secretion saturated with a 6.2-fold increase at 15 mm glucose, showing a 50% effective concentration of 10.4 mm. Secretory responses to amino acids and sulfonylurea were similar to those of islets. Moreover, INS-1E cells retained the amplifying pathway, as judged by glucose-evoked augmentation of insulin release in a depolarized state. Regarding metabolic parameters, INS-1E cells exhibited glucose dose-dependent elevations of NAD(P)H, cytosolic Ca(2+), and mitochondrial Ca(2+) levels. In contrast, mitochondrial membrane potential, ATP levels, and cell membrane potential were all fully activated by 7.5 mm glucose. Using the perforated patch clamp technique, 7.5 and 15 mm glucose elicited electrical activity to a similar degree. A K(ATP) current was identified in whole cell voltage clamp using diazoxide and tolbutamide. As in native beta-cells, tolbutamide induced electrical activity, indicating that the K(ATP)conductance is important in setting the resting potential. Therefore, INS-1E cells represent a stable and valuable beta-cell model.

Accumulating evidence suggests that the cytosolic calcium-independent phospholipase A(2) (iPLA(2)beta) manifests a signaling role in insulin-secreting (INS-1) beta-cells. Earlier, we reported that insulin-secretory responses to cAMP-elevating agents are amplified in iPLA(2)beta-overexpressing INS-1 cells (Ma Z, Ramanadham S, Bohrer A, Wohltmann M, Zhang S, and Turk J. J Biol Chem 276: 13198-13208, 2001). Here, immunofluorescence, immunoaffinity, and enzymatic activity analyses are used to examine distribution of iPLA(2)beta in stimulated INS-1 cells in greater detail. Overexpression of iPLA(2)beta in INS-1 cells leads to increased accumulation of iPLA(2)beta in the nuclear fraction. Increasing glucose concentrations alone results in modest increases in insulin secretion, relative to parental cells, and in nuclear accumulation of the iPLA(2)beta protein. In contrast, cAMP-elevating agents induce robust increases in insulin secretion and in time-dependent nuclear accumulation of iPLA(2)beta fluorescence, which is reflected by increases in nuclear iPLA(2)beta protein content and specific enzymatic activity. The stimulated effects are significantly attenuated in the presence of cell-permeable inhibitors of protein phosphorylation and glycosylation. These findings suggest that conditions that amplify insulin secretion promote translocation of beta-cell iPLA(2)beta to the nuclei, where it may serve a crucial signaling role.

We and others have previously shown that insulin-secreting cells of the pancreas express high levels of SNAP-25 (synaptosomal-associated protein of 25 kDa), a 206-amino acid t-SNARE (target soluble N-ethylmaleimide-sensitive factor attachment protein receptors) implicated in synaptic vesicle exocytosis. In the present study, we show that SNAP-25 is required for insulin secretion by transient transfection of Botulinum Neurotoxin A (BoNT/A) into insulin-secreting HIT-T15 cells. Transient expression of BoNT/A cleaved the endogenous as well as overexpressed SNAP-25 proteins and caused significant reductions in K+ and glucose-evoked secretion of insulin. To determine whether the inhibition of release was due to the depletion of functional SNAP-25 or the accumulation of proteolytic by-products, we transfected cells with SNAP-25 proteins from which the C-terminal nine amino acids had been deleted to mimic the effects of the toxin. This modified SNAP-25 (amino acids 1-197) remained bound to the plasma membrane but was as effective as the toxin at inhibiting insulin secretion. Microfluorimetry revealed that the inhibition of secretion was due neither to changes in basal cytosolic Ca2+ levels nor in Ca2+ influx evoked by K(+)-mediated plasma membrane depolarization. Electron microscopy revealed that cells transfected with either BoNT/A or truncated SNAP-25 contained significantly higher numbers of insulin granules, many of which clustered close to the plasma membrane. Together, these results demonstrate that functional SNAP-25 proteins are required for insulin secretion and suggest that the inhibitory action of BoNT/A toxin on insulin secretion is in part caused by the production of the plasma membrane-bound cleavage product, which itself interferes with insulin granule docking and fusion.

The arylpiperazine L-686,398 was described as an oral hypoglycemic agent and is shown to be an insulin secretagogue in vitro. The characteristics of its activity were similar to those of the incretin glucagon-like peptide I (GLP-I). We demonstrate that both the peptide and L-686,398 increase the accumulation of cAMP in isolated ob/ob mouse pancreatic islet cells, but by different mechanisms. Although GLP-I activates adenylate cyclase, the arylpiperazine has no effect on this enzyme or on the binding of 125I-labeled GLP-I to its receptor on RINm5F rat insulinoma cell membranes. However, L-686,398 inhibits the total cAMP phosphodiesterase (PDE) activity in homogenates of ob/ob mouse pancreatic islets with an EC50 of approximately 50 mumol/l. To determine the mechanism of PDE inhibition by the arylpiperazine and to examine its specificity, we studied the kinetics of arylpiperazine inhibition of two recombinant PDEs. The arylpiperazine is a competitive inhibitor of both a human heart type III PDE and a rat type IV-D PDE. Inhibition of the type III and IV isozymes are characterized by Ki values of 27 and 5 mumol/l, respectively. Although not extremely potent, the arylpiperazine does exhibit modest selectivity between these PDEs. The observation that L-686,398 acts as a PDE inhibitor suggests that exploration for beta-cell-specific PDE isoforms may reveal novel PDEs as targets for the development of therapeutically useful glucose-dependent insulin secretagogues.

The differentiating agent sodium butyrate inhibits proliferation and stimulates cell-specific hormone expression in rat insulinoma cells. In this study, we investigated the effect of sodium butyrate on neuroendocrine cytodifferentiation in the rat insulinoma subclone, RINm5F. The cells were cultured with 0.5, 1, or 1.5 mM sodium butyrate for up to 72 h. Ultrastructurally, cells cultured with 1 mM sodium butyrate revealed a more differentiated appearance with an induction of cellular compartments involved in regulated insulin secretion. Morphometric analysis showed a significant elevation of neuroendocrine granule density. The total area of the specific granules was increased after incubation with 1 mM sodium butyrate for 48 and 72 h. Proliferation of RINm5F cells was inhibited by sodium butyrate in a dose-dependent manner. DNA production ceased completely within 24 h at 1.5 mM sodium butyrate. This concentration of sodium butyrate increased the cellular insulin content 8.9-fold and the insulin production 2-fold after 72 h. The insulin release was reduced from 79 +/- 3.5% in controls to 37 +/- 5.6% of total in a 24-h incubation period after 3 days of culture with 1.5 mM sodium butyrate. Insulin mRNA levels increased to a maximum of 324% compared with controls after 48 h of culture with 1.5 mM sodium butyrate. Chromogranin A mRNA levels increased to a similar extent (368 +/- 26%), whereas sodium butyrate did not stimulate the expression of synaptophysin, a major membrane component of small neuroendocrine vesicles. In conclusion, our data suggest the selective induction of neuroendocrine cytodifferentiation by sodium butyrate in RINm5F cells.(ABSTRACT TRUNCATED AT 250 WORDS)

The cis-acting sequences that mediate insulin gene expression exclusively in pancreatic islet beta-cells are localized within the 5'-flanking region between nucleotides -340 and -91. We have identified an evolutionarily conserved, A+T-rich element at -201/-196 basepairs in the rat insulin II gene that is essential for efficient expression in beta-cells. Affinity-purified antibody to the XIHbox 8 protein super-shifted the major beta-cell-activator factor complex binding to the -201/-196 element. XIHbox 8 is a Xenopus endoderm-specific homeodomain protein whose expression is restricted to the nucleus of endodermal cells of the duodenum and developing pancreas. Antibody to XIHbox 8 specifically interacts with a 47-kilodalton protein present in this DNA complex. Immunohistochemical studies revealed XIHbox 8-like proteins within the nucleus of almost all mouse islet beta-cells and a subset of islet alpha- and beta-cells. These results are consistent with the proposal that an XIHbox 8-related homeoprotein of 47 kilodalton is required for expression of the mammalian insulin gene in beta-cells. Experiments conducted with antiserum raised to somatostatin transcription factor-1 (STF-1), a recently isolated mammalian XIHbox 8-related homeoprotein, indicate that the STF-1 protein is the mammalian homolog of Xenopus XIHbox 8.

Hamster islet tumor (HIT) cells retain much of the capacity of normal beta cells to act as glucose sensors. When stimulated with glucose or glucose plus forskolin, HIT cells release much more insulin than unstimulated cells. Ultrastructural analysis reveals that the secretory product of these cells is stored in membrane-bound granules that associate with microtubules under certain circumstances. Immunofluorescence studies using insulin antibody confirm the presence of insulin in granular structures in these cells. The microtubule inhibitor Nocodazole reduces the number of polymerized microtubules and inhibits the sustained phase of insulin secretion in HIT cells. Thus, the structural integrity of microtubules is important for the sustained phase of the insulin secretion to occur. The microtubule stabilizing drug taxol does not decrease insulin secretion. Since taxol blocks microtubule depolymerization, microtubule polymerization-depolymerization alone does not appear to be responsible for insulin granule transport. The increased use of these drugs in cancer research and therapy makes it important to understand their effects on insulin secretion.

Nontumoral endocrine pancreas from three patients with malignant vasoactive intestinal polypeptide (VIP)-omas and the Verner-Morrison (watery diarrhea, hypokalemia, and hypoachlorhydria) syndrome was studied immunocytochemically, ultrastructurally, and morphometrically. Compared with normal islets from control subjects, those of the VIPoma-associated pancreas showed a decrease of immunoreactive insulin in B-cells associated with cytological features indicative of enhanced insulin synthesis and secretion and an increase in the number of immunoreactive somatostatin- and pancreatic polypeptide-containing cells, in the absence of ultrastructural signs of modified secretory activity. No substantial alterations of A-cells were observed. In addition, images of diffuse de novo formation of ducts and islet tissue were often found. Possible mechanisms involved in determining the above changes are discussed.

New insulin-secreting cell lines (INS-1 and INS-2) were established from cells isolated from an x-ray-induced rat transplantable insulinoma. The continuous growth of these cells was found to be dependent on the reducing agent 2-mercaptoethanol. Removal of this thiol compound caused a 15-fold drop in total cellular glutathione levels. These cells proliferated slowly (population doubling time about 100 h) and, in general, showed morphological characteristics typical of native beta-cells. Most cells stained positive for insulin and did not react with antibodies against the other islet hormones. The content of immunoreactive insulin was about 8 micrograms/10(6) cells, corresponding to 20% of the native beta-cell content. These cells synthesized both proinsulin I and II and displayed conversion rates of the two precursor hormones similar to those observed in rat islets. However, glucose failed to stimulate the rate of proinsulin biosynthesis. In static incubations, glucose stimulated insulin secretion from floating cell clusters or from attached cells. Under perifusion conditions, 10 mM but not 1 mM glucose enhanced secretion 2.2-fold. In the presence of forskolin and 3-isobutyl-1-methylxanthine, increase of glucose concentration from 2.8-20 mM caused a 4-fold enhancement of the rate of secretion. Glucose also depolarized INS-1 cells and raised the concentration of cytosolic Ca2+. This suggests that glucose is still capable of eliciting part of the ionic events at the plasma membrane, which leads to insulin secretion. The structural and functional characteristics of INS-1 cells remained unchanged over a period of 2 yr (about 80 passages). Although INS-2 cells have not been fully characterized, their insulin content was similar to that of INS-1 cells and they also remain partially sensitive to glucose as a secretagogue. INS-1 cells retain beta-cell surface antigens, as revealed by reactivity with the antigangloside monoclonal antibodies R2D6 and A2B5. These findings indicate that INS-1 cells have remained stable and retain a high degree of differentiation which should make them a suitable model for studying various aspects of beta-cell function.

The cytokine interleukin-1 beta may have an important role in the autoimmune mediated damage of pancreatic Beta cells in insulin-dependent diabetes mellitus. In the present study we have investigated the effects of an interleukin-1 receptor antagonist protein, a blocker of the type I interleukin-1 receptor, on the suppressive actions of recombinant interleukin-1 beta on insulin-producing cells. Brief exposure (1-2 h) of rat and mouse pancreatic islets to 10 ng/ml recombinant interleukin-1 beta induced an 70-80% inhibition of insulin response to glucose after 12 h. These effects were completely counteracted by co-incubation with 100 ng/ml interleukin-1 receptor antagonist protein. When rat islets were cultured for 48 h in the presence of recombinant interleukin-1 beta (5 ng/ml) higher concentrations of interleukin-1 receptor antagonist protein (5000 ng/ml) were required to protect Beta-cell function. Interleukin-1 receptor antagonist protein also counteracted the inhibitory effects of recombinant interleukin-1 beta on the growth of the rat insulinoma cell line RINm5F. These data suggest that interleukin-1 receptor antagonist protein can protect insulin-producing cells from the deleterious effects of recombinant interleukin-1 beta, and that these cells possess type I interleukin-1 receptors.

T LYMPHOCYTES reactive to pancreatic beta-cells are thought to have a central role in the autoimmune process leading to type 1 (insulin-dependent) diabetes, but the molecular targets of these T cells have not yet been defined. As identification of such antigens may enable measures to be developed to prevent the disease, we have characterized an antigen that is recognized by insulinoma membrane-reactive T-cell clones established from a newly diagnosed type-1 diabetes patient. Subcellular fractionation studies using rat insulinoma indicate that the antigenic determinant recognized by one of these clones is an integral membrane component of the insulin secretory granule. After a 5,000-fold purification, we have defined the antigen as a monomer of relative molecular mass 38,000. As granular membrane proteins are transiently exposed on the cell surface during exocytosis, their accessibility to components of the immune system may be a function of the secretory activity of beta-cells.

Cytokine effects on permanent cell lines of transformed mouse pancreatic alpha- and beta-cells were compared. The beta-tumor cell 1 (beta TC1) line (from an adenoma created in transgenic mice expressing the SV40 large T-antigen oncogene under control of the rat insulin II promoter) produced insulin predominantly, although small quantities of intracellular glucagon (100:1 insulin to glucagon) were detectable by radioimmunoassay. The alpha TC1 line (from an adenoma created in transgenic mice expressing the SV40 large T-antigen oncogene under control of the rat preproglucagon promoter) produced not only glucagon but also considerable quantities of insulin (4:1 glucagon to insulin) and preproinsulin mRNA. We therefore cloned alpha TC1 cells and obtained 12 glucagon-producing clonal cell lines that did not produce levels of insulin detectable by radioimmunoassay. Analysis by Northern blotting of total RNA from two lines, alpha TC1 clones 6 and 9, confirmed the absence of preproinsulin mRNA. No somatostatin or pancreatic polypeptide was detected by immunohistochemical staining in alpha TC1 clones 6 or 9 or beta TC1 cells. Rat recombinant gamma-interferon (IFN-gamma; 5-250 U/ml) or mouse recombinant interleukin 1 (IL-1; 1-25 U/ml) individually inhibited DNA synthesis in beta TC1 cells after 3 days of treatment. The two cytokines in combination acted synergistically to further depress DNA synthesis and increase cytotoxicity. In contrast, alpha TC1 clone 9 cells were not sensitive to inhibition of DNA synthesis by each cytokine individually, although glucagon synthesis was inhibited. The combination of these cytokines caused marked inhibition of DNA and glucagon syntheses in alpha TC1 clone 9 cells. alpha TC1 clone 9 cells were somewhat more resistant to the cytotoxic action of the combined cytokines than were beta TC1 cells. Incubation with 50 U/ml IFN-gamma induced class II MHC molecules (I-Ab, I-Ad, and I-Ed) and enhanced the constitutive expression of class I molecules (H-2Kb and H-2Kd) on the cell surfaces of beta TC1, uncloned alpha TC1, and alpha TC1 clones 6 and 9. Thus, these cell lines are heterozygous for MHC alleles derived from both parental strains used in the construction of the transgenic mice [C57BL/6J (H-2b) and DBA/2J (H-2d)]. Class II gene transcription induced by IFN-gamma was confirmed in beta TC1 and alpha TC1 clone 9 cells by Northern blot analysis with A alpha-, A beta-, E alpha, and E beta-DNA probes.(ABSTRACT TRUNCATED AT 400 WORDS)

In view of reports that prostaglandins influence insulin and glucagon secretion, we have studied PGE2, insulin and glucagon release from fragments (15-20 mg) of human insulinoma tissue incubated in vitro in the absence or presence of indomethacin (100 mumol/liter) an inhibitor of prostaglandin synthesis. Acid-ethanol extraction of this tissue showed the following hormonal contents : insulin : 7.17 U and glucagon 84.4 ng per g of tissue (wet weight). In the absence of indomethacin, the mean release of PGE2, insulin and glucagon into the incubation medium was 3.65 +/- 1.3 pmol, 10.5 +/- 1.2 mU and 708.4 +/- 141.8 pg in two hours (mean of 5 vials containing 2 fragments of 15-20 mg of tissue). PGE2 release was significantly inhibited in the presence of indomethacin (0.89 +/- 0.23 pmol). This effect was associated with a significantly higher insulin (16.8 +/- 1.9 mU/2 hours) and lower glucagon (176 +/- 19.7 pg/2 hours) release. These results support the view that insular tissue possesses a prostaglandin synthesis system which positively modulates glucagon secretion whereas it negatively influences insulin release.

Nineteen spontaneous pancreatic islet cell tumors were studied in 26-month-old control Fischer 344 rats. Seventeen of 19 (89%) tumors demonstrated insulin reactivity using peroxidase-antiperoxidase immunocytochemistry. None of ten tumors had positive glucagon immunoreactivity. Immunocytochemical and ultrastructural observations suggested that spontaneous occurring islet cell tumors of F344 rats were composed exclusively of beta cells.

A female patient is described with a single pancreatic tumour producing vasoactive intestinal polypeptide (VIP), insulin, and pancreatic polypeptide. The initial presentation was with diarrhoea and hypokalaemia and a raised plasma VIP was demonstrated. Her symptoms improved with metoclopramide administration and absolute concentrations of 28 aminoacid (peak IV) VIP were found to have fallen. She then developed hypoglycaemia with hyperinsulinism. All symptoms resolved after surgical excision. This case emphasises the potential of these tumours to contain more than one endocrine cell type synthesising different biologically active peptides.

Density gradient centrifugation techniques, using iso-osmotic colloidal silica suspensions (Percoll), were developed for the isolation of insulin secretory granules from a transplantable rat islet cell tumour. These procedures were readily completed within 7 h and from each animal yielded approximately 1 mg of granule protein. The isolated granules were essentially free of other subcellular organelles as evaluated by their contents of marker proteins, electron microscopy and by electrophoretic analyses. Their susceptibilities to lysis at low osmotic strength, at pH values above 7 or in media containing sodium ions were similar to those of granules partially purified from islets. Insulin comprised 50-60% of the total granule protein when determined by immunoassay or by densitometry of electrophoretic profiles. The proinsulin content was marginally higher than that of islets, as was the ratio of insulins I to II. Electrophoretic analyses revealed that the secretory granules contained 150 or more proteins besides insulin-related peptides. The majority of these had acidic isoelectric points and were located both within the granule interior and its enveloping membrane.

Dissociated human insulinoma cells were plated onto plastic multiwell dishes. Cells were maintained for 1 mo on plastic with three passages. Cultures consisted of small colonies with some areas of stratification and few intercellular spaces. Ultrastructural studies indicated that cultured cells had epithelial features with desmosomes at cell-to-cell contacts and intermediate filaments in addition to secretory granules in the cytoplasm. Insulin and C-peptide were released in equimolar amounts in culture media. When challenged for 30 min with 16.7 mM glucose, 1 mM 3-isobutyl-1-methylxanthine, 4 mM tolbutamide, or 10(-6) M glucagon, insulinoma cells responded by a 1.5-, 1.5-, 2-, or 3-fold increase, respectively, in insulin release above baseline levels. A 15-min challenge with 10(-5) M isoproterenol increased insulin secretion by 1.85-fold. By indirect immunofluorescence, an anti-insulin antibody reacted positively with cell cytoplasm, whereas anti-somatostatin and anti-glucagon antibodies did not. Insulinoma cell surface expressed class I MHC molecules but not class II molecules. Immediately after isolation, crude insulinoma cells were contaminated by 2% of DR+ cells from nonislet components that disappeared after several weeks in culture. The ability of insulinoma cells to stimulate allogenic T-lymphocyte proliferation was assessed by [3H]thymidine incorporation in mixed culture combinations. Crude insulinoma cells elicited a strong lymphoproliferative response with a stimulation index ranging between 3.5 and 7, whereas no stimulation was found after 1 mo in culture. It is postulated that absence of class II-positive cells in the stimulatory cell preparation conditioned this immune tolerance across the major histocompatibility barrier.(ABSTRACT TRUNCATED AT 250 WORDS)

It has been shown, by using the immunogold technique, that C-peptide and insulin are co-localized in the mature granules of human pancreatic beta cells and insulinomas with typical granules. The mean gold bead densities of both C-peptide and insulin were at least twice as high in the normal pancreas when compared with the insulinomas. The mean granule diameter of the insulinoma cells (D = 0.30 +/- 0.12 micron) was smaller than that of human pancreatic cells (D = 0.45 +/- 0.15 micron). The morphometric data indicate that each of the antigens (C-peptide and insulin) is distributed similarly in the halos and the dense cores of the beta granules. Thus, no topological segregation of these two antigens occurs within the beta granules of either normal human pancreas or insulinomas.

In a 46-year old Caucasian woman, the authors report a B-cell adenoma with plasma immunoreactive insulin (IRI) ranging from 10 to 32 microunits/ml, despite severe spontaneous hypoglycemia. In a peroperative sample withdrawn from the portal vein, normal IRI (40 micromicron/ml) in the presence of high insulin-like activity (290 microunits/ml) was observed by using a biological assay performed on rat epididymal fat tissue. Furthermore, this material did not cross-react with insulin antibodies and was undetectable in systemic venous samples. Although further identification by chromatographic extraction was not performed, the substance secreted by the tumor is probably identical to the non-suppressible insulin-like activity (NSILA) isolated by Froesch and responsible for hypoglycemia in a few cases of extrapancreatic tumors. The absence of this material in systemic samples indicates an immediate removal by a single passage through the liver.

Amyloid-containing islets of Langerhans from the pancreas of 75 diabetics and one case of an amyloid-containing insulinoma were investigated. By light and electron microscopy, amyloid deposits were observed between the B cells and adjacent capillaries. The cytoplasm of B cells and extracellular amyloid display immunohistological binding of anti-insulin antibody. Correspondingly, ferritin-labeled anti-insulin antibody was found by electron microscopy on and between the amyloid fibrils. Insulin or proinsulin (or a protein closely related to insulin) thus appears to be a component of the protein which constitutes the amyloid. The molecular weight and mode of deposition of this form of amyloid are in agreement with other amyloidoses of the hormonal type (AH-type).

Subtotal pancreatectomy specimens from one case of nesidioblastosis, one case of focal adenomatosis, and two cases of insulin-producing islet-cell tumours were studied with special reference to their production of pro-insulin, C-peptide and insulin, and their contents of argyrophil parenchymal cells. Specific immunostaining revealed the presence of abundant cells reacting with pro-insulin, C-peptide, and insulin antiserum; at least the great majority of them were obviously non-argyrophil cells. The content of extractable immunoreactive insulin (IRI) was higher in the cases of nesidioblastosis and focal adenomatosis than in the two insulomas. Molar ratios of IRI to C-peptide immunoreactivity (CPR) varied between 7 and 100. Gel filtration analysis of the extracts revealed two peaks of CPR, corresponding to 3,000 and 10,000 daltons, respectively. Ultrastructurally, the insulin cells in cases of nesidioblastosis and focal adenomatosis contained numerous typical beta granules. In the islet-cell neoplasms some "polycrine" islet cells were also found, containing typical as well as atypical granules with electron dense or pale cores. Some cells even showed a mixture of apparent beta and alpha granules. Despite structural differences and variable contents of IRI and CPR, the predominance of cells reactive with antibodies to pro-insulin, C-peptide, and insulin, and the absence of argyrophil pro-insulin cells in adenomatosis and insulomas indicates that the hormonal products of these parenchymal cells are not any chemically modified insulin or any other member of the insulin family.

This paper reports on an ultrastructural and electron-microscopic immunocytochemical study of pancreatic B cells from normal mice, pancreatic B cells and derivative tumors from transgenic mice, and tissue from human pancreatic B-cell tumors. In normal and neoplastic B cells from both species, typical immature and mature beta-granules (with spherical cores of variable density) were observed, whereas typical beta-granules with a crystalloid core were only present in human B cells (normal and tumor). A small number of atypical granules were found in distinct neoplastic cells which contained no typical beta-granules. The atypical granules were smaller (100-200 nm diameter) than typical beta-granules (250-450 nm diameter) seen in other cells. Immunoreactivity for proinsulin was localized only to immature granules, whereas insulin and C-peptide immunoreactivities were demonstrated in atypical, immature, and mature granules. In transgenic mouse and human B-cell tumors, insulin immunoreactivity was consistently weaker than the immunostaining for C-peptide. An intragranular, topographic segregation of immunoreactive C-peptide was observed in a population of transgenic tumor cells. Our results showed similarities in antigenic distribution and only slight differences in morphology between human and mouse B cells. Therefore, the transgenic mouse system may prove to be an effective model for studying mammalian B-cell tumorigenesis.

Five cell lines have been derived from a rat transplantable islet cell tumour using two different methods. The lines differ in morphology and contain and release different amounts of insulin and glucagon (insulin content, 1-90 pmol/10(6) cells; insulin release, 6-250 pmol/10(6) cells per 24 h; glucagon content, less than 0.005-35 pmol/10(6) cells; glucagon release, less than 0.05-10 pmol/10(6) cells per 24 h). All the lines responded to the presence of the secretagogues leucine (20 mmol/l) plus theophylline (5 mmol/l) by increasing the rate of release of insulin approximately twofold. A high extracellular concentration of potassium (40 mmol/l) caused a three- to tenfold calcium-dependent increase in release of insulin and a parallel release of glucagon. Increasing the concentration of glucose from 2.8 to 16.7 mmol/l did not alter the rate of insulin release by any of the cell lines.