Islet Cell Nuclei Research Articles

Differential expression of regulators of the canonical Wnt pathway during the compensatory beta-cell hyperplasia in prediabetic mice

We previously reported that the canonical Wnt signaling pathway is activated during compensatory islet hyperplasia in prediabetic mice. Here, we aimed to expand our knowledge concerning the Wnt signaling partners and modulators involved in this process. We report here that Axin1, Axin2, and DACT1, inhibitors of the canonical Wnt signaling pathway, displayed no change in their expression, while GSK-3β, a multi-functional kinase that acts as a negative regulator of this pathway as well as affects insulin secretion/action, was up-regulated in hyperplastic islets of prediabetic mice. We also observed that COUP-TFII, a protein that acts positively on Wnt-target genes related to cell proliferation, displays a significant increase in gene expression and protein content and is highly immunolabeled in islet cell nuclei of prediabetic mice compared to control islets. These findings suggest that GSK-3β and COUP-TFII may play a role in beta-cell dysfunction and hyperplasia during type 2 prediabetes.

Electrofusion of mesenchymal stem cells and islet cells for diabetes therapy: a rat model.

Islet transplantation is a minimally invasive treatment for severe diabetes. However, it often requires multiple donors to accomplish insulin-independence and the long-term results are not yet satisfying. Therefore, novel ways to overcome these problems have been explored. Isolated islets are fragile and susceptible to pro-apoptotic factors and poorly proliferative. In contrast, mesenchymal stem cells (MSCs) are highly proliferative, anti-apoptotic and pluripotent to differentiate toward various cell types, promote angiogenesis and modulate inflammation, thereby studied as an enhancer of islet function and engraftment. Electrofusion is an efficient method of cell fusion and nuclear reprogramming occurs in hybrid cells between different cell types. Therefore, we hypothesized that electrofusion between MSC and islet cells may yield robust islet cells for diabetes therapy. We establish a method of electrofusion between dispersed islet cells and MSCs in rats. The fusion cells maintained glucose-responsive insulin release for 20 days in vitro. Renal subcapsular transplantation of fusion cells prepared from suboptimal islet mass (1,000 islets) that did not correct hyperglycemia even if co-transplanted with MSCs, caused slow but consistent lowering of blood glucose with significant weight gain within the observation period in streptozotocin-induced diabetic rats. In the fusion cells between rat islet cells and mouse MSCs, RT-PCR showed new expression of both rat MSC-related genes and mouse β-cell-related genes, indicating bidirectional reprogramming of both β-cell and MSCs nuclei. Moreover, decreased caspase3 expression and new expression of Ki-67 in the islet cell nuclei suggested alleviated apoptosis and gain of proliferative capability, respectively. These results show that electrofusion between MSCs and islet cells yield special cells with β-cell function and robustness of MSCs and seems feasible for novel therapeutic strategy for diabetes mellitus.

Stanniocalcin-1 Co-Localizes with Insulin in the Pancreatic Islets

The polypeptide hormone stanniocalcin-1 (STC-1) is widely expressed in mammals and signals both locally and systemically. In many tissues STC-1 ligand is sequestered by target cell organelles (mitochondria, nuclei, and cholesterol lipid droplets) to exert diverse biological effects. Most notably, STC-1 serves as an uncoupler of oxidative phosphorylation in liver, muscle, and kidney mitochondria. The present paper describes the identification of STC-1 receptors in mouse pancreatic β cells and the discovery that the ligand co-localizes with insulin in pancreatic β cells. In situ hybridization (ISH) analysis subsequently revealed that pancreatic β cells were the source of the ligand. Intriguingly however, all ISH signal was localized over putative islet cell nuclei as opposed to the cell cytoplasm. Real-time qPCR and agarose gel electrophoresis revealed that the STC-1 amplicon generated from islet cell total RNA was the same size as that from kidney. However, relative levels of STC-1 gene expression were >100-fold lower in islets than those in kidney tissue. Collectively, these findings are indicative of a local STC-1 signalling pathway in pancreatic β cells. The role of STC-1 in this context remains to be established, but it could very well entail the regulation of β cell mitochondria membrane potential which is an integral aspect of regulated insulin release. Interestingly, STC-1 immunoreactivity was not evident in embryonic pancreatic islets, suggesting that ligand synthesis may only commence postnatally.

Transgenic Expression of Insulin-Response Element Binding Protein-1 in β-Cells Reproduces Type 2 Diabetes

Recent evidence supports the idea that insulin signaling through the insulin receptor substrate/phosphatidyl-inositol 3-kinase/Akt pathway is involved in the maintenance of beta-cell mass and function. We previously identified the insulin-response element binding protein-1 (IRE-BP1) as an effector of insulin-induced Akt signaling in the liver, and showed that the 50-kDa carboxyl fragment confers the transcriptional activity of this factor. In this investigation we found that IRE-BP1 is expressed in the alpha, beta, and delta-cells of the islets of Langerhans, and is localized to the cytoplasm in beta-cells in normal rats, but is reduced and redistributed to the islet cell nuclei in obese Zucker rats. To test whether IRE-BP1 modulates beta-cell function and insulin secretion, we used the rat insulin II promoter to drive expression of the carboxyl fragment in beta-cells. Transgenic expression of IRE-BP1 in FVB mice increases nuclear IRE-BP1 expression, and produces a phenotype similar to that of type 2 diabetes, with hyperinsulinemia, hyperglycemia, and increased body weight. IRE-BP1 increased islet type I IGF receptor expression, potentially contributing to the development of islet hypertrophy. Our findings suggest that increased gene transcription mediated through IRE-BP1 may contribute to beta-cell dysfunction in insulin resistance, and allow for the hypothesis that IRE-BP1 plays a role in the pathophysiology of type 2 diabetes.

Genotype-Phenotype Associations in Patients with Severe Hyperinsulinism of Infancy

In hyperinsulinism of infancy (HI), unregulated insulin secretion causes hypoglycemia. Pancreatectomy may be required in severe cases, most of which result from a defect in the beta-cell KATP channel, encoded by ABCC8 and KCNJ11. Pancreatic histology may be classified as diffuse or focal disease (the latter associated with single paternal ABCC8 mutations), indicated by the presence of islet cell nuclear enlargement in areas of diffuse abnormality. We investigated genotype-phenotype associations in a heterogeneous Australian cohort. ABCC8 and KCNJ11 genes were sequenced and case histology was reviewed in 21 infants who had pancreatectomy. Ninety-eight control DNA samples were tested by single nucleotide polymorphism analysis. Eighteen ABCC8 mutations were identified, 10 novel. Eleven patients (4 compound heterozygote, 4 single mutation, 3 no mutation detected) had diffuse hyperinsulinism. Nine patients had focal hyperinsulinism (6 single paternal mutation, 2 single mutation of undetermined parental origin, 1 none found) with absence of islet cell nuclear enlargement outside the focal area, although centroacinar cell proliferation and/or nesidiodysplasia was present in 7 cases. Regeneration after near-total pancreatectomy was documented in 4 patients, with aggregates of endocrine tissue observed at subsequent operations in 3. Although the absence of enlarged islet cell nuclei is a useful discriminant of focal hyperinsulinism associated with a paternal ABCC8 mutation, further research is needed to understand the pathophysiology of other histological abnormalities in patients with HI, which may have implications for mechanisms of ductal and islet cell proliferation. Previous surgery should be taken into account when interpreting pancreatic histology.

Cyclin-dependent kinase (cdk6) and p16 in pancreatic endocrine neoplasms

p16 and p27, the inhibitors of cyclin-dependent kinases, have been reportedly decreased in certain human tumours, including a few endocrine tumours. The current study used immunocytochemical staining to compare the staining intensity of cdk6 and its inhibitor, p16, in pancreatic endocrine neoplasms with normal pancreatic islets. Twenty-four primary pancreatic endocrine neoplasms, consisting of 12 insulinomas, one glucagomoma, three pancreatic polypeptide (PP)-omas, five gastgrinomas and three non-fuctioning tumours, were immunocytochemically studied for cdk6 and p16 compared with the adjacent non-neoplastic islets. In the normal islets, cdk6 staining was strongly positive for islet cell nuclei and cytoplasms, whereas p16 was strongly positively stained for islet cell cytoplasms. Insulinomas, glucagonoma, PP-omas and non-functioning tumours were weakly stained for cdk6 and p16. Among five gastrinomas, three tumours were moderately stained and two tumours were more weakly stained for cdk6 and p16. Thus, tumour cells were weaker stained for cdk6 and p16 compared with the strong staining of normal islet cells. No distinct immunostaining difference was observed among five kinds of pancreatic endocrine neoplasms. The decreased immunocytochemical staining for cdk6 and p16 is consistently observed in five kinds of pancreatic endocrine neoplasms. This decreased cdk6 and p16 in pancreatic endocrine neoplasms may be a part of the cell cycle event in tumour transformation and progression, and the same process may involve other endocrine tumours.

Co-localization of nestin and PDX-1 in small evaginations of the main pancreatic duct in adult rats

Pancreatic stem cells are powerful tools for future gene/cell therapy for diabetes, pancreatic carcinoma and chronic pancreatitis. However, it is unclear where the pancreatic stem cells exist in adult pancreas. To identify the pancreatic stem cells, we have focused on a homeodomain-containing transcription factor, pancreatic/duodenal homeobox-1 (PDX-1), and an intermediate filament protein, nestin, which are important candidates of pancreatic stem cell markers. We investigated the immunohistological localization for PDX-1 and nestin in adult rat pancreas. PDX-1 staining was detected in small evaginations of the main pancreatic duct and in nuclei of islet cells. Nestin staining was also detected in small evaginations of the main duct, islets and spindle-shaped cells in the connective tissue around the main duct. These spindle-shaped cells were also positive for alpha-smooth muscle actin, the marker of myofibroblasts. Confocal laser microscopy study showed that nestin and PDX-1 are co-localized not only in a subset of cells in small evaginations of the main duct but also in a few cells in the islets. These cells of the main duct were also positive for the ductal cell marker, cytokeratin 19. Our results suggest that the double positive cells for PDX-1 and nestin might be important candidates for pancreatic stem cells in adult rats.

Role of Oxidative Stress in Pancreatic β‐Cell Dysfunction

Abstract: Oxidative stress is produced under diabetic conditions and is likely involved in progression of pancreatic β‐cell dysfunction found in diabetes. Possibly caused by low levels of antioxidant enzyme expressions, pancreatic β‐cells are vulnerable to oxidative stress. When β‐cell‐derived HIT‐T15 cells or isolated rat islets were exposed to oxidative stress, insulin gene expression was markedly decreased. To investigate the significance of oxidative stress in the progression of pancreatic β‐cell dysfunction in type 2 diabetes, we evaluated the effects of antioxidants in diabetic C57BL/KsJ‐db/db mice. According to an intraperitoneal glucose tolerance test, the treatment with antioxidants retained glucose‐stimulated insulin secretion and moderately decreased blood glucose levels. Histological analyses of the pancreata revealed that the β‐cell mass was significantly larger in the mice treated with the antioxidants, and the antioxidant treatment suppressed apoptosis in β‐cells without changing the rate of β‐cell proliferation. The antioxidant treatment also preserved the amounts of insulin content and insulin mRNA, making the extent of insulin degranulation less evident. As possible mechanism underlying the phenomena, expression of pancreatic and duodenal homeobox factor‐1 (also known as IDX‐1/STF‐1/IPF1), an important transcription factor for the insulin gene, was more clearly visible in the nuclei of islet cells after the antioxidant treatment. Under diabetic conditions, JNK is activated by oxidative stress and involved in the suppression of insulin gene expression. This JNK effect appears to be mediated in part by nucleocytoplasmic translocation of PDX‐1, which is also downstream of JNK activation. Taken together, oxidative stress and consequent activation of the JNK pathway are involved in progression of β‐cell dysfunction found in diabetes. Antioxidants may serve as a novel mechanism‐based therapy for type 2 diabetes.

Over-expression of sterol-regulatory-element-binding protein-1c (SREBP1c) in rat pancreatic islets induces lipogenesis and decreases glucose-stimulated insulin release: modulation by 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR).

Accumulation of intracellular lipid by pancreatic islet beta-cells has been proposed to inhibit normal glucose-regulated insulin secretion ('glucolipotoxicity'). In the present study, we determine whether over-expression in rat islets of the lipogenic transcription factor SREBP1c (sterol-regulatory-element-binding protein-1c) affects insulin release, and whether changes in islet lipid content may be reversed by activation of AMPK (AMP-activated protein kinase). Infection with an adenovirus encoding the constitutively active nuclear fragment of SREBP1c resulted in expression of the protein in approx. 20% of islet cell nuclei, with a preference for beta-cells at the islet periphery. Real-time PCR (TaqMan) analysis showed that SREBP1c up-regulated the expression of FAS (fatty acid synthase; 6-fold), acetyl-CoA carboxylase-1 (2-fold), as well as peroxisomal-proliferator-activated receptor-gamma (7-fold), uncoupling protein-2 (1.4-fold) and Bcl2 (B-cell lymphocytic-leukaemia proto-oncogene 2; 1.3-fold). By contrast, levels of pre-proinsulin, pancreatic duodenal homeobox-1, glucokinase and GLUT2 (glucose transporter isoform-2) mRNAs were unaltered. SREBP1c-transduced islets displayed a 3-fold increase in triacylglycerol content, decreased glucose oxidation and ATP levels, and a profound inhibition of glucose-, but not depolarisation-, induced insulin secretion. Culture of islets with the AMPK activator 5-amino-4-imidazolecarboxamide riboside decreased the expression of the endogenous SREBP1c and FAS genes, and reversed the effect of over-expressing active SREBP1c on FAS mRNA levels and cellular triacylglycerol content. We conclude that SREBP1c over-expression, even when confined to a subset of beta-cells, leads to defective insulin secretion from islets and may contribute to some forms of Type II diabetes.

Role of oxidative stress in pancreatic beta-cell dysfunction.

Oxidative stress is produced under diabetic conditions and is likely involved in progression of pancreatic beta-cell dysfunction found in diabetes. Possibly caused by low levels of antioxidant enzyme expressions, pancreatic beta-cells are vulnerable to oxidative stress. When beta-cell-derived HIT-T15 cells or isolated rat islets were exposed to oxidative stress, insulin gene expression was markedly decreased. To investigate the significance of oxidative stress in the progression of pancreatic beta-cell dysfunction in type 2 diabetes, we evaluated the effects of antioxidants in diabetic C57BL/KsJ-db/db mice. According to an intraperitoneal glucose tolerance test, the treatment with antioxidants retained glucose-stimulated insulin secretion and moderately decreased blood glucose levels. Histological analyses of the pancreata revealed that the beta-cell mass was significantly larger in the mice treated with the antioxidants, and the antioxidant treatment suppressed apoptosis in beta-cells without changing the rate of beta-cell proliferation. The antioxidant treatment also preserved the amounts of insulin content and insulin mRNA, making the extent of insulin degranulation less evident. As possible mechanism underlying the phenomena, expression of pancreatic and duodenal homeobox factor-1 (also known as IDX-1/STF-1/IPF1), an important transcription factor for the insulin gene, was more clearly visible in the nuclei of islet cells after the antioxidant treatment. Under diabetic conditions, JNK is activated by oxidative stress and involved in the suppression of insulin gene expression. This JNK effect appears to be mediated in part by nucleocytoplasmic translocation of PDX-1, which is also downstream of JNK activation. Taken together, oxidative stress and consequent activation of the JNK pathway are involved in progression of beta-cell dysfunction found in diabetes. Antioxidants may serve as a novel mechanism-based therapy for type 2 diabetes.

Oxidative stress and the JNK pathway as a potential therapeutic target for diabetes

Oxidative stress is produced under diabetic conditions and is likely involved in progression of pancreatic beta-cell dysfunction found in diabetes. Possibly due to low levels of antioxidant enzyme expressions, beta-cells are vulnerable to oxidative stress. When beta-cell-derived cell lines or isolated rat islets were exposed to oxidative stress, insulin gene expression was markedly decreased. Furthermore, when diabetic C57BL/ KsJ-db/db mice were treated with antioxidants, glucose tolerance was ameliorated. Histological analyses of the pancreata revealed that the beta-cell mass is significantly larger in the mice treated with the antioxidants. The antioxidant treatment also preserved the amounts of insulin content and insulin mRNA. As a possible mechanism underlying the phenomena, expression of pancreatic and duodenal homeobox factor-1 (PDX-1), an important transcription factor for the insulin gene, was more clearly visible in the nuclei of islet cells after the antioxidant treatment. Furthermore, oxidative stress induces nucleocytoplasmic translocation of PDX-1 through activation of the c-Jun N-terminal kinase (JNK) pathway, which leads to suppression of insulin gene expression. Taken together, oxidative stress and consequent activation of the JNK pathway are involved in progression of beta-cell dysfunction found in diabetes, and thus are a therapeutic target for diabetes.

Hypoglycemia after Pancreas Transplantation: Association with Allograft Nesidiodysplasia and Expression of Islet Neogenesis-Associated Peptide

We report a case of severe hypoglycemia occurring in a 35-yr-old woman, 6 yr after pancreas transplantation for type 1 diabetes mellitus. Extensive preoperative and intraoperative exploration failed to disclose the presence of a focal adenomatous lesion. Partial allograft pancreatectomy was performed initially, but it failed to control the hypoglycemic symptoms, leading to complete removal of the pancreas allograft. Histopathological examination of the resected pancreas allograft showed the presence of nesidioblastosis, characterized by foci of islet cells budding off ducts, accompanied by an increase in the number of islets, numerous small intralobular islet cell aggregates, and nesidiodysplasia (large, hyperchromatic islet cell nuclei). Islet neogenesis-associated protein-positive islets and ducts were seen by immunofluorescence. Insulin-positive islets ranged from very small to large, with isolated insulin-positive cells diffusely scattered, consistent with islet neogenesis. Very little glucagon staining was identified. Reported cases of adult nesidioblastosis are reviewed. The significance of nesidioblastosis in the context of pancreas transplantation and possible mechanisms of posttransplant hypoglycemia are discussed.

Beneficial effects of antioxidants in diabetes: possible protection of pancreatic beta-cells against glucose toxicity.

Oxidative stress is produced under diabetic conditions and possibly causes various forms of tissue damage in patients with diabetes. The aim of this study was to examine the involvement of oxidative stress in the progression of pancreatic beta-cell dysfunction in type 2 diabetes and to evaluate the potential usefulness of antioxidants in the treatment of type 2 diabetes. We used diabetic C57BL/KsJ-db/db mice, in whom antioxidant treatment (N-acetyl-L-cysteine [NAC], vitamins C plus E, or both) was started at 6 weeks of age; its effects were evaluated at 10 and 16 weeks of age. According to an intraperitoneal glucose tolerance test, the treatment with NAC retained glucose-stimulated insulin secretion and moderately decreased blood glucose levels. Vitamins C and E were not effective when used alone but slightly effective when used in combination with NAC. No effect on insulin secretion was observed when the same set of antioxidants was given to nondiabetic control mice. Histologic analyses of the pancreases revealed that the beta-cell mass was significantly larger in the diabetic mice treated with the antioxidants than in the untreated mice. As a possible cause, the antioxidant treatment suppressed apoptosis in beta-cells without changing the rate of beta-cell proliferation, supporting the hypothesis that in chronic hyperglycemia, apoptosis induced by oxidative stress causes reduction of beta-cell mass. The antioxidant treatment also preserved the amounts of insulin content and insulin mRNA, making the extent of insulin degranulation less evident. Furthermore, expression of pancreatic and duodenal homeobox factor-1 (PDX-1), a beta-cell-specific transcription factor, was more clearly visible in the nuclei of islet cells after the antioxidant treatment. In conclusion, our observations indicate that antioxidant treatment can exert beneficial effects in diabetes, with preservation of in vivo beta-cell function. This finding suggests a potential usefulness of antioxidants for treating diabetes and provides further support for the implication of oxidative stress in beta-cell dysfunction in diabetes.

Neural cell adhesion molecule (N-CAM) is required for cell type segregation and normal ultrastructure in pancreatic islets.

Classical cell dissociation/reaggregation experiments with embryonic tissue and cultured cells have established that cellular cohesiveness, mediated by cell adhesion molecules, is important in determining the organization of cells within tissue and organs. We have employed N-CAM-deficient mice to determine whether N-CAM plays a functional role in the proper segregation of cells during the development of islets of Langerhans. In N-CAM-deficient mice the normal localization of glucagon-producing alpha cells in the periphery of pancreatic islets is lost, resulting in a more randomized cell distribution. In contrast to the expected reduction of cell-cell adhesion in N-CAM-deficient mice, a significant increase in the clustering of cadherins, F-actin, and cell-cell junctions is observed suggesting enhanced cadherin-mediated adhesion in the absence of proper N-CAM function. These data together with the polarized distribution of islet cell nuclei and Na+/K+-ATPase indicate that islet cell polarity is also affected. Finally, degranulation of beta cells suggests that N-CAM is required for normal turnover of insulin-containing secretory granules. Taken together, our results confirm in vivo the hypothesis that a cell adhesion molecule, in this case N-CAM, is required for cell type segregation during organogenesis. Possible mechanisms underlying this phenomenon may include changes in cadherin-mediated adhesion and cell polarity.

DNA-damage and NAD(+)-depletion are initial events in oxygen radical induced islet cell death.

Previous studies in vitro and in animal models indicate that reactive oxygen intermediates (ROI) contribute to the destruction of pancreatic islet cells in the pathogenesis of insulin dependent diabetes (Mendola et al., 1989; Rabinovitch et al., 1992). During islet inflammation endothelial cells and infiltrating macrophages are thought to release cyto-toxic amounts ROI (Zweier et al., 1988; Brenner et al., 1993). In addition it was shown, that islet cells are highly susceptible to ROI since they possess only low levels of oxygen radical defense systems (Grankvist et al., 1981; Malaisse et al., 1982). We recently have described an in vitro model of oxygen-radical induced islet cell death where ROI are generated by xanthine oxidase in the presence of hypoxanthine (Burkart et al., 1992). In the present study we investigated the initial events of ROI-induced pancreatic islet cell destruction. We observed that DNA strand breaks and the activation of poly(ADP-ribose) polymerase in islet cell nuclei are prominent early consequences of ROI action. The subsequent islet cell death occured several hours after ROI treatment, which appeared to result from depletion of cellular NAD+ pools. Inhibition of poly(ADP-ribose) polymerase by nicotinamide resulted in the preservation of the NAD+ pool and in islet cell survival.

Analysis of oxygen radical toxicity in pancreatic islets at the single cell level.

Despite extensive studies on streptozotocin, alloxan and nitric oxide toxicity in pancreatic islets the mechanism of oxygen radical induced islet cell death has not been determined. The present study shows at the level of single cells that following exposure to oxygen radicals generated from xanthine oxidase DNA strand breaks occur in cell nuclei within 5-60 min and precede cell death by several hours. Similar kinetics were seen when treating islet cells with the alkylating agent streptozotocin. Immunofluorescence studies demonstrated the endogenous formation of ADP-ribose polymers in nearly all islet cell nuclei within minutes of treatment with xanthine oxidase, indicating activation of the enzyme poly(ADP-ribose) polymerase (PARP). Concomitantly, cellular NAD+ depletion was noted. Nicotinamide largely prevented NAD+ depletion and in parallel resulted in islet cell survival. These findings identify islet cell nuclear DNA as a primary target of oxygen radical toxicity and suggest related pathways of oxygen radical, nitric oxide and streptozotocin toxicity.

Pancreatic islet lesions of autoimmune origin, and ICA and ICSA.

CHBB male rats, 120-150 g in weight, were used both as animals to be sensitized and as donors of homologous islets as antigen. At no time did sensitized animals give a positive reaction for islet-cell antibodies or islet-cell surface bound antibodies at any of the dilutions tested. None of the frozen sections of the pancreas were positive for fluorescence specific for IgG or C3. Marked fibrosis and cell infiltration of pancreatic islets, a high degree of pyknosis of parenchymatous cells of islets, phagocytosis of fragmented islet cell nuclei by histiocytes, and a marked reduction in the number of beta cells were noted.

Alloxan cytotoxicity in vitro. Microscope photometric analyses of Trypan Blue uptake by pancreatic islet cells in suspension

Suspensions of islet cells were prepared by shaking pancreatic islets from non-inbred ob/ob mice in a Ca2+-free buffer. The cells were incubated with or without 20 mM-alloxan, and subsequently with Trypan Blue. The uptake of Trypan Blue by cell nuclei was analysed by microscope photometry and by counting the frequency of cells appearing stained on visual inspection. Cells classified as stained or unstained by inspection showed no overlap in nuclear absorbance. Suspensions not exposed to alloxan contained 70-80% of unstained cells. Alloxan markedly decreased the frequency of unstained cells, an effect counteracted by 5 or 20 mM-D-glucose. The spectrum of Trypan Blue in islet-cell nuclei was red-shifted by about 20 nm. A similar red-shift was observed on adding the dye to solutions of albumin or histones, but not on mixing the dye with DNA. Binding to basic proteins may explain the concentrative uptake of Trypan Blue in dead cells and contribute to the oncogenic transformation of phagocytotically active cells. Beta-Cells in vitro are killed by alloxan and hence represent a valid model for studying the diabetogenic action of the drug.

Diploid, tetraploid and octaploid beta cells in the islets of Langerhans of the normal human pancreas.

The nuclear DNA content of normal human endocrine and exocrine pancreatic cells was determined by Feulgen microdensitometry. Exocrine cell nuclei consistently yielded only diploid DNA values, while islet cell nuclei showed distinct diploid, tetraploid and octaploid classes. The existence of a strict positive correlation between nuclear size and DNA content in individual nuclei permitted the assignment of islet cell nuclei to one of three polyploid classes after staining by Gomori's modification of the Mallory-Heidenhain azan technic. It was thus determined that polyploidy is confined entirely to beta cells, while alpha and delta cells remain diploid. Although a complete analysis was made on only six adult specimens, the unequivocal volume-DNA relationship established permits the assumption, based on the observation of numerous other human pancreases, that the phenomenon is a generalized one.