Rat Insulinoma Cell Line INS-1 Research Articles

Over-expression of CDK11p58 gene suppresses the proliferation of rat insulinoma cell line INS-1

Over-expression of CDK11p58 gene suppresses the proliferation of rat insulinoma cell line INS-1

Cytokines regulate miRNA expression in islet beta cells (129.41)

Abstract Type 1 diabetes (T1D) results from the autoimmune destruction of insulin-secreting beta cells. The immune system utilizes diverse mediators, including the cytokines, to kill pancreatic beta cells. Accumulating evidence suggests that apoptosis is themain form of beta cell death in spontaneousT1D and islet transplantation, and that the cytokines are key contributors in triggering beta cell apoptosis.MicroRNAs (miRNAs) are a recently discovered class of evolutionarily conserved small noncoding RNAs that lead to post-transcriptional repression, which is involved in the regulation of cell proliferation and cell death. To test the role of miRNAs in cytokine-mediated apoptosis, we compared the miRNA expression profile in rat insulinoma cell line INS-1 (834/40) with or without cytokine treatment, and between cytokine-resistant (INS-1res 833/15) and cytokine-sensitive (INS-1 834/40) cells, using miRNA array. We found that miRNAs were differentially regulated in cytokine-treated INS-1 834/40 compared to untreated cells, and between INS-1 834/40 and INS-1res 833/15 before or after cytokine treatments. Some of these changed miRNAs were recently reported to be linked to control cell growth and apoptosis. These results are the first evidence, to our knowledge, that cytokines can regulate miRNA expression, which suggests that these cytokine-regulated miRNAs could be involved in cytokine-mediated beta cell apoptosis. (JDRF, 5-2006-386)

The Concept of Functional Peptidomics for the Discovery of Bioactive Peptides in Cell Culture Models

Detection and purification of novel bioactive peptides from biological sources is a scientific task that led to a substantial number of important discoveries. One major laborious approach used is the repetitive stepwise separation of the test sample into several fractions followed by the determination of their bioactivity, until purity allows for sequence identification. We tested whether functional peptidomics, a combination of biological read-outs with differential peptide display (DPD) is a suitable strategy to isolate bioactive peptides at lower workload and with improved success. Additionally, we evaluated the use of DPD to monitor the processing status of proinsulin by inhibition of the insulin processing pathway. The rat insulinoma cell line INS-1 stimulated either with 2 mmol/l or 10 mmol/l glucose was used as model to generate differential peptide displays. In parallel, the bioactivity of the supernatants from the INS-1 cells was measured by glucose uptake and lipolysis assays using the adipocyte cell line 3T3-L1. We were able to quickly and elegantly trace the known activity of insulin to increase glucose uptake and inhibit lipolysis. Following re-chromatography of selected fractions, relevant peptides were identified by DPD and bioassays: the rat insulin-1 precursor and two different insulin peptides. We demonstrated in a semi-quantitative fashion that inhibition of proinsulin processing leads to accumulation of the insulin precursor, and reduced secretion of insulin-1. Thus, we conclude that DPD is an attractive support technology in peptide purification strategies aiming to identify bioactive compounds, and is superior to ELISA in discriminating between the processing status of insulin and its precursor.

Glucagon production of the rat insulinoma cell line INS-1—A quantitative comparison with primary rat pancreatic islets

The rat insulinoma cell line INS-1 is the most commonly used clonal cell model in pancreatic β-cell research. Considering the multihormonality of many insulinomas we examined as to how INS-1 cells comply with the notion of resembling a pure β-cell line. Glucagon immunoassays revealed that INS-1 cells secrete glucagon in a similar range as islets. By immunohistochemistry we detected a cytoplasmic glucagon signal in INS-1 cells which colocalized with C-peptide. Cellular content of preproglucagon-mRNA and glucagon protein in INS-1 cells was less than two percent of the respective values in islets, which probably reflects differences in the intracellular metabolism and/or secretory pathways. Taken together, it is obvious that INS-1 cells do not represent an exclusively insulin producing β-cell line.

Pattern of genes influenced by conditional expression of the transcription factors HNF6, HNF4 and HNF1 in a pancreatic -cell line

Using the rat insulinoma cell line INS-1 we generated beta-cell clones that are most efficient for gene transfer, as they contain an FRT site for Flp recombinase-mediated, site-directed integration of a single copy transgene. Therefore, the gene-of-interest can be introduced by DNA transfection without the need to select individual cell clones. Additionally, the clones contain the tetracycline repressor allowing tetracycline induction of the transgene. By oligonucleotide microarray we define the beta-cell specific phenotype of the Flp-In T-REx cell clones. Using a clone expressing the HNF6, HNF4alpha and HNF1beta transcription factors at a limited level, we introduced the expression vectors encoding these factors. We show efficient tetracycline induction of these transcription factors by western blots and immunocytochemistry. Microarrays reveal that these three factors affect a similar number of genes with only few genes regulated in common. Statistical analysis reveals that the three transcription factors affect genes categorized to different biological processes. Furthermore, we document the usefulness of these Flp-In T-REx cells for the functional analysis of mutated HNF1beta transcription factors found in human MODY5 patients. We show that the expression of the mutant P328L329del and A263insGG affects only very few transcripts and these are predominantly distinct from those induced by wild-type HNF1beta.

A constitutive nitric oxide synthase modulates insulin secretion in the INS-1 cell line

We provide immunocytochemical evidence that the neuronal isoform of constitutive NO synthase (cNOS) is expressed in the rat insulinoma cell line INS-1. Furthermore, using Nω-nitro- l-arginine methyl ester ( l-NAME), a pharmacological inhibitor of cNOS activity, we show that this enzyme is implicated in the modulation of insulin secretion in INS-1 cells. Indeed, in the presence of 2.8 mM glucose, l-NAME induced a specific and dose-dependent increase in insulin release, suggesting that cNOS exerts an inhibitory tone on basal insulin secretion. Moreover, l-arginine, the physiological substrate of cNOS, significantly reduced the marked enhancing effect of l-NAME on insulin release and to a lesser extent, at low concentrations, that of 10 mM KCl. l-NAME also potentiated the insulin secretion stimulated by 5.5 and 8.3 mM glucose, but in this case, its effect was not reduced by l-arginine. In conclusion, our data show that the neuronal isoform of cNOS exerts a negative modulation on insulin secretion in INS-1 cells, confirming the previous results obtained in the isolated perfused rat pancreas or pancreatic islets.

Establishment of a tet-on gene expression system in glucose-responsive and -unresponsive MIN6 cells.

The tetracycline (or doxycycline)-inducible gene expression (Tet-On) system developed by Gossen and Bujard (1) is useful for controlling the expression of targeted genes and for determining the roles of the gene products in cellular functions. The Tet-On system has recently been applied to the rat insulinoma cell line INS-1 to study the roles of wild-type and mutant genes in insulin secretion (2,3). We have recently established two cell lines, MIN6-m9, and MIN6-m14, derived from the mouse insulinoma-derived cell line MIN6. MIN6-m9 and MIN6-m14 cells are glucose-responsive and glucose-unresponsive, respectively. To study the roles of newly identified genes in pancreatic � -cell functions, we have established a Tet-On system in these MIN6 sublines.

Growth factor-mediated proliferation and differentiation of insulin-producing INS-1 and RINm5F cells: identification of betacellulin as a novel beta-cell mitogen.

It is not clear which growth factors are crucial for the survival, proliferation, and differentiation of pancreatic beta-cells. We used the relatively differentiated rat insulinoma cell line INS-1 to elucidate this issue. Responsiveness of the DNA synthesis of serum-starved cells was studied to a wide variety of growth factors. The most potent stimulators were PRL, GH, and betacellulin, a member of the epidermal growth factor (EGF) family that has not previously been shown to be mitogenic for beta-cells. In addition to these, only vascular endothelial growth factor, insulin-like growth factor-1 and -2, had significant mitogenic activity, whereas hepatocyte growth factor, nerve growth factor-beta, platelet-derived growth factors, basic fibroblast growth factor, EGF, transforming growth factor-alpha (TGF-alpha), neu differentiation factor, and TGF-beta were inactive. None of these factors affected the insulin content of INS-1 cells. In contrast, certain differentiation factors, including nicotinamide, sodium butyrate, activin A, and 1,25-dihydroxyvitamin D3 inhibited the DNA synthesis and increased the insulin content. Also all-trans-retinoic acid had an inhibitory effect on cell DNA synthesis but no effect on insulin content. From these findings betacellulin emerges as a novel growth factor for the beta-cell. Half-maximal stimulation of INS-1 DNA synthesis was obtained with 25 pM betacellulin. Interestingly, betacellulin had no effect on RINm5F cells, whereas both EGF and TGF-alpha were slightly mitogenic. These effects may possibly be explained by differential expression of the erbB receptor tyrosine kinases. In RINm5F cells a spectrum of erbB gene expression was detected (EGF receptor/erbB-1, erbB-2/neu, and erbB-3), whereas INS-1 cells showed only expression of EGF receptor. Expression of the erbB-4 gene was undetectable in these cell lines. In summary, our results suggest that the INS-1 cell line is a suitable model for the study of beta-cell growth and differentiation because the responses to previously identified beta-cell mitogens were essentially similar to those reported in primary cells. In addition, we have identified betacellulin as a possible modulator of beta-cell growth.

Role of the Prohormone Convertase PC3 in the Processing of Proglucagon to Glucagon-like Peptide 1

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.

Glucose-induced insulin secretion in INS-1 cells depends on factors present in fetal calf serum and rat islet-conditioned medium.

To study the regulation of growth and differentiated function of insulin-secreting cells, the rat insulinoma cell line INS-1 was cultured in a defined serum-free medium containing prolactin, IGF-I, and triiodothyronine, which was originally reported to maintain insulin secretion of islet cells. Growth and viability, as well as cellular insulin content of INS-1 cells in the defined medium, were comparable to the control cells cultured in the complete medium containing 10% fetal calf serum. However, after a 3-day culture in this medium, insulin secretion in response to glucose, pyruvate, and leucine was markedly blunted compared with the control cells (-78, -68, and -56%, respectively), whereas the response to 30 mmol/l K+ was only slightly decreased. In these cells: 1) nutrient metabolism assessed by tetrazolium salt reduction was reduced in response to pyruvate and leucine, which are mainly metabolized in the mitochondria; 2) oxidation of both [3,4-(14)C]glucose and [1-(14)C]pyruvate was decreased (-22 and -32%, respectively); 3) glucose failed to depolarize the membrane potential, whereas tolbutamide was fully active; 4) video imaging analysis of cytosolic Ca2+ showed a decrease in the population of glucose-responsive cells, while the response to 30 mmol/l K+ was preserved; 5) serum replenishment for 3 days restored glucose-induced insulin secretion. Interestingly, conditioned serum-free medium from rat islets maintained the insulin secretory function of INS-1 cells, although glucagon, somatostatin, and some other factors failed to restore the function. In contrast, conditioned media from HepG2, PC12, and human umbilical vein endothelial cells did not substitute for serum. Thus, the impaired insulin secretion of the cells cultured in the defined medium is best explained by defective mitochondrial metabolism. Islet cells, but not INS-1 cells, produce factors required for normal signal generation by nutrient secretagogues.