Rat Islet Cell Tumor Research Articles

MiR-320a induces pancreatic β cells dysfunction in diabetes by inhibiting MafF

A variety of studies indicate that microRNAs (miRNAs) are involved in diabetes. However, the direct role of miR-320a in the pathophysiology of pancreatic β cells under diabetes mellitus remains unclear. In the current study, islet transplantation and hyperglycemic clamp assays were performed in miR-320a transgenic mice to explore the effects of miR-320a on pancreatic β cells in vivo. Meanwhile, β cell-specific overexpression or inhibition of miR-320a was delivered by adeno-associated virus (AAV8). In vitro, overexpression or downregulation of miR-320a was introduced in cultured rat islet tumor cells (INS1). RNA immunoprecipitation sequencing (RIP-Seq), luciferase reporter assay, and western blotting were performed to identify the target genes. Results showed that miR-320a was increased in the pancreatic β cells from high-fat-diet (HFD)-treated mice. Overexpression of miR-320a could not only deteriorate the HFD-induced pancreatic islet dysfunction, but also initiate pancreatic islet dysfunction spontaneously in vivo. Meanwhile, miR-320a increased the ROS level, inhibited proliferation, and induced apoptosis of cultured β cells in vitro. Finally, we identified that MafF was the target of miR-320a that responsible for the dysfunction of pancreatic β cells. Our data suggested that miR-320a could damage the pancreatic β cells directly and might be a potential therapeutic target of diabetes.

Effects of Akkermansia muciniphila on the Proliferation, Apoptosis and Insulin Secretion of Rat Islet Cell Tumor Cells

To investigate the effects of Akkermansia muciniphila ( A. muciniphila) on the proliferation, apoptosis and insulin secretion of rat pancreatic islet cell tumor cells (INS-1). INS-1 cells were divided into three groups, normal, repair, and protect groups, and subsequently every group was subjected with A. muciniphila metabolites, live A. muciniphilaorpasteurized A. muciniphila for 48 h. A group that did not treat with anything was set as blank control. After intervention, the cell viability was determined by MTT method, the insulin secretion level stimulated by glucose was determined by ELISA, the expressions of the genes involved in insulin secretion and apoptosis were tested by qRT-PCR, and the expression of apoptosis related protein Bax was evaluated by Western blot. There was no significant change in INS-1 cell morphology after co-incubation with 3 types of A. Muciniphila interventions for 48 h. The proliferative activity of INS-1 cells was decreased in the repair group that treated with live A. muciniphila than that of control ( P<0.005). A. muciniphila intervention had no effect on insulin secretion in INS-1 cells in normal, repair or protection group ( P>0.05). A. muciniphila secretions promoted the expression of glucose transporter 2 ( Glut2) in 3 groups and the expression of glucokinase ( GCK) in repair group ( P<0.05). The expression of Baxof the INS-1 cell in the normal group was decreased after intervented with 3 kinds of A. muciniphila intervention materials ( P<0.001).The expression of Bax gene of the INS-1 cell in the repair group that treated with dead A. muciniphilawas decreased ( P<0.05). The expression of Bax protein of INS-1 cells that treated with A. muciniphila interventions was decreased. A. muciniphila can promote the expression of insulin secretion-related genes in INS-1 cells, inhibit the expression of apoptotic genes and apoptosis protein Bax.This research provides a new direction for applying A. muciniphila in improving type 2 diabetes.

Induction of Pancreatic Phenotypes in Central Nervous System Derived Pluripotential Progenitor Cells

Fetal rat brain stem cells (RSCs) have been induced to express pituitary properties when exposed to pituitary cells (U et al., 2002). In this study, we explored whether these RSCs could also be influenced to acquire properties characteristic of the pancreas. To this end, RSCs in culture were exposed to media conditioned by rat islet tumor cells and media containing Exendin-4 and nicotinamide since both have been shown to induce pancreatic phenotypes in embryonic stem cells. Lastly, an expression construct for pdx-1 was introduced into RSCs. The expression of pancreatic markers was analyzed using RT-PRC and immunocytochemistry. When RSCs were exposed to rat islet tumor cell conditioned media and media containing Exendin-4 and nicotinamide, the expression of pdx-1, insulin and somatostatin were observed. They also acquired a spherical shape typical of pancreatic cells in culture. Under these varied conditions, transcriptional factors essential to pancreatic development such as pdx-1 and Isl-1 were induced. The critical role of pdx-1 in stimulating certain endocrine pancreatic properties in RSCs was further confirmed upon the introduction of an expression construct for pdx-1 which markedly induced insulin and somatostatin. Taken together, these findings suggests that fetal brain stem cells are pluripotent and can be reprogrammed to acquire pancreatic properties through pathways which involved the transcription factor Pdx1.

Abstract C48: The role of the neuregulins and their receptors in the islets of Langerhans

Abstract The Neuregulins are a sub-class of the Epidermal Growth Factor (EGF) family of mitogenic ligands. This research aims to investigate their expression in individual cell types within the Islets of Langerhans in order to determine whether they play a role in regulating islet cell growth or hormone secretion. This information may be useful in the future in treating diabetic patients or in rare instances of islet cell cancer. An expression profile of all the EGF receptors and ligands, including the four NRG genes and some of their splice variants, was determined in rat islets using immunohistochemical staining with specific mouse monoclonal and rabbit polyclonal antibodies. Double immunofluorescent staining with antibodies to the ligands or receptors and to the main islet cell hormones, insulin, glucagon and somatastatin was used to identify which cell types were expressing each isoform. The rat islet tumor cell line CRI-G1 was selected as a model system to determine cellular responses to addition of the different ligands. Immunofluorescent staining and RT-PCR was used to construct a receptor and ligand expression profile. Immunohistochemistry demonstrated that some ligands such as NRG3 or the alpha and beta isoforms of NRG1 were expressed in restricted regions of the islets. Through double immunofluorescence it was found that NRG-1α was present in β-cells and NRG-1β and NRG-3 were mainly present in α-cells. The same restricted pattern of expression was also found in human islets. In contrast, staining for HER-3 and HER-4 receptors in rat islets was much more homogenous. In using single immunofluorescence with the CRI-G1 cell line both HER-3 and HER-4 as well as with two of the NRG-4 splice variants were detected. PCR using cDNA from the cell line confirmed the expression of both NRG-1 and NRG-2 at the mRNA level. Differential expression of NRG-1α and 1 in or cells due to alternative splicing may play a role in regulating cell proliferation, differentiation or hormone secretion in individual islet cell types across a range of species. Differences in localisation between the three Neuregulin forms and their two receptors suggests that both autocrine and paracrine neuregulin-mediated signalling occurs. The expression of NRG-1, NRG-2, NRG-4 and the receptors HER-3 and HER-4 within the CRI-G1 cell line provides a model system to explore the activity of this complex system in vitro. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C48.

Molecular Characterization of Size and Copy Number of the Human Insulin Gene in Transfected Rat Islet Tumor Cell Clones

Two stable transfectants, NHI-6F and NHI-5B, obtained by cotransfection of the human insulin gene (a 15 kb EcoRI genomic fragment) with pSV2-neo into MSL-cells (pluripotent rat islet tumor cells), were characterized with regard to copy number and size of the inserted human sequences by Southern blot analyses using appropriate restriction enzymes. Digestion of total genomic DNA from the NHI-6F clone with EcoRI, SacI and BglII gave in all cases single human insulin gene fragments of 25 kb, 28 kb and 11 kb, respectively. From these data we were able to conclude that NHI-6F carries a single copy of the human insulin gene and that this copy has at least retained the 5' BglI and the 3' BglII flanking sites of human origin. Digestion of NHI-5B DNA gave a different banding pattern compared to NHI-6F (EcoRI: 20 kb; SacI: 7.8 kb; BglII: 11 kb). These data indicate the presence of a single human insulin gene copy in clone NHI-5B with the preservation of the 5' SacI and 3' BglII human flanking sites. In conclusion, the two transfected rat islet tumor clones, NHI-6F and -5B, carry a single copy of the human insulin gene. Each copy is presumably functionally active since known promoter and enhancer elements reside within 400 bp 5' to the gene. NHI-6F and -5B carry 2,500 bp and 4,500 bp 5' to the gene, respectively.

Monoclonal Antibodies as Probes to the Differentiated Exocrine Pancreas React to Monoclonal Islet Tumor Tissue

Pancreatic exocrine and ductal reacting monoclonal antibodies were derived from a mouse immunized with fixed pluripotent rat islet tumor cells (MSL) and boosted in vitro with fixed and concentrated tumor cell culture supernatant. Antibodies were obtained against duct cells, intercalated ducts, acinar cells and zymogen granules as well as against parietal cells. Unexpectedly, no monoclonal antibodies were directed against the endocrine pancreas, whereas six out of seven exocrine reacting antibodies stained total or subpopulations of cells in sections of monoclonal hypoglycemic MSL-tumors. These data may support the hypothesis of a common endodermal origin of the exocrine and endocrine pancreas.

Somatostatin-14, somatostatin-28, and prosomatostatin [1-10] are independently and efficiently processed from prosomatostatin in the constitutive secretory pathway in islet somatostatin tumor cells (1027B 2)

We have characterized the biosynthetic origin of somatostatin-14 (SS-14), SS-28, and pro-SS [1-10] from pro-SS (PSS) in 1027B 2 rat islet tumor cells. Because these cells lack regulated secretion and show unresponsiveness of the SS gene to cAMP, we have additionally carried out morphological and functional studies to elucidate the molecular defect in cAMP signalling and to localize the sites of PSS maturation along the secretory pathway. Cell extracts and secretion media were analysed by high performance liquid chromatography and specific C- and N-terminal radioimmunoassays. Electron microscopic sampling of 1027B 2 cell cultures showed that most cells had very few dense core secretory granules for heterogeneous sizes. The cells expressed the endoproteases furin, PC1, and PC2 and contained large quantities of fully processed SS-14 and SS-28 with very little unprocessed PSS (ratio SS-14:SS-28:PSS=39:51:10%). They secreted high concentrations of SS-14, SS-28, and PSS [1-10] constitutively along with PC1 and PC2. Pulse-chase studies demonstrated that PSS is rapidly (within 15 min), and efficiently processed to SS-14, SS-28, and PSS [1-10] via separate biosynthetic pathways: PSS→SS-14+8 kDa; PSS→SS-28+7 kDa; PSS→PSS [1-10]. Monensin reduced intracellular SS-like immunoreactivity without altering processing efficiency. Transfection with the catalytic subunit of protein kinase A (PKA-C) activated SS promoter-CAT activiting indicating that the defect in cAMP-dependent signaling in 1027B 2 cells lies at the level of PKA-C. PKA-C overexpression failed to alter the ratio of processed SS-14 and SS-28. These results demonstrate that SS-14, SS-28, and PSS [1-10] are independently synthesized from PSS and that efficient precursor processing can occur within the constitutive secretory pathway in the relative absence of dense core secretory vesicles.

Glucocorticoids inhibit somatostatin gene expression through accelerated degradation of somatostatin messenger ribonucleic acid in human thyroid medullary carcinoma (TT) cells.

We previously reported that dexamethasone (DEX) induces dose-dependent biphasic effects on steady state somatostatin (SS) messenger RNA (mRNA) levels in normal rat islet and islet SS-producing tumor cells (1027B2), characterized by stimulation at low doses and marked inhibition at high doses. The stimulatory effect is transcriptionally mediated, whereas the molecular mechanism underlying DEX-induced suppression of SS mRNA levels is unknown. In the present study, we investigated these mechanisms in human thyroid medullary carcinoma (TT) cells, which exhibit only inhibition of SS mRNA with DEX. Cultured TT cells synthesized and secreted large quantities of SS-like immunoreactivity (content, 90 ng/10(6) cells; release, 18 ng/10(6) cells/24h). DEX produced a dose-dependent reduction of both SS-like immunoreactivity secretion and SS mRNA levels, with a maximum inhibition of 60% at 10(-6) M at 48 h. In time-course studies, DEX inhibition of SS function occurred after a lag period of about 12 h, suggesting a posttranscriptional mechanism. To exclude a transcriptional effect of DEX on the SS gene, chloramphenicol acetyltransferase (CAT) activity was determined in TT cells acutely transfected with SS promoter (-750 base pairs) ligated to the receptor CAT gene. No inhibition of CAT activity occurred with DEX (10(-6) M) for 48 h. Furthermore, DEX did not influence the rate of SS gene transcription determined by nuclear run-on assay compared to approximately 2-fold stimulation by cAMP. Actinomycin D (inhibitor of mRNA synthesis) reduced the size of the SS mRNA transcript and rendered it resistant to DEX-induced degradation when coincubated with DEX, but not when it was added after a delay of 12 h, indicating that DEX destabilizes SS mRNA by an active process requiring ongoing gene transcription. Cycloheximide (inhibitor of protein synthesis) reduced SS mRNA levels to the same level as DEX, suggesting that the two agents promote SS mRNA degradation through a common pathway. We conclude that glucocorticoids inhibit steady state SS mRNA levels in TT cells. This effect is not mediated through direct transcriptional inhibition of the SS gene. It requires transcription of another gene(s) whose product(s) accelerates SS mRNA degradation.

Glucocorticoids inhibit somatostatin gene expression through accelerated degradation of somatostatin messenger ribonucleic acid in human thyroid medullary carcinoma (TT) cells

We previously reported that dexamethasone (DEX) induces dose-dependent biphasic effects on steady state somatostatin (SS) messenger RNA (mRNA) levels in normal rat islet and islet SS-producing tumor cells (1027B2), characterized by stimulation at low doses and marked inhibition at high doses. The stimulatory effect is transcriptionally mediated, whereas the molecular mechanism underlying DEX-induced suppression of SS mRNA levels is unknown. In the present study, we investigated these mechanisms in human thyroid medullary carcinoma (TT) cells, which exhibit only inhibition of SS mRNA with DEX. Cultured TT cells synthesized and secreted large quantities of SS-like immunoreactivity (content, 90 ng/10(6) cells; release, 18 ng/10(6) cells/24h). DEX produced a dose-dependent reduction of both SS-like immunoreactivity secretion and SS mRNA levels, with a maximum inhibition of 60% at 10(-6) M at 48 h. In time-course studies, DEX inhibition of SS function occurred after a lag period of about 12 h, suggesting a posttranscriptional mechanism. To exclude a transcriptional effect of DEX on the SS gene, chloramphenicol acetyltransferase (CAT) activity was determined in TT cells acutely transfected with SS promoter (-750 base pairs) ligated to the receptor CAT gene. No inhibition of CAT activity occurred with DEX (10(-6) M) for 48 h. Furthermore, DEX did not influence the rate of SS gene transcription determined by nuclear run-on assay compared to approximately 2-fold stimulation by cAMP. Actinomycin D (inhibitor of mRNA synthesis) reduced the size of the SS mRNA transcript and rendered it resistant to DEX-induced degradation when coincubated with DEX, but not when it was added after a delay of 12 h, indicating that DEX destabilizes SS mRNA by an active process requiring ongoing gene transcription. Cycloheximide (inhibitor of protein synthesis) reduced SS mRNA levels to the same level as DEX, suggesting that the two agents promote SS mRNA degradation through a common pathway. We conclude that glucocorticoids inhibit steady state SS mRNA levels in TT cells. This effect is not mediated through direct transcriptional inhibition of the SS gene. It requires transcription of another gene(s) whose product(s) accelerates SS mRNA degradation.

Transcriptional regulation of the human insulin gene is dependent on the homeodomain protein STF1/IPF1 acting through the CT boxes.

Insulin gene transcription is a unique feature of the pancreatic beta cells and is increased in response to glucose. The recent cloning of insulin promoter factor 1 (IPF1) and somatostatin transcription factor 1 (STF1) unexpectedly revealed that these are mouse and rat homologues of the same protein mediating transactivation through binding of CT box-like elements in rat insulin 1 and somatostatin promoter/enhancer regions, respectively. By using oligonucleotides representing each of the three CT boxes of the human insulin (HI) gene enhancer and nuclear extracts from the mouse islet tumor cell lines beta TC3 and alpha TC1, we have identified a beta-cell-specific binding activity as reported for IPF1, which has maximal affinity toward the CT2 box. However, in pluripotent, HI-transfected rat islet tumor cells, NHI-6F, this binding activity is present prior to induction of (human) insulin gene transcription. Its migration is identical to that of in vitro translated STF1 in electrophoretic mobility-shift assays; it is specifically recognized by anti-STF1 antibodies and has an apparent molecular mass of 46 kDa. Mutation of the CT2 box decreases transcriptional activity of a HI reporter plasmid by approximately 65% in beta TC3 cells and blocks the glucose response in isolated newborn rat islet cells. Furthermore, cotransfection with STF1 cDNA into the glucagon-producing alpha TC1 cells increases the activity of the HI enhancer 4- to 5-fold, suggesting that STF1/IPF1 can confer on alpha TC1 cells the ability to transcribe the HI gene. We conclude that STF1/IPF1 is a necessary but not sufficient key regulator of insulin gene activity, possibly also involved in glucose-regulated transcription.

Glucocorticoids regulate somatostatin peptide and steady state messenger ribonucleic acid levels in normal rat tissues and in a somatostatin-producing islet tumor cell line (1027B2).

Although a number of previous studies have suggested that glucocorticoids influence somatostatin (SS) function at the peptide level in some tissues and SS mRNA levels in thyroid tumor cells, there has been no systematic investigation of the effects of glucocorticoids on SS gene expression in normal tissues. In the present study, we have examined the effect of dexamethasone (DEX) on SS secretion and gene expression in rat tissues using as models DEX-treated rats in vivo, primary cultures of rat islet and cerebrocortical cells, and a SS-producing rat islet tumor cell line (1027B2). In vivo DEX administration (0.5 mg/kg) for 3 or 8 days augmented SS-mRNA 2- to 3-fold in peripheral tissues (stomach, pancreas, and jejunum), but reduced it by 50-60% in brain. The DEX effect was time dependent, being more pronounced after 8 days than after 3 days of treatment. In all tissues, SS mRNA levels returned to control values 2 weeks after cessation of DEX. Changes in tissue content of immunoreactive SS paralleled those in SS mRNA. In cultured rat islet cells, 18-h incubation with DEX induced dose-dependent biphasic effects on immunoreactive SS and SS mRNA; low doses (10(-10) M) were stimulatory, and high doses (10(-8)-10(-5) M) were inhibitory. Insulin secretion displayed dose-dependent stimulation by DEX, whereas glucagon release was inhibited. The effect of DEX on SS mRNA levels in primary cultures of brain cells was solely inhibitory. 1027B2 cells responded to DEX with augmented immunoreactive SS secretion and SS mRNA levels at low concentration (10(-10) M), followed by a dose-dependent inhibition of both parameters with increasing DEX concentrations. We conclude that glucocorticoids exert significant effects on SS peptide production and steady state mRNA levels in normal SS-producing tissues in vivo, in vitro, and in cultured 1027B2 cells. The glucocorticoid effect is time and dose dependent, tissue specific, and at least in part due to a direct action of the steroid hormone on SS-producing cells.

Growth hormone action in rat insulinoma cells expressing truncated growth hormone receptors.

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.

A clonal rat pancreatic δ cell line (Rin 4B) expresses a high number of galanin receptors negatively coupled to a pertussis‐toxin‐sensitive cAMP‐production pathway

Galanin, an ubiquitous neuropeptide, was recently shown to inhibit somatostatin release by the rat islet tumor cell line, Rin-m. By using the clonal pancreatic delta cell line Rin14B, originating from Rin-m cells, we were able to identify the presence of one type of specific galanin-binding site of high affinity (Kd = 1.6 nM; maximal binding capacity = 270 fmol/mg protein) and high specificity for the peptide. Binding of 125I-galanin to these receptors was time-dependent and highly sensitive to guanine nucleotides. Using the cross-linker disuccinimidyl tartrate, covalent linking of the galanin receptor to 125I-galanin in membranes from Rin14B cells, followed by SDS/PAGE analysis of membrane proteins, indicated that the galanin receptor is a protein of 54 kDa. 0.1-100 nM galanin also exerted a marked inhibitory effect on the cAMP-production system under basal conditions, as well as in the presence of the pancreatic peptide glucagon. At a maximal dose, galanin induces a 90-100% decrease of basal and glucagon-stimulated cAMP production levels, with a median inhibition concentration (IC50) of 3 nM galanin. The direct inhibitory effect of galanin on the adenylate cyclase activity in Rin14B cell membranes was also demonstrated (IC50 = 3 nM galanin). The inhibitory effect of galanin on the basal and glucagon-stimulated cAMP production in Rin14B cells was reversed by pertussis toxin. The toxin was also shown to specifically ADP-ribosylate a protein of 41 kDa in membranes from Rin14B cells. Taken together, these data show that the pancreatic delta cell line Rin14B expresses high affinity galanin receptors negatively coupled to a pertussis-toxin-sensitive cAMP-production system.

Galanin inhibits somatostatin release by the rat islet cell tumor in culture, Rin-m

Using a rat islet cell tumor in culture. Rin-m, we studied the effects of the neuropeptide, galanin, on somatostatin release. Galanin applied to the incubation medium inhibited pancreatic hormone release rapidly and dose dependently with an IC 50 at 4 nM and the maximal effect (40% inhibition) was elicited by 100 nM peptide. Pretreatment of Rin-m cells with pertussis toxin abolished the inhibitory effect of galanin on somatostatin release. The results suggest that galanin probably controls the function of the pancreatic delta cell through a pertussis toxin-sensitive pathway.

Novel Islet, Duct, and Acinar Cell Markers Defined by Monoclonal Autoantibodies from Prediabetic BB Rats

Novel islet cell, duct cell, and acinar cell markers have been identified by monoclonal autoantibodies (Maab) derived from prediabetic BB rats. Spleen cells from two rats that both developed diabetes after splenectomy were fused with mouse myeloma cells. A cellular immunoradiometric assay for differential reactivity toward the surface of two closely related, insulin- and non-insulin-producing rat islet tumor cell lines was used to select and clone several IgM-producing hybridomas. The supernatants were finally characterized by two-color immunofluorescence with islet hormone antisera on frozen sections of human, monkey, and rat pancreas. Maab EB52 stained PP cells, but also few A cells on rat pancreas. Maab CA812 identified a subpopulation of islet D cells on rat, human and monkey pancreas. Although the CA812-reactive antigen and somatostatin were coexpressed in most D cells in adult rat pancreas, only a few islet D cells were stained in the newborn pancreas. The CA812-reactive antigen was not detected in somatostatin-producing cells in the duct epithelium. Maab H37 and IF5 selectively stained acinar cells in rat, human, and monkey pancreas, whereas Maab DA39 identified the rat ductal epithelium including the scattered endocrine cells of the ducts. In summary, B lymphocytes producing autoantibodies to pancreatic endocrine, exocrine, and ductal markers are present in prediabetic BB rats and can be detected by use of transformed pluripotent islet cells as target. Such B lymphocytes can be immortalized to produce monoclonal antibodies to study their role in insulin-dependent diabetes mellitus pathogenesis and to clarify the development of the pancreas.

Multiple growth hormone-binding proteins are expressed on insulin-producing cells.

The insulin-producing rat islet tumor cell line, RIN-5AH, expresses somatogen binding sites and responds to GH by increased proliferation and insulin production. Affinity cross-linking shows that RIN-5AH cells contain two major GH-binding subunits of Mr 100-130K (110K), which appear to exist as disulfide-linked multimers of Mr 270-350K (300K). In addition, a minor Mr 180K GH-binding protein is identified which does not appear to be associated with other proteins by disulfide bridges. A plasma membrane-enriched fraction accounts for 86% of the RIN-cell GH-binding activity while cytosol and intracellular organelles are low in GH-binding activity. The plasma membrane-bound activity is soluble in Triton X-100 with intact hormone binding characteristics. The apparent KD in detergent solution is estimated to 18 ng/ml (8 x 10(-10) M). 125I-hGH-affinity cross-linking to intact and detergent-solubilized membranes as well as hGH-affinity purified protein reveals labeled proteins of Mr 180K and Mr 285-350K. In contrast to the cross-linked Mr 300K complexes of intact cells those of disintegrated cellular material are resistant to reduction with dithiothreitol, and it is speculated that this is due to intersubunit cross-linking of the disulfide-linked Mr 110K GH-binding subunits. The GH-binding proteins are purified approximately 100-fold by one cycle of hGH-affinity chromatography and five major proteins of Mr 180K, 94K, 86K, 64K, and 54K are identified by silver staining in the purified fraction. It is concluded that the RIN-5AH cells have multiple GH-binding proteins which may mediate signals for either proliferation and/or insulin production.

Effects of sulphonylureas and diazoxide on insulin secretion and nucleotide‐sensitive channels in an insulin‐secreting cell line

1. The effects of various sulphonylureas and diazoxide on insulin secretion and the activity of various channels have been studied using tissue culture and patch-clamp methods in an insulin-secreting cell line derived from a rat islet cell tumour. 2. Tolbutamide, glibenclamide and HB699 increased the rate of insulin release by 2-5 fold. The concentrations of tolbutamide and glibenclamide giving half-maximum effects on insulin secretion were approximately 40 microM and 0.2 microM, respectively. 3. Diazoxide (0.6-1.0 mM) per se, had either no effect or produced a small increase in insulin secretion, whereas when secretion was maximally stimulated by the combination of glucose (3 mM) and leucine (20 mM), it produced inhibition. Tolbutamide-induced release was also inhibited by diazoxide. 4. Tolbutamide, glibenclamide, HB699 and HB985 reduced the open-state probability of the ATP-K+ channel in a dose-dependent manner. Tolbutamide and glibenclamide were shown to be effective regardless of which side of the membrane they were applied. 5. In whole cell recording, in which the total ATP-sensitive K+ conductance of the cell could be measured, dose-inhibition curves for tolbutamide and glibenclamide were constructed, resulting in Ki values of 17 microM and 27 nM, respectively. The value of Ki for tolbutamide was unchanged when ATP (0.1 mM) was present in the electrode. 6. Diazoxide (0.6 mM) activated the ATP-K+ channels only when they had first been inhibited by intracellular ATP (0.1 mM) or bath applied tolbutamide (3-30 microM). The inhibition produced by glibenclamide could not be reversed by diazoxide. 7. Neither tolbutamide (1.0 mM) nor glibenclamide (10 microM) altered the open-state probability of the Ca2+-activated K+ channel or the Ca2+-activated non-selective cation channel which are present in this cell line. 8. It is concluded that the sulphonylureas and related hypoglycaemic drugs and diazoxide regulate insulin secretion by direct effects on the ATP-K+ channel or a protein closely associated with this channel.

Tissue-specific expression of transfected human insulin genes in pluripotent clonal rat insulinoma lines induced during passage in vivo.

The pluripotent rat islet tumor cell line MSL-G2 expresses primarily glucagon or cholecystokinin and not insulin in vitro but changes phenotype completely after prolonged in vivo cultivation to yield small-sized hypoglycemic tumors composed almost entirely of insulin-producing beta cells. When a genomic DNA fragment containing the coding and upstream regulatory regions of the human insulin gene was stably transfected into MSL-G2 cells no measurable amounts of insulin or insulin mRNA were detected in vitro. However, successive transplantation of two transfected clones resulted in hypoglycemic tumors that efficiently coexpressed human and rat insulin as determined by human C-peptide-specific immunoreagents. These results demonstrate that cis-acting tissue-specific insulin gene enhancer elements are conserved between rat and human insulin genes. We propose that the in vivo differentiation of MSL-G2 cells and transfected subclones into insulin-producing cells reflects processes of natural beta-cell ontogeny leading to insulin gene expression.

Presence of complement-dependent cytotoxic activity against clonally-derived rat islet tumour cells in sera from type 1 (insulin-dependent) diabetic patients and control subjects.

Heat-inactivated sera from newly diagnosed Type 1 (insulin-dependent) diabetic patients and control subjects were tested for the presence of antibodies to islet cell surface antigens by means of a sensitive immunofluorescent, microcytotoxicity assay using two clones of a rat islet cell tumour as antigens. Complement-dependent cytotoxicity was found in 74% of diabetic patient sera and 87% of control sera, and there were no significant differences in titres between diabetic patients and control subjects. A minority of the sera from both patients and controls were cytotoxic for only one of the two clones, suggesting the presence of multiple antigen-antibody systems. Preadsorptions of the sera with rat liver powder, sheep erythrocytes, and/or protein A-conjugated agarose beads were inconsistently effective in decreasing levels of lytic activity in control sera. It is concluded that more information is required concerning the antigens of rat islet cells and islet cell cytotoxic factors present in normal sera before such cells and assays can be reliably used for the detection of islet cell surface antibodies.

Specific binding of the C-peptide of proinsulin to cultured B-cells from a transplantable rat islet cell tumor.

Specific binding of the C-peptide of proinsulin was evaluated using a transplantable NEDH rat islet cell tumour predominantly composed of insulin-secreting B-cells. Cultured tumour B-cells exhibited greater than 90% viability assessed by trypan blue exclusion, and retained the ability to form tumours with accompanying hypoglycaemia and hyperinsulinaemia after reimplantation. During binding experiments with synthetic rat C-peptide I and iodinated tyrosylated rat C-peptide I, tumour B-cells exhibited 54 +/- 6% specific binding. Displacement of tracer increased with increasing concentrations of unlabelled rat C-peptide I (0.25-1,000 ng/ml), and the specificity of binding was substantiated by reduced displacement with human C-peptide. Scatchard analysis of specific C-peptide binding revealed a curvilinear plot with upward concavity. The demonstration of specific C-peptide binding to insulin-secreting B-cells provides evidence for a physiological role of proinsulin C-peptide.