Primary Islet Cell Research Articles

NPC1 is required for postnatal islet β cell differentiation by maintaining mitochondria turnover.

Rationale: NPC1 is a protein localized on the lysosome membrane regulating intracellular cholesterol transportation and maintaining normal lysosome function. GWAS studies have found that NPC1 variants in T2D was a pancreatic islet expression quantitative trait locus, suggesting a potential role of NPC1 in T2D islet pathophysiology. Methods: Two-week-old Npc1-/- mice and wild type littermates were employed to examine pancreatic β cell morphology and functional changes induced by loss of Npc1. Single cell RNA sequencing was conducted on primary islets. Npc1-/- Min6 cell line was generated using CRISPR/Cas9 gene editing. Seahorse XF24 was used to analyze primary islet and Min6 cell mitochondria respiration. Ultra-high-resolution cell imaging with Lattice SIM2 and electron microscope imaging were used to observe mitochondria and lysosome in primary islet β and Min6 cells. Mitophagy Dye and mt-Keima were used to measure β cell mitophagy. Results: In Npc1-/- mice, we found that β cell survival and pancreatic β cell mass expansion as well as islet glucose induced insulin secretion in 2-week-old mice were reduced. Npc1 loss retarded postnatal β cell differentiation and growth as well as impaired mitochondria oxidative phosphorylation (OXPHOS) function to increase mitochondrial superoxide production, which might be attributed to impaired autophagy flux particularly mitochondria autophagy (mitophagy) induced by dysfunctional lysosome in Npc1 null β cells. Conclusion: Our study revealed that NPC1 played an important role in maintaining normal lysosome function and mitochondria turnover, which ensured establishment of sufficient mitochondria OXPHOS for islet β cells differentiation and maturation.

Engineering of Primary Pancreatic Islet Cell Spheroids for Three-dimensional Culture or Transplantation: A Methodological Comparative Study

Three-dimensional (3D) cell culture by engineering spheroids has gained increasing attention in recent years because of the potential advantages of such systems over conventional two-dimensional (2D) tissue culture. Benefits include the ability of 3D to provide a more physiologically relevant environment, for the generation of uniform, size-controlled spheroids with organ-like microarchitecture and morphology. In recent years, different techniques have been described for the generation of cellular spheroids. Here, we have compared the efficiency of four different methods of islet cell aggregation. Rat pancreatic islets were dissociated into single cells before reaggregation. Spheroids were generated either by (i) self-aggregation in nonadherent petri dishes, (ii) in 3D hanging drop culture, (iii) in agarose microwell plates or (iv) using the Sphericalplate 5D™. Generated spheroids consisted of 250 cells, except for the self-aggregation method, where the number of cells per spheroid cannot be controlled. Cell function and morphology were assessed by glucose stimulated insulin secretion (GSIS) test and histology, respectively. The quantity of material, labor intensity, and time necessary for spheroid production were compared between the different techniques. Results were also compared with native islets. Native islets and self-aggregated spheroids showed an important heterogeneity in terms of size and shape and were larger than spheroids generated with the other methods. Spheroids generated in hanging drops, in the Sphericalplate 5D™, and in agarose microwell plates were homogeneous, with well-defined round shape and a mean diameter of 90 µm. GSIS results showed improved insulin secretion in response to glucose in comparison with native islets and self-aggregated spheroids. Spheroids can be generated using different techniques and each of them present advantages and inconveniences. For islet cell aggregation, we recommend, based on our results, to use the hanging drop technique, the agarose microwell plates, or the Sphericalplate 5D™ depending on the experiments, the latter being the only option available for large-scale spheroids production.

Alpha TC1 and Beta-TC-6 genomic profiling uncovers both shared and distinct transcriptional regulatory features with their primary islet counterparts

Alpha TC1 (αTC1) and Beta-TC-6 (βTC6) mouse islet cell lines are cellular models of islet (dys)function and type 2 diabetes (T2D). However, genomic characteristics of these cells, and their similarities to primary islet alpha and beta cells, are undefined. Here, we report the epigenomic (ATAC-seq) and transcriptomic (RNA-seq) landscapes of αTC1 and βTC6 cells. Each cell type exhibits hallmarks of its primary islet cell counterpart including cell-specific expression of beta (e.g., Pdx1) and alpha (e.g., Arx) cell transcription factors (TFs), and enrichment of binding motifs for these TFs in αTC1/βTC6 cis-regulatory elements. αTC1/βTC6 transcriptomes overlap significantly with the transcriptomes of primary mouse/human alpha and beta cells. Our data further indicate that ATAC-seq detects cell-specific regulatory elements for cell types comprising ≥ 20% of a mixed cell population. We identified αTC1/βTC6 cis-regulatory elements orthologous to those containing type 2 diabetes (T2D)-associated SNPs in human islets for 33 loci, suggesting these cells’ utility to dissect T2D molecular genetics in these regions. Together, these maps provide important insights into the conserved regulatory architecture between αTC1/βTC6 and primary islet cells that can be leveraged in functional (epi)genomic approaches to dissect the genetic and molecular factors controlling islet cell identity and function.

Elevated levels of branched‐chain amino acids have little effect on pancreatic islet cells, but l‐arginine impairs function through activation of the endoplasmic reticulum stress response

Recent metabolic profiling studies have identified a correlation between branched-chain amino acid levels, insulin resistance associated with prediabetes and susceptibility to type2 diabetes. Glucose and lipids in chronic excess have been reported to induce toxic effects in pancreatic β-cells, but the effect of elevated amino acid concentrations on primary islet cell function has not been investigated to date. The aim of this study was to investigate the effect of chronic exposure to various amino acids on islet cell function in vitro. Isolated rat islets were incubated over periods of 48h with a range of concentrations of individual amino acids (0.1μm to 10mm). After 48h, islets were assessed for glucose-dependent insulin secretion capacity, proliferation or islet cell apoptosis. We report that elevated levels of branched-chain amino acids have little effect on pancreatic islet cell function or viability; however, increased levels of the amino acid l-arginine were found to be β-cell toxic, causing a dose-dependent decrease in insulin secretion accompanied by a decrease in islet cell proliferation and an increase in islet cell apoptosis. These effects were not due to l-arginine-dependent increases in production of nitric oxide but arose through elicitation of the islet cell endoplasmic reticulum stress response. This novel finding indicates, for the first time, that the l-arginine concentration in vitro may impact negatively on islet cell function, thus indicating further complexity in relationship to in vivo susceptibility of β-cells to nutrient-induced dysfunction.

Autonomous and self-sustained circadian oscillators displayed in human islet cells

Aims/hypothesisFollowing on from the emerging importance of the pancreas circadian clock on islet function and the development of type 2 diabetes in rodent models, we aimed to examine circadian gene expression in human islets. The oscillator properties were assessed in intact islets as well as in beta cells.MethodsWe established a system for long-term bioluminescence recording in cultured human islets, employing lentivector gene delivery of the core clock gene Bmal1 (also known as Arntl)-luciferase reporter. Beta cells were stably labelled using a rat insulin2 promoter fluorescent construct. Single-islet/cell oscillation profiles were measured by combined bioluminescence–fluorescence time-lapse microscopy.ResultsHuman islets synchronised in vitro exhibited self-sustained circadian oscillations of Bmal1-luciferase expression at both the population and single-islet levels, with period lengths of 23.6 and 23.9 h, respectively. Endogenous BMAL1 and CRY1 transcript expression was circadian in synchronised islets over 48 h, and antiphasic to REV-ERBα (also known as NR1D1), PER1, PER2, PER3 and DBP transcript circadian profiles. HNF1A and PDX1 exhibited weak circadian oscillations, in phase with the REV-ERBα transcript. Dispersed islet cells were strongly oscillating as well, at population and single-cell levels. Importantly, beta and non-beta cells revealed oscillatory profiles that were well synchronised with each other.Conclusions/interpretationWe provide for the first time compelling evidence for high-amplitude cell-autonomous circadian oscillators displayed in human pancreatic islets and in dispersed human islet cells. Moreover, these clocks are synchronised between beta and non-beta cells in primary human islet cell cultures.Electronic supplementary materialThe online version of this article (doi:10.1007/s00125-012-2779-7) contains peer-reviewed but unedited supplementary material, which is available to authorised users.

Integrating Radioembolization into the Treatment Paradigm for Metastatic Neuroendocrine Tumors in the Liver

Surgical resection of hepatic metastases from neuroendocrine tumors (mNETs) is controversial because the potential survival benefit of this intervention must be balanced against the risk of surgical morbidity and mortality. In patients with unresectable mNETs in the liver, radioembolization has been used to treat tumors from a range of primary sites, including carcinoid and islet cell carcinomas as well as nonfunctional, asymptomatic tumors. Initial clinical studies and retrospective studies on a large cohort of patients indicate that radioembolization is well tolerated and highly effective in achieving a durable hepatic tumor response and ameliorating symptoms. Radioembolization using Yttrium-90 ((90)Y)-labeled resin or glass microspheres offers effective disease control and possible improved quality of life and thus merits consideration as an option for both functional and nonfunctional mNETs. Benefits of this intervention seem to extend from use in early lines of treatment to salvage of refractory disease. Radioembolization also offers a potential somatostatin analog-sparing effect in symptomatic disease.

A Novel Dual-Color Reporter for Identifying Insulin-Producing Beta- Cells and Classifying Heterogeneity of Insulinoma Cell Lines

Many research studies use immortalized cell lines as surrogates for primary beta- cells. We describe the production and use of a novel “indirect" dual-fluorescent reporter system that leads to mutually exclusive expression of EGFP in insulin-producing (INS+) beta-cells or mCherry in non-beta-cells. Our system uses the human insulin promoter to initiate a Cre-mediated shift in reporter color within a single transgene construct and is useful for FACS selection of cells from single cultures for further analysis. Application of our reporter to presumably clonal HIT-T15 insulinoma cells, as well as other presumably clonal lines, indicates that these cultures are in fact heterogeneous with respect to INS+ phenotype. Our strategy could be easily applied to other cell- or tissue-specific promoters. We anticipate its utility for FACS purification of INS+ and glucose-responsive beta-like-cells from primary human islet cell isolates or in vitro differentiated pluripotent stem cells.

Optimization of condition(s) towards establishment of primary islet cell cultures from WNIN/Ob mutant rat

WNIN/Ob, a mutant rat strain, developed at the National Center for Laboratory Animal Sciences (NCLAS) facility of National Institute of Nutrition (NIN), is a new animal model to study the metabolic syndrome. These animals have 47% fat in their body and isolation of islets from these animals were compounded due to the formation of amorphous viscous and jelly like material which reduced the islet yield. However, islets isolated from WNIN adult (≥12months) control rats gave a good islet recovery, under standard isolation procedures using collagenase digestion. In the present study we optimized culture conditions in WNIN/Ob rats to isolate islets with higher yield, and also established primary islet cell cultures from these mutant rats, retaining cellular integrity and functionality.

Surgical Resection and Multidisciplinary Care for Primary and Metastatic Pancreatic Islet Cell Carcinomas

IntroductionThe role of multidisciplinary management of islet cell cancers (ICC) has not been fully investigated in a population-based setting. MethodsThe Los Angeles County Cancer Surveillance Program was assessed for patients with ICC between the years 1982 to 2006. Patients were stratified by treatment received and clinicopathologic characteristics and survival were compared. ResultsWe identified 236 patients with ICC; 86 patients underwent curative-intent surgery with median survival for local, regional, and distant disease of 17.3, 12.2, and 4.0 years, respectively. In comparison, 102 patients underwent medical management alone; survival was significantly shorter when compared to the surgical cohort for local, regional, and distant disease (p < 0.05). To determine whether adjuvant chemotherapy was associated with improved survival, we compared patients who underwent surgery alone compared to patients who underwent surgery followed by adjuvant chemotherapy. Although patients with metastatic disease had 3-year longer survival with adjuvant chemotherapy, these improvements in survival were not statistically significant. ConclusionSurgical resection was associated with improved survival compared to medical management for any extent of disease in patients with ICC. Furthermore, adjuvant chemotherapy was not associated with survival but does warrant further examination in patients with metastatic disease.

T1656 Surgical Resection is the Only Effective Therapy for Primary and Metastatic Pancreatic Islet Cell Carcinoma

T1656 Surgical Resection is the Only Effective Therapy for Primary and Metastatic Pancreatic Islet Cell Carcinoma

Cyclin dependent kinase 5 (CDK5): a possible link between neuropathologies and diabetes

We study the role of CDK5 in mammalian development. Although a member of the cyclin dependent kinase family, CDK5 has been shown to influence differentiation of non‐dividing nervous tissues. Previously, our lab has used PC12 cells to study CDK5's role in neuronal development. Experiments involving growth factor‐induced (NGF, FGF) differentiation of PC12 cells in the presence of CDK5 inhibitors resulted in drastic morphological changes. Recent reports suggest CDK5 may also play an extra neuronal role. Here we report the impact of CDK5 manipulation in pancreatic tissue using the acinar cell line AR42J. The transdiffereniation of these cells from non‐insulin producing (acinar) to insulin producing (beta‐like) cells was induced by the administration of the chemical exendin‐4. Cells were assayed for the insulin phenotype, with a primary rat islet cell line used as a positive control. To asses the role of CDK5 in the pathway to this phenotype, insulin levels were quantified following simultaneous treatment of these cells with both exendin‐4 and the CDK5 specific inhibitor, olomoucine. Results suggest a role for CDK5 in the acquisition of this insulin phenotype. This data, coupled with the neurological role of CDK5, suggests a correlation between pathologies such as Alzheimer's disease and Type 2 Diabetes.

N-Acetylcysteine derivative inhibits procoagulant activity of human islet cells

The early loss of beta cells after islet cell transplantation has been attributed in part to blood coagulation at the implant site. Tissue factor expressed by beta cells and contaminating duct cells is considered to activate this process. Here, we investigated the ability of N-acetyl-L-cysteine to suppress the in vitro procoagulant activity of duct cells and human islet cell preparations. The effects of Nacystelyn, a salt derivative of N-acetyl-L-cysteine, were first assessed on procoagulant activity induced in human plasma by recombinant tissue factor, human primary duct cells or human islet cell preparations. The influence of Nacystelyn on clot formation, platelet counts and D-dimers were measured in a whole blood tubing loop model. Human beta cell viability and insulin synthesis after Nacystelyn treatment were assessed to exclude cytotoxicity of Nacystelyn. Nacystelyn efficiently inhibited the procoagulant activity of human recombinant tissue factor, primary duct cells and human islet cell preparations at clinically relevant concentrations without cellular toxicity. Nacystelyn is a pharmaceutical candidate to reduce early beta cell loss related to tissue factor-dependent coagulation after islet transplantation.

Group B Coxsackievirus Diabetogenic Phenotype Correlates with Replication Efficiency

Group B coxsackieviruses can initiate rapid onset type 1 diabetes (T1D) in old nonobese diabetic (NOD) mice. Inoculating high doses of poorly pathogenic CVB3/GA per mouse initiated rapid onset T1D. Viral protein was detectable in islets shortly after inoculation in association with beta cells as well as other primary islet cell types. The virulent strain CVB3/28 replicated to higher titers more rapidly than CVB3/GA in the pancreas and in established beta cell cultures. Exchange of 5'-nontranslated regions between the two CVB3 strains demonstrated a variable impact on replication in beta cell cultures and suppression of in vivo replication for both strains. While any CVB strain may be able to induce T1D in prediabetic NOD mice, T1D onset is linked both to the viral replication rate and infectious dose.

PANDER-induced cell-death genetic networks in islets reveal central role for caspase-3 and cyclin-dependent kinase inhibitor 1A (p21)

PANcreatic DERived factor is an islet-specific cytokine that promotes apoptosis in primary islets and islet cell lines. To elucidate the genetic mechanisms of PANDER-induced cell death we performed expression profiling using the mouse PancChip version 5.0 in conjunction with Ingenuity Pathway Analysis. Murine islets were treated with PANDER and differentially expressed genes were identified at 48 and 72 h post-treatment. 64 genes were differentially expressed in response to PANDER treatment. 22 genes are associated with cell death. In addition, the genes with the highest fold change were linked with cell death or apoptosis. The most significantly affected gene at 48 h was the downregulated cyclin-dependent kinase inhibitor 1A (CDKN1A or p21). Approximately half of the genes impacted at 72 h were linked to cell death. Cell death differentially expressed genes were confirmed by quantitative RT-PCR. Further analysis identified cell death genetic networks at both time points with 21 of the 22 cell death genes related in various biological pathways. Caspase-3 (CASP3) was biologically linked to CDKN1A in several genetic networks and these two genes were further examined. Elevated cleaved CASP3 levels in PANDER-treated β-TC3 insulinoma cells were found to abrogate CDKN1A expression. Levels of CDKN1A were not affected in the absence of cleaved CASP3. PANDER-induced downregulation of CDKN1A expression coupled with induced CASP3-activation may serve a central role in islet cell death and offers further insight into the mechanisms of cytokine-induced β-cell apoptosis.

Novel porous matrix and bioreactors for high density cultures of insulinoma cell lines: insulin secretion and response to glucose

This paper describes the use of a novel porous matrix, Porocell, for high density, tissue-like culture of two insulinoma cell lines, CRI-D2 and CRI-D11. Both these cell lines have previously been shown not to secrete insulin in response to glucose. Porocell is a macro-porous, polymeric material manufactured in the shape of discs that are 6·2 mm in diameter and 2 mm in thickness. Insulinoma cells were cultured in two different mini-bioreactors, each containing six Porocell discs inoculated with 2·5 × 106 cells per disc. In surface aerated, stirred bioreactors, the insulinoma cells grew as closely packed dense cell sheets penetrating deep into the pores of Porocell. In a second type of system, a packed-bed perfused mini-bioreactor, flat, extended monolayers of cells were observed growing throughout the Porocell matrix. In both bioreactor configurations, viable cell populations were maintained for 30 days because of the excellent oxygen and nutrient transfer properties of Porocell. CRI-D2 insulinoma cells cultured in static flasks and on Porocell did not show any insulin secretion in response to 30 min exposures in media supplemented with 5·5–16·7 mmol dm−3 glucose. However, in long term (14–19 day) cultures, CRI-D2 cells growing in Porocell secreted low, but measurable amounts (25–35 pmol dm−3) of insulin in medium supplemented with elevated (14·5 mmol dm−3) glucose concentrations. The glucose uptake rates of cells cultured in 4·0 mmol dm−3 glucose increased linearly from 1·0 to 2·3 mmol dm−3 day−1 over a period of 19 days. At 14·5 mmol dm−3 glucose concentration, the uptake rate increased from 1·0 to 7·05 mmol dm−3 day−1 over the same period of culture. Contrary to previous studies, we have demonstrated that the CRI-D2 cell line cultured at high cell density in Porocell is capable of secreting insulin when exposed to prolonged and elevated concentrations of glucose. The Porocell mini-bioreactors are easy to use, robust systems that can be used for long-term studies of primary and tumorgenic islet cell function and response to secretagogues. © 1998 SCI.

The use of somatostatin receptor scintigraphy in the differential diagnosis of pancreatic duct cancers and islet cell tumors.

In the present study, the diagnostic value of somatostatin receptor scintigraphy (SRS) was evaluated in the preoperative workup in patients with pancreatic duct cancers and islet cell tumors, as well as in the follow-up of these patients. Twenty-six patients with suspected primary pancreatic duct cancers and 48 patients with islet cell tumors were studied. The SRS was performed using the radionuclide-labeled somatostatin analogue 111In-octreotide. Another group of 12 patients who were still alive more than 3 years after pancreaticoduodenectomy for pancreatic duct adenocarcinomas also underwent SRS. In 31 (65%) of 48 patients, the primary pancreatic islet cell tumor as well as its often previously not yet recognized metastases could be visualized. In contrast, none of the 26 pancreatic adenocarcinomas or their metastases could be seen. In 5 of 12 patients who were alive more than 3 years after pancreaticoduodenectomy for pancreatic duct adenocarcinomas, metastatic lesions were visualized at SRS. In retrospect, these patients were not operated on for adenocarcinomas but for "nonfunctioning" islet cell tumors. The present study supports the concept that SRS has a place in the preoperative differential diagnosis of islet cell tumors and pancreatic duct cancers as well as in the follow-up, especially in those cases in which no tumor histologic analysis was obtained, or the pathologic examination of the tumor tissue had not included special staining procedures for neuroendocrine characteristics. Our results also indicate that the evaluation of the results of investigations on the role of surgery or radiation therapy and chemotherapy or both in pancreatic duct cancer have to be interpreted with caution, if no histologic analysis and staining for neuroendocrine characteristics was performed.

Diabetic diarrhea. An underdiagnosed complication?

Diarrhea is a common gastrointestinal problem in diabetes, and its prevalence has been underestimated. The cause of diabetic diarrhea is unknown, but it is probably related to gastrointestinal motility disturbances secondary to diabetic autonomic neuropathy. Other causes (especially primary malabsorption syndromes and islet cell tumors) must be excluded. Treatment of diabetic diarrhea is largely symptomatic and only moderately effective. Antidiarrheal agents may ameliorate acute episodes. Broad-spectrum antibiotics and clonidine hydrochloride (Catapres) have had some success in long-term control. Most recently, subcutaneous administration of somatostatin analogues has been shown to be helpful, the main side effects being drowsiness and vomiting.

Islet Amyloid Polypeptide and Insulin Expression Are Controlled Differently in Primary and Transformed Islet Cells

The pancreatic beta-cell is a major site of islet amyloid polypeptide (IAPP) biosynthesis, and the peptide is coreleased with insulin. We have analyzed the expression of IAPP (mRNA and protein) in various cell types in normal and transformed murine islet cell cultures by Northern blot analyses and immunocytochemistry. IAPP is primarily coexpressed with insulin in the beta-cell of GH-promoted primary rat islet cell cultures. Additionally, a small population of non-beta-cells exhibited a prominent IAPP expression, and double staining experiments showed colocalization with glucagon or somatostatin in some of these cells. IAPP mRNA was confined to the beta-cell phenotype when analyzing the phenotypically stable in vivo tumor lines, MSL-G2-IN (insulinoma) and MSL-G-AN (glucagonoma), and the transgenic mouse islet cell lines, beta-Tc and alpha-Tc. However, IAPP and insulin expression were completely uncoupled in unstable heterogeneous clones such as NHI-6F. This clone is composed of primarily glucagon-producing cells in vitro, but insulin gene expression becomes dominant after passage in vivo. Interestingly, IAPP was hyperexpressed with glucagon under in vitro conditions in this clone. We conclude that the tissue specificity of expressions of IAPP and insulin are controlled differently, and that coexpression of IAPP with hormones different from insulin may be a marker for pluripotent transformed rat islet cell clones, which are able to activate insulin gene transcription during passage in vivo.

The Rat Glucagon Gene Is Regulated by a Protein Kinase A-Dependent Pathway in Pancreatic Islet Cells*

A cAMP response element (CRE) has been identified in the proximal 5'-flanking region of the rat glucagon gene, and activation of the cAMP-dependent pathway in fetal rat intestinal cells leads to an increase in the levels of glucagon mRNA transcripts. In contrast, the human glucagon gene does not contain a similar CRE, and the results of studies using immortalized rat and hamster islet cell lines have suggested that glucagon gene expression may not be regulated by cAMP. To reconcile these observations, we have studied the control of glucagon gene expression. Incubation of primary rat islet cell cultures with forskolin in the presence of low (0.5 g/liter) or high (2.0 g/L) glucose resulted in a 2- to 3-fold increase in the levels of glucagon mRNA transcripts. Forskolin also stimulated the secretion and synthesis of immunoreactive glucagon. The importance of the protein kinase-A-dependent pathway in the regulation of glucagon gene expression was also examined in hamster islet InR1-G9 cells. Cotransfection of a glucagon-chloramphenicol acetyltransferase (CAT) fusion gene containing the glucagon CRE and a cDNA encoding the catalytic subunit of protein kinase-A resulted in stimulation of glucagon-CAT activity in hamster islet cells. Catalytic subunit cotransfection also activated somatostatin-CAT, but no activation of RSVCAT was detected. The results of these experiments suggest that the rat glucagon gene is regulated by a protein kinase-A-dependent pathway in the endocrine pancreas.

Studies in vivo and in vitro on chemically-induced primary islet cell tumours and non-tumour endocrine pancreatic tissue.

Rat islet cell tumours induced by injection of streptozotocin and nicotinamide have been studied in vivo and after the establishment of monolayer cultures of tumour cells. During an intravenous glucose tolerance test, tumour-bearing rats had increased release of immunoreactive insulin, with a high proportion of proinsulin, as well as accelerated glucose disposal relative to control rats. The tumours were rich in immunoreactive insulin and somatostatin, poor in glucagon. Non-tumour pancreatic tissue or isolated islets contained 10% or less of the corresponding normal amounts of insulin whereas the islet content of somatostatin was unchanged and that of glucagon increased. This is best interpreted as a selective suppression of non-tumour B cells, further supported by the observation that the initially reduced insulin release and content of non-tumour islets were partially restored after 2 days in tissue culture. In monolayer culture, tumour cells maintained insulin production and acute responsiveness to glucose for prolonged periods. There was no sign of cell proliferation. It is concluded that primary, chemically-induced insulin-producing pancreatic islet cell tumours retain several features characteristic to normal B cells and continue to influence glucose homeostasis in vivo.