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Chronic hyperglycemia causes pancreatic β-cell dysfunction, impaired insulin secretion and suppression of insulin gene expression, referred to as glucotoxicity. Insulin gene expression is regulated by several protein kinases and protein phosphatases. However, the molecular mechanisms of the suppressed insulin gene expression in glucotoxicity are not fully understood. In this study, we employed rat insulinoma INS-1 cells as a model of pancreatic glucotoxicity. In INS-1 cells, insulin gene expression is up-regulated by incubation with 11.2 mM glucose for 7 days and down-regulated by incubation with 22.4 mM glucose for the same period. To identify the protein kinases and protein phosphatases involved in the suppression of insulin gene expression, we analyzed gene expression in INS-1 cells cultured with 11.2 mM or 22.4 mM glucose for 7 days using microarray analysis and real-time PCR. The expression levels of nine protein kinases were affected by glucotoxic conditions. In particular, CPG16 expression level was increased in INS-1 cells under these conditions. Transfection of CPG16 decreased insulin promoter activity, whereas kinase-dead mutant of CPG16 did not affect this. These results suggest that CPG16 plays a role in the suppression of insulin gene expression in pancreatic β-cells under glucotoxic conditions.

The islet-specific zinc transporter ZnT8 is a major self-antigen found in insulin granules of pancreatic β-cells. Frequent insulin secretion exposes ZnT8 to the cell surface, but the humoral antigenicity of the surface-displayed ZnT8 remains unknown. Here we show that a membrane-embedded human ZnT8 antigen triggered a vigorous immune response in ZnT8 knock-out mice. Approximately 50% of serum immunoreactivities toward ZnT8 were mapped to its transmembrane domain that is accessible to extracellular ZnT8 antibody (ZnT8A). ZnT8A binding was detected on live rat insulinoma INS-1E cells, and the binding specificity was validated by a CRISPR/Cas9 mediated ZnT8 knock-out. Applying established ZnT8A assays to purified serum antibodies from patients with type 1 diabetes, we detected human ZnT8A bound to live INS-1E cells, whereas a ZnT8 knock-out specifically reduced the surface binding. Our results demonstrate that ZnT8 is a cell surface self-antigen, raising the possibility of a direct involvement in antibody-mediated β-cell dysfunction and cytotoxicity.

Our previous work revealed that in the pancreatic β cell line, geniposide modulated ATP production and glucose-stimulated insulin secretion (GSIS) induced by the acute stimulation of high glucose concentration. However, the effects of geniposide on functional impairment and the mass of β-cells exposed to elevated levels of glucose remains unknown. In the present study, impaired GSIS and restrained proliferation were observed in the prolonged culture of insulinoma INS-1 cells with 33mM of glucose (high glucose). Our results indicate that the glucose-induced impairment of insulin release was significantly reverted by the inclusion of 1 or 10μM of geniposide. Moreover, induction of the phosphorylation of AMP-activated protein kinase (AMPK) was observed, which promoted the utilization of nutrient stores for energy production. AMPK phosphorylation was enhanced by an increased number of INS-1 cells, and the increased expression of AMPK downstream target heme oxygenase 1 (HO-1), under high glucose concentration. Furthermore, geniposide protected rat insulinoma cells from apoptosis in high-glucose concentrations. We have shown that these effects were associated with an increased apoptosis-related Bcl-2/BAX protein ratio. In conclusion, geniposide dose dependently improves β-cell function and increases the proliferation of β-cells exposed to prolonged hyperglycemia.

Di-(2-ethylhexyl) phthalate (DEHP) and dibutyl phthalate (DBP) are representative phthalic acid esters (PAEs), a class of environmental endocrine disruptors used as plasticizers. PAEs exposure is associated with glucose metabolism, insulin resistance, and glucose tolerance; however, the mechanism and various PAE effects on human glucose metabolism remain largely unknown. In this study, we investigated the effects of DEHP, DBP, and their mixture on rat insulinoma (INS-1) cell apoptosis and the mechanism involved in vitro. The INS-1 cells were cultured in RPMI-1640 + 10% fetal bovine serum for 24 h and pretreated with dimethyl sulfoxide (vehicle

Microcystins (MCs) are the most frequent cyanobacterial toxins observed in freshwater systems. Accumulating evidence suggests that MCs pose a serious threat to public health. However, the contributions of the exposure of MCs to the occurrence of human diseases remain largely unknown. This study provides the evidence of the effects of MC-LR on pancreatic β-cell function through the exposure of rat insulinoma (INS-1) cells to 0, 10, 20, or 40μM MC-LR for 72h and explores the underlying molecular mechanisms. Our results demonstrate that exposure to MC-LR for 72h suppresses cell viability, disturbs glucose-stimulated insulin secretion (GSIS), and decreases the expression of insulin protein. Moreover, MC-LR disrupts the cell cycle distribution and increases cell apoptosis at 20 or 40μM for 72h, respectively, indicating that the β-cell mass would be decreased by MC-LR exposure. A transcriptomic analysis revealed several key genes (e.g., Pdx-1, Neurod1, and Abcc8) involved in insulin secretion are significantly differentially expressed in INS-1 cells in response to MC-LR exposure. In addition, several signal transduction pathways associated with diabetes (e.g., type 1 and 2 diabetes) were also identified compared with the control cells. We recommend that MC be considered as a new environmental factor that promotes diabetes development. The identified key genes or pathways may potentially contribute to the future therapies in the environmental contaminants induced β-cell damage.

The inducible nitric oxide synthase (iNOS) is a target of great research interest due to its importance in a number of diseases, for example, septic shock and inflammatory lung diseases. A variety of 3-substituted [1,2,4]triazolo[1,2-a]pyridazine derivatives was synthesized by ring closure with hexahydropyridazine-1-carbothioamide by using aliphatic and aromatic aldehydes. The activity of the new substances was tested on the insulin-secreting rat insulinoma cell line RINm5F. iNOS was expressed through exposure to interleukin-1β (IL-1β) and interferon-γ (IFN-γ). A number of the investigated compounds were more active than the reference inhibitor aminoguanidine (AG). Structure-activity relationships showed that a phenyl substituent in position 3 is apparently essential for inhibition.

Metabolic syndrome, a collection of metabolic abnormalities including diabetes and obesity, affects hundreds of millions of people, and poses a major financial burden to public healthcare around the world. There are two major types of diabetes: Type-1, which is caused by a lack of insulin production and Type-2, which results from a relative deficiency of insulin secretion. Electrical activity of various ion channels in beta (β)-cells plays an important role in the stimulus-secretion of insulin in Type-2 diabetes. Here, we utilize a silicon based lateral patch clamp device to conduct whole cell patch clamp measurements in insulin secreting rat insulinoma (INS-1) cells. High gigaohm seals were achieved between the microfabricated glass capillary apertures and INS-1 cells resulting in picoampere current recordings from various potassium and calcium ion channels. Steady state I–V plots elicited characteristic ion channel properties and longevity of the whole cell mode could be maintained for 1 h without any breakage of the gigaohm seal. Glucose dose responses were also recorded and showed proportional current responses in voltage gated potassium channels. Delivery of multiple ion channel blockers (sulfonylurea drugs) resulted in suppression of currents in potassium channels. The results showcase the capability of our device in performing whole cell measurements and pave a way for developing a cheap platform using our silicon based approach.

Sustained elevations of glucose and free fatty acid concentration have deleterious effects on pancreatic beta cell function. One of the hallmarks of such glucolipotoxicity is a reduction in insulin gene expression, resulting from decreased insulin promoter activity. Sterol regulatory element binding protein-1c (SREBP-1c), a lipogenic transcription factor, is related to the development of beta cell dysfunction caused by elevated concentrations of glucose and free fatty acid. Small heterodimer partner (SHP) interacting leucine zipper protein (SMILE), also known as Zhangfei, is a novel protein which interacts with SHP that mediates glucotoxicity in INS-1 rat insulinoma cells. Treatment of INS-1 cells with high concentrations of glucose and palmitate increased SREBP-1c and SMILE expression, and decreased insulin gene expression. Adenovirus-mediated overexpression of SREBP-1c in INS-1 cells induced SMILE expression. Moreover, adenovirus-mediated overexpression of SMILE (Ad-SMILE) in INS-1 cells impaired glucose-stimulated insulin secretion as well as insulin gene expression. Ad-SMILE overexpression also inhibited the expression of beta-cell enriched transcription factors including pancreatic duodenal homeobox factor-1, beta cell E box transactivator 2 and RIPE3b1/MafA, in INS-1 cells. Finally, in COS-1 cells, expression of SMILE inhibited the insulin promoter activity induced by these same beta-cell enriched transcription factors. These results collectively suggest that SMILE plays an important role in the development of beta cell dysfunction induced by glucolipotoxicity.

Rat insulinoma cells (INS-1), an immortalized pancreatic beta cell line, were cultured on low-fouling carboxymethyl-dextran (CMD) layers bearing fibronectin, the tripeptide Arg–Gly–Asp (RGD) or CDPGYIGSR, a laminin nonapeptide. INS-1 cells were non-adherent on CMD and RGE but adhered to fibronectin- and peptide-coated CMD surfaces and to tissue culture polystyrene (TCPS). On CMD bearing fibronectin and the peptides, INS-1 cells showed higher glucose-stimulated insulin secretion compared to those on TCPS, bare CMD and RGE. INS-1 cells experienced a net cell growth, with the lowest found after 7 days on CMD and the highest on fibronectin. Similarly, cells on RGD and CDPGYIGSR showed lower net growth rates than those on fibronectin. Expression of E-cadherin and integrins α v β 3 and α 5 were similar between the conditions, except for α 5 expression on fibronectin, RGD and CDPGYIGSR. Larger numbers of Ki-67-positive cells were found on CDPGYIGSR, TCPS, fibronectin and RGD. Cells in all conditions expressed Pdx1.

To evaluate the role of sphingosine kinase 1 (SphK1) in insulin secretion, we used stable transfection to knock down the expression of the Sphk1 gene in the rat insulinoma INS-1 832/13 cell line. Cell lines with lowered Sphk1 mRNA expression and SphK1 enzyme activity (SK11 and SK14) exhibited lowered glucose- and 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid (BCH) plus glutamine-stimulated insulin release and low insulin content associated with decreases in in the mRNA of the insulin 1 gene. Overexpression of the rat or human Sphk1 cDNA restored insulin secretion and total insulin content in the SK11 cell line, but not in the SK14 cell line. The Sphk1 cDNA-transfected SK14 cell line expressed significantly less SphK1 activity than the Sphk1 cDNA-transfected SK11 cells suggesting that the shRNA targeting SK14 was more effective in silencing the exogenous rat Sphk1 mRNA. The results indicate that SphK1 activity is important for insulin synthesis and secretion.

Repeated fluctuation in plasma glucose levels, as well as chronic hyperglycemia, is an important phenomenon frequently observed in diabetic patients. Recently, several studies have reported that glucose fluctuation, compared to chronic hyperglycemia, mediates more adverse effects due to induced oxidative and/or endoplasmic reticulum (ER) stress. In type 2 diabetes, stimulation of insulin secretion by glucagon-like peptide-1 (GLP-1) has been found to be reduced, and the results of recent studies have shown that the expression of the GLP-1 receptor (GLP-1R) is reduced by chronic hyperglycemia. However, GLP-1R signaling in glucose fluctuation has not been elucidated clearly. In this study, we hypothesized that intermittent high glucose (IHG) conditions also reduced GLP-1-mediated cellular signaling via reduction in GLP-1R expression. To evaluate this hypothesis, rat insulinoma cells (INS-1) were exposed for 72 h to either sustained high glucose (SHG) conditions (30 mM glucose) or IHG conditions (11 and 30 mM glucose, alternating every 12 h). In comparison to both the SHG and control groups, IHG conditions induced a more significant impairment of insulin release and calcium influx in response to 1 nM GLP-1 treatment. In addition, the activity of caspase 3/7 as well as the gene expression of binding protein (Bip) and C/EBP homologous protein (CHOP), molecular markers of ER stress, was significantly higher in IHG-treated cells than in SHG-treated cells. Interestingly, the expression level of GLP-1R was significantly lower under IHG conditions than under SHG conditions. Collectively, these findings indicated that glucose fluctuation reduces GLP-1R expression through ER stress more profoundly than sustained hyperglycemia, which may contribute to the diminished response of GLP-1.

Insulin-producing pancreatic beta cells are known to be extremely susceptible to the oxidative stress and hypoxia generated following islet transplantation in diabetic patients. We hereby present a novel in vivo selection strategy based on the isolation of insulin-producing cells with enhanced protection after repeated rounds of encapsulation and xenotransplantation. Rat insulinoma INS-1 cells were encapsulated in alginate macrobeads and transplanted in the peritoneal cavity of mice. After 2 days the beads were retrieved and cells were recovered from alginate and propagated in vitro until submitted to a second round of encapsulation and transplantation. Three days later, the surviving cells, named INS-1m2, were isolated from the alginate beads and their protection and functional activity examined. Compared to parental INS-1 cells, the selected INS-1m2 cells were more resistant to hydrogen peroxide, nitric oxide, alloxan and hypoxia. This enhanced protection of the selected cells correlated with the increased level of catalase and poly (ADP-ribose) polymerase expression. Although selected cells expressed more insulin than parental cells, no change in their insulin response to glucose was observed. We conclude that the in vivo selection strategy is a powerful tool for the engineering of insulin producing cells with a broad spectrum of defense properties.

cAMP is a key modulator for glucose-dependent insulin secretion (GDIS). Members of the phosphodiesterase (PDEs) gene family regulate intracellular levels of cAMP by hydrolyzing cAMP to the corresponding inactive 5'AMP derivative. These studies examined the expression and function of all 18 cAMP-specific PDEs in the rat insulinoma derived INS-1 (832/13) cell and isolated rat islets using quantitative PCR and siRNA-mediated gene-specific knockdown. PDE1C, PDE3B, PDE4C, PDE8B, PDE10A, and PDE11A were significantly expressed in rat islets and INS-1 (832/13) cells at the mRNA level. PDE1C, PDE10A and PDE11A were also expressed in brain, along with PDE3B, PDE4C and PDE8B which were also highly expressed in liver, and PDE3B was present in adipose tissue and PDE4C in skeletal muscle. siRNA mediated knockdown of PDE1C, PDE3B, PDE8B and PDE4C, but not PDE10A and PDE11A, significantly enhanced GDIS in rat INS-1 (832/13) cells. Also, selective inhibitors of PDE3 (trequinsin) and PDE4 (roflumilast and L-826,141) significantly augmented GDIS in both INS-1 (832/13) cells and rat islets. The combination of PDE3 and PDE4 selective inhibitors demonstrate that these enzymes comprise a significant proportion of the cAMP metabolizing activity in INS-1 cells and rat islets.

One of the goals in the treatment for diabetes is to enhance pancreatic beta cell function, proliferation, and survival. This study explores the role of lactogenic hormones, prolactin (PRL) and placental lactogen (PL), in beta cell survival. We have previously shown that transgenic mice expressing mouse placental lactogen-1 (mPL1) in beta cells under the rat insulin II promoter (RIP) are resistant to the diabetogenic and cytotoxic effects of streptozotocin (STZ) in vivo. The current study demonstrates that lactogens protect rat insulinoma (INS-1) cells and primary mouse beta cells against two distinct beta cell death inducers, STZ and dexamethasone (DEX), in vitro. Further, we identify the mechanism through which lactogens protect beta cells against DEX-induced death. The signaling pathway mediating this protective effect is the janus-activated-kinase-2/signal transducer and activator of transcription-5 (JAK2/STAT5) pathway. This is demonstrated in INS-1 cells and primary mouse beta cells using three separate approaches, pharmacological inhibitors, JAK2-specific siRNAs and a dominant-negative STAT5 mutant. Furthermore, lactogens specifically and significantly increase the anti-apoptotic protein Bcl-XL in insulinoma cells and mouse islets. Bcl-XL-specific siRNA significantly inhibits lactogen-mediated protection against DEX-induced beta cell death. We believe this is the first direct demonstration of lactogens mediating their protective effect through the JAK2/STAT5 pathway in the beta cell and through Bcl-XL in any cell type.

Both unacylated ghrelin (UAG) and acylated ghrelin (AG) exert metabolic effects. To investigate the interactions between AG and UAG on ghrelin receptors we evaluated the effects of AG and UAG on INS-1E rat insulinoma cells, using insulin secretion after 30min static incubation as a read-out. A possible involvement of the growth hormone secretagogue receptor type 1a (GHS-R1a) or the corticotropin-releasing factor 2 (CRF2) receptor (CRF2R), as a putative receptor for UAG, was also studied determining their mRNA expression and the functional effects of receptor antagonists on insulin release. Both UAG and AG stimulated insulin release dose-dependently in the nanomolar range. The AG-induced insulin output was antagonized by two GHS-R1a antagonists ([d-Lys(3)]GHRP-6 and BIM28163), which did not block UAG actions. These effects occurred in the presence of low levels of GHS-R1a mRNA. Neither CRF2R expression nor effects of the CRF2R antagonist (astressin(2)B) on insulin output were observed. In conclusion, we provide a sensitive and reproducible assay for specific effects of UAG, which in this study is responsible for insulin release by INS-1E cells. Our data support the existence of a specific receptor for UAG, other than the CRF2R and GHS-R1a. The stimulatory effect on insulin secretion by AG in this cell line is mediated by the GHS-R1a.

Cytokines produced by immune cells in pancreatic islets infiltrating are important mediators of beta-cell destruction in insulin-dependent diabetes mellitus. In this study, the effects of retinoic acid (RA) on cytokine-induced beta-cell dysfunction were examined. RA significantly protected interleukin-1 beta (IL-1) and interferon-gamma (IFN-gamma)-mediated cytotoxicity of rat insulinoma cell (RINm5F), and also reduced in IL-1 and IFN-gamma-induced nitric oxide (NO) production, which correlated well with reduced levels of the inducible form of NO synthase (iNOS) mRNA and protein. The molecular mechanism, by which RA inhibited iNOS gene expression, appeared to involve the inhibition of NF-kappa B activation. Our results suggest possible therapeutic value of RA for the prevention of diabetes mellitus progression.

We previously showed that Amomum xanthoides extract prevented alloxan-induced diabetes through the suppression of NF-kappaB activation. In this study, the preventive effects of A. xanthoides extract on cytokine-induced beta-cell destruction were examined. Cytokines produced by immune cells infiltrating pancreatic islets are important mediators of beta-cell destruction in insulin-dependent diabetes mellitus. A. xanthoides extract completely protected interleukin-1beta (IL-1beta) and interferon-gamma (IFN-gamma)-mediated cytotoxicity in rat insulinoma cell line (RINm5F). Incubation with A. xanthoides extract resulted in a significant reduction in IL-1beta and IFN-gamma-induced nitric oxide (NO) production, a finding that correlated well with reduced levels of the inducible form of NO synthase (iNOS) mRNA and protein. The molecular mechanism by which A. xanthoides extract inhibited iNOS gene expression appeared to involve the inhibition of NF-kappaB activation. Our results revealed the possible therapeutic value of A. xanthoides extract for the prevention of diabetes mellitus progression.

Beta cell and insulin antibodies are involved in the pathogenesis of diabetes in human patients. Beta cell antibodies have also been found in about 50% of newly diagnosed diabetic dogs. This study's objective was to examine these antibodies' role in feline diabetes. The serum of 26 newly diagnosed untreated diabetic cats, 29 cats on insulin therapy, 30 cats with diseases other than diabetes, and 30 healthy cats was examined for beta cell and insulin antibodies. For beta cell antibody testing, purified beta cells from a radiation-induced transplantable rat insulinoma were used. Serum from cats in which anti-beta cell antibodies were induced by injecting a purified beta cell suspension subcutaneously was used as a positive control. Following incubation with test sera, fluorescein-labeled anti-cat immunoglobulins were used to visualize binding between the beta cells and cat gamma globulins. Each serum was tested on two different tumor preparations. For the detection of insulin antibodies, a charcoal separation method was used. It was found that none of the healthy cats, none of the newly diagnosed, untreated diabetic cats and none of the cats with diseases other than diabetes had antibodies against beta cells or against endogenous insulin. Four diabetic cats (14%) that had been treated with different insulin preparations had insulin antibodies. It is concluded that immune-mediated processes are not causing diabetes in the cat. Further studies are needed to evaluate if antibodies directed against exogenous insulin alter the response of diabetic cats to insulin.

Cytokines and nitric oxide (NO) have been implicated in the pathogenesis of insulin-dependent diabetes mellitus (IDDM). We have shown that the spin-trapping agent phenyl N-tert-butylnitrone (PBN) protects against streptozotocin (STZ)-induced IDDM in mice. In order to gain more insights into the mechanism(s) of the protective action of PBN against IDDM, we have investigated the effect of this compound on the cytokine-induced NO generation (measured as nitrite) in rat insulinoma RIN-5F cells. Our results demonstrate that PBN cotreatment prevents the generation of nitrite by RIN-5F cells induced by treatment with tumor necrosis factor-alpha, interleukin 1beta, and interferon-gamma in a dose-dependent fashion. The generation of NO as a result of cytokine treatment and the inhibitory effect of PBN were further confirmed by electron paramagnetic resonance spectroscopy. Aminoguanidine, a selective inhibitor of inducible nitric oxide synthase (iNOS), abolished the cytokine-induced nitrite generation whereas N-nitro-l-arginine, an inhibitor more selective for other NOS isoforms, was significantly less effective. Western and Northern analyses demonstrated that PBN inhibits the cytokine-mediated expression of iNOS at the transcriptional level. Cytokine-induced nitrite formation was also inhibited by the two antioxidant agents alpha-lipoic acid and N-acetylcysteine. These results indicate that PBN protects against IDDM at least in part by prevention of cytokine-induced NO generation by pancreatic beta-cells.

To identify genes that might play a role in growth and differentiation of pancreatic beta-cells, we have applied the technique of differential mRNA display to the lineage-related, but functionally distinct rat insulinoma (RIN) cell lines RIN-5AH and RIN-A12. Direct comparison of PCR-generated RIN-5AH and RIN-A12 cDNAs on DNA sequencing gels revealed 31 differentially expressed bands. By Northern blot hybridization, authentic differential expression was confirmed for three cDNAs derived from RIN-5AH cells and four cDNAs from RIN-A12 cells. Nucleotide sequences were determined for these cDNAs and database searches identified one known gene that encoded heat stable antigen CD24. Of the remaining six genes, three matched with established sequence tags from fetal tissue, and three were potentially novel. By RT-PCR analysis, five of the seven genes were expressed in normal fetal and/or adult pancreas. In a detailed survey of CD24 protein expression in the pancreas using the CD24-specific monoclonal antibody J11d, CD24 was predominantly expressed in ductal epithelial cells (E13.5-15.5), developing endocrine (alpha, beta and delta) and exocrine cells (E15.5-20.5) and mature exocrine and peripheral islet delta-cells post E20.5. The retention of CD24 expression in a large proportion of delta-cells but only in a minority of alpha- and beta-cells leads us to hypothesize that CD24 may mark a pool of precursor endocrine cells within adult islets.

Background Microencapsulation of pancreatic islets with polymeric compounds constitutes an attractive alternative therapy for type 1 diabetes mellitus. The major limiting factor is the availability of a biocompatible and mechanically stable polymer. We investigated the potential of Biodritin, a novel polymer constituted of alginate and chondroitin sulfate, for islet microencapsulation. Methods Biodritin microcapsules were obtained using an air jet droplet generator and gelated with barium or calcium chloride. Microencapsulated rat insulinoma RINm5F cells were tested for viability using the [3-(4,5-dimetyl-thiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide] [MTT] colorimetric assay. Microencapsulated rat pancreatic islets were coincubated with macrophages derived from mouse peritoneal liquid to assess the immunomodulatory potential of the microcapsules, using quantitative real time-PCR (qPCR). Biodritin biocompatibility was demonstrated by subcutaneous injection of empty microcapsules into immunocompetent Wistar rats. Insulin secretion by microencapsulated human pancreatic islets was evaluated using an electrochemoluminescent assay. Microencapsulated human islets transplanted into chemically induced diabetic mice were monitored for reversal of hyperglycemia. Results The metabolic activity of microencapsulated RINm5F cells persisted for at least 15 days. Interleukin-1β expression by macrophages was observed during coculture with islets microencapsulated with Biodritin-CaCl2, but not with Biodritin-BaCl2. No statistical difference in glucose-stimulated insulin secretion was observed between nonencapsulated and microencapsulated islets. Upon microencapsulated islet transplantation, the blood glucose level of diabetic mice normalized; they remained euglycemic for at least 60 days, displaying normal oral glucose tolerance tests. Conclusion This study demonstrated that Biodritin can be used for islet microencapsulation and reversal of diabetes; however, further investigations are required to assess its potential for long-term transplantation.

Cholecystokinin (CCK) stimulates insulin secretion. It is, however, not established whether CCK receptors are expressed in insulin-producing cells. We therefore investigated, by in situ hybridization, whether CCK-A or CCK-B receptor mRNA could be detected in normal rat pancreatic islets and in the rat insulinoma cell line, RINm5F. The CCK-A, but not the CCK-B, receptor transcript was detected in both islets and RINm5F cells. Islet CCK-A receptors were mostly confined to the center of the islets corresponding to the distribution of the B cells. In RINm5F cells, insulin release was not significantly affected by cholecystokinin (CCK)-8-S (10(-13) to 10(-7) M), which is in contrast to the insulinotropic effect of CCK-8-S in normal rat islets. Similarly, in FURA-2AM-loaded cells, CCK-8-S (10(-11) to 10(-7) M) was without effect on the intracellular Ca2+ concentration ([Ca2+]ic) in RINm5F cells, whereas CCK-8-S (10(-7) M) markedly increased [Ca2+]ic (by 366+/-2 nM (P0.001) in normal rat islet cells. We conclude that the CCK-A, but not the CCK-B, receptor subtype is expressed in both normal rat islets and in the rat insulinoma-derived cell line RINmS5F. There is, however, a functional difference between normal islets and the RINm5F cells with respect to effects of CCK-8-S on insulin release and [Ca2+]ic.

Proglucagon is processed differentially in pancreatic alpha-cells and intestinal endocrine L cells to release either glucagon or glucagon-like peptide-1-(7-36amide) (tGLP-1), two peptide hormones with opposing biological actions. Previous studies have demonstrated that the prohormone convertase PC2 is responsible for the processing of proglucagon to glucagon, and have suggested that the related endoprotease PC3 is involved in the formation of tGLP-1. To understand better the biosynthetic pathway of tGLP-1, proglucagon processing was studied in the mouse pituitary cell line AtT-20, a cell line that mimics the intestinal pathway of proglucagon processing and in the rat insulinoma cell line INS-1. In both of these cell lines, proglucagon was initially cleaved to glicentin and the major proglucagon fragment (MPGF) at the interdomain site Lys70-Arg71. In both cell lines, MPGF was cleaved successively at the monobasic site Arg77 and then at the dibasic site Arg109-Arg110, thus releasing tGLP-1, the cleavages being less extensive in INS-1 cells. Glicentin was completely processed to glucagon in INS-1 cells, but was partially converted to oxyntomodulin and very low levels of glucagon in AtT-20 cells in the face of generation of tGLP-1. Adenovirus-mediated co-expression of PC3 and proglucagon in GH4C1 cells (normally expressing no PC2 or PC3) resulted in the formation of tGLP-1, glicentin, and oxyntomodulin, but no glucagon. When expressed in alphaTC1-6 (transformed pancreatic alpha-cells) or in rat primary pancreatic alpha-cells in culture, PC3 converted MPGF to tGLP-1. Finally, GLP-1-(1-37) was cleaved to tGLP-1 in vitro by purified recombinant PC3. Taken together, these results indicate that PC3 has the same specificity as the convertase that is responsible for the processing of proglucagon to tGLP-1, glicentin and oxyntomodulin in the intestinal L cell, and it is concluded that this enzyme is thus able to act alone in this processing pathway.

In addition to its interaction at hypothalamic sites to affect feeding and energy expenditure, leptin has been shown to exhibit a proliferative response in erythropoietic cells. The functional leptin receptor is also present in pancreatic islets and we now demonstrate that a commonly used clonal insulin secreting beta-cell line, RINm5F, expresses high levels of the Ob-Rb mRNA. Leptin causes an increase in tyrosine phosphorylation of a number of intracellular proteins and a dose related (10 nM-200 nM) increase in expression of the immediate-early gene, c-fos. This precedes a leptin induced proliferative response in serum-deprived RINm5F cells, which suggests that leptin might be involved in the complex regulation of proliferation of the pancreatic beta-cell.

The purpose of this study was to evaluate the effects of interferon-gamma (IFN-gamma) alone and in combination with interleukin 1beta (IL-1beta) on inducible nitric-oxide synthase (iNOS) mRNA and protein expression, nitrite production, and insulin secretion by islets of Langerhans. Treatment of rat islets with IL-1beta results in a concentration-dependent increase in the production of nitrite that is maximal at 5 units/ml. Individually, 0. 1 unit/ml IL-1beta or 150 units/ml rat IFN-gamma do not stimulate iNOS expression or nitrite production by rat islets; however, in combination, these cytokines induce the expression of iNOS and the production of nitrite to levels similar in magnitude to the individual effects of 5 units/ml IL-1beta. The islet beta-cell, selectively destroyed during insulin-dependent diabetes mellitus, appears to be one islet cellular source of iNOS as 150 units/ml rat IFN-gamma and 0.1 unit/ml IL-1beta induced similar effects in primary beta-cells purified by fluorescence-activated cell sorting and in the rat insulinoma cell line, RINm5F. iNOS expression and nitrite production by rat islets in response to 150 units/ml rat IFN-gamma and 0.1 unit/ml IL-1beta are correlated with an inhibition of insulin secretion and islet degeneration that are prevented by the iNOS inhibitor aminoguanidine. The mechanism by which IFN-gamma increases the sensitivity of beta-cells for IL-1-induced iNOS expression appears to be associated with an increase in the stability of iNOS mRNA. Last, cellular damage during physical dispersion of islets results in the release of sufficient amounts of IL-1beta to induce iNOS expression and nitrite production in the presence of exogenously added rat IFN-gamma. The cellular source of IL-1beta under these conditions is believed to be resident islet macrophages as depletion of macrophages prior to dispersion prevents IFN-gamma-induced iNOS expression and nitrite formation by dispersed islet cells. These studies show that the T-lymphocyte cytokine, IFN-gamma, increases the sensitivity of rat islets to the effects of IL-1beta on iNOS expression and nitrite production by 10-fold, in part, through the stabilization of iNOS mRNA. Our studies also support an effector role for IFN-gamma, in concert with resident islet macrophage release of IL-1beta, in mediating beta-cell destruction during the development of autoimmune diabetes.

Several immortalized cell lines serve as models for procholecystokinin (pro-CCK) processing. Rin5F cells, derived from a rat insulinoma, and STC-1 cells, derived from a murine intestinal tumor, process pro-CCK mainly to amidated CCK 8. Both also make significant quantities of amidated CCK 22, a slightly larger form found in the gut. Many modifications are necessary during pro-CCK processing including cleavages performed by endoproteases, the identities of which are unknown. A candidate endoprotease is prohormone convertase 1 (PC1) also known as PC3, a Ca2+-dependent serine endoprotease of the subtilisin family. Constitutive expression of antisense PC1 message in stably transfected Rin5F cells resulted in a significant reduction of the cellular content of CCK 8 as measured by radioimmunoassay. Several affected cell lines displayed about 80% reduction in CCK content in early passages after transfection. Expression of antisense PC1 message in these cell lines resulted in a selective depletion of CCK 8 and a comparative sparing of CCK 22. The induction of antisense PC1 message within a single subclone of Rin5F cells using the Lac Switch system also resulted in a significant inhibition of CCK content. Expression of antisense PC1 message in a stably transfected STC-1 cell line also resulted in a decrease in CCK content and in PC1 protein expression, and the specific depletion of CCK 8 with comparative sparing of CCK 22. These observations support the hypothesis that PC1 is necessary for pro-CCK processing in Rin5F and STC-1 cells and suggests a role for PC1 endoprotease in the biosynthesis of CCK 8 in vivo.

A critical aspect of type II diabetes is the dysfunction and death of insulin-secreting β-cells. A 37-residue peptide hormone, islet amyloid polypeptide (IAPP), plays a central role in this pathology since it is known that rodent models transgenic for human IAPP show spontaneous development of β-cell dysfunction and diabetic symptoms. Recent evidence suggests that a subset of several alternative α-helical membrane-bound oligomers on-pathway to amyloid fiber formation, and not the amyloid fibrils themselves, are the toxic species in disease. We recently showed that IAPP adopts cell and membrane penetrating properties that are likely correlated with its toxicity towards β-cells; IAPP localizes to lysosomes of rat insulinoma pancreas β-cells (INS-1) at peptide concentrations determined by cell viability assays to be nontoxic, and relocates to mitochondria at toxic peptide concentrations (M. Magzoub and A. D. Miranker, Faseb J., 2011, 26, 1228-1238). Here, we evaluate the ability of small molecules to agonize and antagonize IAPP toxicity towards INS-1 cells via large-scale screens of cell viability. Localization of IAPP to subcellular compartments of INS-1 cells in the presence of these molecules is also investigated. We further describe the activity modulation and subcellular localization of the peptide in the presence of small molecules from libraries at Yale Small Molecule Discovery Center that have been shown to slow lipid-catalyzed amyloid fiber formation of IAPP.

Islet transplantation is a promising treatment for diabetes. Serum is a necessary supplement in islet cultures, but it has various disadvantages including the risk of contamination by several pathogens. Results of this study suggest that sericin is a useful alternative supplement. Sericin accelerated the proliferation of the rat insulinoma cell line RIN-5F and improved the serum-free culture of rat islets.

The mitochondrial glycerol phosphate dehydrogenase (mGPD) is one of several proteins that are abundant in the pancreatic islet. Hormonal and nutritional influences confer tissue-specific patterns of expression on many of these proteins and the primary amino acid sequence of these proteins in the islet often differs from those in other tissues. However, the deduced amino acid sequence of the rat islet mGPD was identical to that of testis and liver. (The islet mGPD also possesses calmodulin-like calcium-binding sequences.) Islet mGPD activity and amount of protein were not changed by culturing islets at various concentrations of the insulin secretagogues, glucose, leucine, glutamine, or methyl succinate, which are conditions that alter the amounts of other enzymes in the islet. Unlike mGPD in tissues, such as liver, where mGPD activity is low, the high amount of islet mGPD was not further induced in hyperthyroid rats or by adding T3 to cultured islets or rat insulinoma cells. This suggests that the islet mGPD is under different regulation than the enzyme in tissues where its activity is low.

The rat insulinoma cell line RIN 1046-38 loses glucose-stimulated insulin secretion as a function of time in culture. We found that the loss of glucose sensing in these cells was correlated with the loss of expression of GLUT-2 and glucokinase. Stable transfection of RIN cells with a plasmid containing the GLUT-2 cDNA conferred glucose-stimulated insulin release in intermediate but not high passage cells, with the near-maximal 3-fold increase occurring at 50 microM glucose. GLUT-2 expressing cells also exhibited a larger response to the combination of 5 mM glucose + 1 microM forskolin than untransfected cells (7.9 versus 1.6-2.7-fold, respectively). GLUT-2 expressing intermediate passage, but not high passage, RIN cells exhibited a 4-fold increase in glucokinase enzymatic activity relative to nonexpressing controls. Glucokinase activity was also increased by transfer of the GLUT-2 gene into intermediate passage RIN cells via recombinant adenovirus. Preincubation of GLUT-2 expressing intermediate passage RIN cells with 2-deoxyglucose to inhibit low Km hexokinases resulted in a glucose-stimulated insulin secretion response that was shifted toward the physiologic range. These studies indicate that GLUT-2 expression confers both a high and low affinity glucose-stimulated insulin secretion response to intermediate passage RIN cells.

Gastrin gene expression in the pancreatic islets is developmentally regulated and occurs largely during fetal life. Deletional analysis of transiently transfected rat insulinoma cells with gastrin 5'-flanking sequences in luciferase reporter genes demonstrated that the gastrin promoter sequence proximal to -111 base pairs (bp) contains the cis-regulatory elements necessary for maximal transcription. Mutational analysis identified the sequence CCCCACCCCA (-109 to -100 bp) as a positive cis-regulatory element (CACC) located 5' to a previously described negative element (-100 to -90 bp) and E-box positive element at -82 bp. Multimers of the CACC element in a heterologous promoter activated transcription independent of the other cis-regulatory elements. CACC binding proteins were purified from insulinoma cell nuclear extracts by cation exchange and affinity chromatography. Southwestern blot of nuclear extracts identified a 70-kDa CACC-binding protein. Mutational analysis of the CACC element showed a close correlation between DNA binding of this protein and transcriptional activation. Transcriptional activation by multimers of the CACC element in a heterologous promoter was detected in a variety of cell lines but was strongest in those of islet lineage. Likewise, the presence of the 70-kDa CACC-binding protein was found in many cell lines but was most abundant in the insulinoma cells. The CACC-binding protein has not been previously identified among the known pancreatic regulatory factors and may have an important role in the developmental expression of gastrin.

The specific binding of a novel tritiated K+ channel opener, [3H]BAY X 9228, has been characterized in a rat insulinoma (RINm5F) cell line. The KD was 2.1 nM and Bmax 50 fmol/mg total protein as determined by saturation analysis. The high affinity binding to intact cells was inhibited by pinacidil and by a series of BAY X 9228 analogs with an activity sequence correlating well with that for producing glyburide-reversible relaxation of partially depolarized rat aorta. This represents the first report of the specific binding of a K+ channel opener to cultured cells.

Octreotide (SMS), a somatostatin analogue, is an established antigrowth peptide, but it does not effectively inhibit the growth of insulinoma cells. In order to study the mechanisms that underlie this apparent lack of an antiproliferative effect on insulinoma tumor cells we established the rat insulinoma cell line, RINm5F, in culture. Cells in culture were tested by incubation in media with and without SMS. To study tritiated [3H]-thymidine incorporation into extracted DNA (TTID), 2 muCi/well of 3H was added for 24 hr, and cells were harvested and assayed for TTID (cpm/microgram DNA). Insulin (IRI) and intracellular cAMP (cAMPi) were measured by RIA. To study the effects of SMS on insulin secretion, conditioned media were sampled after 24 hr. To study the effects of cAMPi, conditioned medium was used to extract cAMPi following incubation with SMS for 15 min. Increasing concentrations of SMS had no significant effect on TTID in the presence of 1% FBS. Trypan blue exclusion tests showed90% viable cells throughout all stages of these experiments. There were no significant differences in cell numbers and protein content in the presence of SMS. There was a significant decrease in the secretion of insulin and intracellular cAMP levels in response to 50 nM SMS. However, SMS significantly inhibited TTID in RINm5F cells following a 4-hr pretreatment with pertussis toxin (PT) (23553 +/- 1747 vs 20635 [cpm/microgram DNA] +/- 1983 [SEM], P0.01). We conclude that the inhibition of insulin secretion by SMS is associated with an attenuation of cAMP formation.(ABSTRACT TRUNCATED AT 250 WORDS)

Insulin-secreting cells (RINm5F) have successfully been grown on a large scale on poly- l -lysine coated-polystyrene microcarriers, providing a high cell number in a restricted volume under conditions that respect the metabolic integrity of these anchorage-dependent cells. The energetic metabolism of the perfused cells hae been followed non-invasively by phosphorus-31 nuclear magnetic resonance spectroscopy. Glucose starvation induced a rapid decrease in nucleoside triphosphates (mainly ATP) pools, correlated with an increase in Pi level. The initial ATP level was rapidly recovered when the cells were refed with glucose or wth mannose, but not with galactose, even after 2 h of perfusion. These differential effects of hexoses on energetic metabolism might be related to their various insulin-release actions on tumor islet cells.

(Na+K+)ATPase is necessary for the maintenance of the membrane potential. The activity of this enzyme was studied in purified plasma membranes from a glucose-responsive rat insulinoma. Ouabain-sensitive (Na+K+)ATPase activity showed expected ATP dependency with a Km of 0.4 m m . It was also dependent on Mg2+ (Km range 70–80 μM). In the presence of Mg and ATP, half-maximal activity was obtained at a Na concentration of 30 m m and the enzyme activity increased sigmoidally with a Hill coefficient of 1.5. No direct effect on enzyme activity was observed with the insulin secretagogues glucose, fructose, glyceraldehyde, and ketoisocaproate, or with dibuturyl-cAMP and the phosphodiesterase-inhibitor isobutyl methyl xanthine. It is concluded that (Na+K+)ATPase is not directly influenced by known secretagogues associated with insulin release by the β cell.

The rat insulinoma RIN 5F and the mouse pituitary AtT-20 cell line, which are known to express several biologically active peptides, were found to express CCK mRNA, to correctly process, and to release immunoreactive cholecystokinin (CCK) peptides. They expressed low levels of these peptides (about 0.4 and 0.2 ng/mg protein, respectively) and both cell lines processed pro-CCK to a form which co-eluted with CCK 8 sulfate on Sephadex gel filtration chromatography and HPLC. The major CCK 8 immunoreactive peptide which they secreted co-eluted with CCK 8 on Sephadex G-50 chromatography. The secretion of CCK from both cell lines was significantly enhanced by treatment for 24 h with forskolin + IBMX (3-isobutyl-1-methyl-xanthine, a phosphodiesterase inhibitor). This treatment also doubled the CCK content of the AtT-20 cells. It appears that the ability of different endocrine tumor cells to express and process CCK is not as uncommon as previously thought. These cells should be useful for future studies of CCK expression, processing, and regulation of secretion.

Transfection of the insulin-producing rat islet tumor cell line RIN-5AH with a full length cDNA of the rat hepatic growth hormone (GH) receptor (GH-R1-638) augments the GH-responsive insulin synthesis in these cells. Using this functional system we analyzed the effect of COOH-terminal truncation of the GH receptor. Two mutated cDNAs encoding truncated GH receptors, GH-R1-294 and GH-R1-454, respectively, were generated by site-directed mutagenesis and transfected into the RIN cells. Both receptor mutants were expressed on the cell surface and displayed normal GH binding affinity. Whereas GH-R1-638 had a molecular mass of about 110 kDa, GH-R1-294 and GH-R1-454 showed molecular masses of 49 and 80 kDa, respectively. Cells expressing GH-R1-454 internalized GH to a similar extent as cells transfected with the full length receptor and the parent cell line, but GH-R1-294-expressing cells showed a markedly reduced capability of GH internalization. In contrast to cells transfected with GH-R1-638, none of the cell lines expressing truncated GH receptors exhibited any increase of the GH-stimulated insulin production. We conclude that domains within the COOH-terminal half of the cytoplasmic part of the GH receptor are required for transduction of the signal for GH-stimulated insulin synthesis, whereas cytoplasmic domains proximal to the transmembrane region are involved in receptor-mediated GH internalization.

The BB rat provides an excellent animal model for type 1 (insulin-dependent) diabetes mellitus. Cytotoxic autoantibodies against pancreatic beta cells have been found in the sera of both patients with type 1 (insulin-dependent) diabetes and BB rats. These antibodies have been implicated in the pathogenesis of the disease. In this study, a monoclonal autoantibody, designated KT1, has been developed by the fusion of spleen cells from a BB rat and a mouse myeloma cell line. KT1 was found to be of the immunoglobulin M isotype and reacted specifically with islet cells. In microcytotoxicity assays KT1 was shown to mediate complement-dependent lysis of approximately 30% of a rat insulinoma cell line and 25% of rat pancreatic islet cells in culture. It did not cause lysis of the other cell lines tested. KT1 has been demonstrated, by indirect immunofluorescence, to bind specifically to a cell surface antigen on live and acetone-fixed islet cell cultures from Wistar rat neonates and to rat insulinoma cells. Western blotting experiments revealed reaction to a 68-kDa protein from rat insulinoma cell extracts. This monoclonal antibody may have clinical relevance as it exhibits properties similar to the islet cell surface antibodies present in the sera of BB rats.

The clinical significance of cytoplasmic islet cell autoantibodies (ICA) has been studied since their discovery by Bottazzo et al. in 1974 [1]. Some ICAs destroy pancreatic B cells in the presence of complement [2], whereas others take no part in this destruction [3]. This suggests that islet function varies with the amount of ICA produced. In the present investigation we report the heterogeneity of monoclonal islet cell antibodies produced by one of us in terms of insulin release from isolated rat islets as well as from rat insulinoma cells (RINr).

Type I diabetes is the result of an autoimmune destruction of pancreatic beta cells. T cells appear to play a key role in this process. Thus far little information is available on the beta cell antigen or antigens recognized by auto-reactive T cells. Previously, we identified a 38 kD T cell antigen that appears to be localized in the membrane of insulin secretory granules and that is recognized by T cells from newly diagnosed type I diabetes patients. Other groups have reported T cell reactivity against glutamic acid decarboxylase (GAD). To obtain an indication of whether or not beta-cell reactive T cells from type I diabetes patients recognize a limited number of beta-cell antigens, we cloned T-cell lines reactive with rat insulinoma (RIN) membranes from two patients and analysed their antigen specificity. We also studied the antigen specificity of one RIN membrane reactive T-cell clone (1C5), previously isolated from a third patient. From the first patient two identical RIN membrane reactive T-cell clones (7A13) were isolated. The second patient yielded two identical (23A19) RIN membrane reactive T-cell clones, and one that was different (234A33). All clones were CD4+ and saw antigen in the context of different HLA class II alleles. The reactivity of the clones was, however, not restricted to beta cells: all clones showed cross-reactivity with one or more rat tissues, with some preference for those of neuroendocrine origin, but the cross-reactivity patterns were all different. All four clones recognized different fractions electro-eluted from RIN membranes: 29-36 kDa (7A13), 120-170 (23A19), 29-41 (23A33) and 56-72 kDa (1C5). The 23A33 clone reacted with the same 38 kDa fraction electro-eluted from insulinoma membranes as a beta-cell reactive clone (1C6) published previously, but none of the other known beta cell antigen preparations tested were recognized by the T-cell clones. Finally, the subcellular localization of the antigens recognized showed at least two different patterns. These data indicate that beta-cell reactive T cells from the peripheral blood of type I diabetes patients are not necessarily beta-cell specific and may be heterogeneous in regard to their antigen specificity and HLA class II restriction.

We have isolated rat rig/ribosomal protein S15 gene from a DNA library derived from a rat insulinoma and determined the complete nucleotide sequence. The rat rig/S15 gene is composed of four exons and three introns spanning 2 kbp and exhibits distinctive structural features unique for a ribosomal protein gene.

Background: Promoting islet cell viability in the face of cytokine-induced oxidative stress will prove valuable in prolonging islet allograft survival. Gene transfer to pancreatic islet beta cells may prove beneficial in enhancing islet graft survival following cytokine exposure.

Objective: To investigate the effect of single and combined antioxidant gene overexpression on β-cell viability following cytokine exposure in a rat insulinoma (RIN) cell line.

We have isolated a novel gene, rig (rat insulinoma gene) from rat insulinomas. In the present study, rig was found to be expressed in rat regenerating liver and in primary cultured rat hepatocytes. The level of rig mRNA was increased at the proliferative phase of liver regeneration. In synchronously cultured hepatocytes, the rig mRNA level was elevated at the G1 phase of the cell cycle and the rig-protein was accumulated in the nuclei during the S phase. These results indicated that rig could be involved in a more general way in growth or cell replication.

The monoclonal IgM 3G5, which reacts with the surface membranes of rat insulinoma cells RINm5F, was purified by HPLC and labeled with 125I using Protag-125; bovine IgG (bIgG) was similarly radiolabeled, and used as a control. 125I-3G5 was incubated with RINm5F cells either at 4 degrees C or at 37 degrees C. 125I-3G5 bound onto RINm5F cells growing in Petri dishes remained approx. constant over 44 h when incubated at 4 degrees C, whereas at 37 degrees C radioactivity was released back in the medium starting at 3 h (plateau at approx. 20 h). At the end of incubation at 37 degrees C, activity in the medium included a high percentage of free 125I (15.69 vs 2.62% for bIgG, and 1% for 3G5 at 4 degrees C). In a cell suspension experiment, cell-bound 125I-3G5 also remained constant over a 24 h incubation at 4 degrees C, whereas at 37 degrees C it decreased to 37.5% of its initial value (64.1% at 4 h). Concomitant microautoradiography showed diffuse radioactive deposits within the RINm5F cells following incubation with 125I-3G5 (but not 125I-bIgG) at 37 degrees C. These results indicate that 3G5-IgM reacts with a surface antigen on the RINm5F cells, but is rapidly internalized by the cells: within the cells, this antibody undergoes some metabolic processing which results in the release of free 125I outside the cells.

Regulation of endoplasmic reticulum (ER) Ca2+ cycling by inositol 1,4,5-trisphosphate (IP3) was studied in saponin-permeabilized RINm5F insulinoma cells. Cells were incubated with mitochondrial inhibitors, and medium Ca2+ concentration established by nonmitochondrial pool(s) (presumably the ER) was monitored with a Ca2+ electrode. IP3 degradation accounted for the transience of the Ca2+ response induced by pulse additions of the molecule. To compensate for degradation, IP3 was infused into the medium. This resulted in elevation of [Ca2+] from about 0.2 microM to a new steady state between 0.3 and 1.0 microM, depending on both the rate of IP3 infusion and the ER Ca2+ content. The elevated steady state represented a bidirectional buffering of [Ca2+] by the ER, as slight displacements in [Ca2+], by small aliquots of Ca2+ or the Ca2+ chelator quin 2, resulted in net uptake or efflux of Ca2+ to restore the previous steady state. When IP3 infusion was stopped, [Ca2+] returned to its original low level. Ninety per cent of the Ca2+ accumulated by the ER was released by IP3 when the total Ca2+ content did not exceed 15 nmol/mg of cell protein. Above this high Ca2+ content, Ca2+ was accumulated in an IP3-insensitive, A23187-releasable pool. The maximal amount of Ca2+ that could be released from the ER by IP3 was 13 nmol/mg of cell protein. The data support the concept that in the physiological range of Ca2+ contents, almost all the ER is an IP3-sensitive Ca2+ store that is capable of finely regulating [Ca2+] through independent influx (Ca2+-ATPase) and efflux (IP3-modulated component) pathways of Ca2+ transport. IP3 may continuously modulate Ca2+ cycling across the ER and play an important role in determining the ER Ca2+ content and in regulating cytosolic Ca2+ under both stimulated and possibly basal conditions.

Experiments aimed at the partial reconstitution of the intracellular transport systems regulating the cytosolic free Ca2+ homeostasis are reported. Rat insulinoma subcellular fractions enriched in mitochondria, endoplasmic reticulum (microsomes), and secretory granules were studied. The ambient free Ca2+ concentration maintained by the separate or combined organelles was determined with a Ca2+-selective minielectrode. The data demonstrate that ambient [Ca2+] is established by the microsomes, not by the mitochondria or the secretory granules, in the range of resting cytosolic Ca2+ concentrations (0.1-0.2 microM Ca2+). Furthermore, the microsomes are able to deplete largely the mitochondria of their exchangeable calcium. Nonetheless, both mitochondria and microsomes, but not secretory granules, function as a coordinated unit to restore the previous ambient [Ca2+] following its perturbation. Thus, mitochondria play a major role in bringing down rapidly ambient [Ca2+] to the submicromolar range, whereas the endoplasmic reticulum acts as a relay in the transport mechanisms which lower [Ca2+] to the resting level.

1. 1. The effects of cytotoxic drugs and inhibitors of insulin secretion were examined in vivo in rats with a radiation-induced transplantable insulinoma, and in vitro using cultured rat insulinoma cells and the derived RINm5F insulin-secreting cell line. 2. 2. Administration of diazoxide to insulinoma-bearing rats resulted in a transient decrease of plasma insulin with a temporary rise of glucose concentrations. Mannoheptulose and somatostatin failed to affect the marked hyperinsulinaemia and hypoglycaemia. 3. 3. Streptozotocin produced a rapid and sustained decrease of insulin concentrations in insulinoma-bearing rats, accompanied by a progressive elevation of plasma glucose. Administration of alloxan failed to affect circulating insulin or glucose concentrations. 4. 4. In vitro , streptozotocin and alloxan exerted approximately equipotent time-dependent and concentration-dependent cytotoxic effects on insulinoma cells and RINm5F cells as established by cell staining with trypan blue. The cytotoxic actions of both drugs were decreased by agents believed to scavenge free radicals or to act as inhibitors of poly(ADP-ribose) synthetase. 5. 5. The results suggest that the cytotoxic actions of streptozotocin and alloxan on rat insulinoma cells and RINm5F cells are mediated by the generation of hydroxyl free radicals and DNA strand breaks. The ineffectiveness of alloxan in insulinoma-bearing rats probably reflects the high rate of decomposition of the drug in vivo .