Beta TC3 Research Articles

Members of the large Maf transcription family regulate insulin gene transcription in islet beta cells.

The C1/RIPE3b1 (-118/-107 bp) binding factor regulates pancreatic-beta-cell-specific and glucose-regulated transcription of the insulin gene. In the present study, the C1/RIPE3b1 activator from mouse beta TC-3 cell nuclear extracts was purified by DNA affinity chromatography and two-dimensional gel electrophoresis. C1/RIPE3b1 binding activity was found in the roughly 46-kDa fraction at pH 7.0 and pH 4.5, and each contained N- and C-terminal peptides to mouse MafA as determined by peptide mass mapping and tandem spectrometry. MafA was detected in the C1/RIPE3b1 binding complex by using MafA peptide-specific antisera. In addition, MafA was shown to bind within the enhancer region (-340/-91 bp) of the endogenous insulin gene in beta TC-3 cells in the chromatin immunoprecipitation assay. These results strongly suggested that MafA was the beta-cell-enriched component of the RIPE3b1 activator. However, reverse transcription-PCR analysis demonstrated that mouse islets express not only MafA but also other members of the large Maf family, specifically c-Maf and MafB. Furthermore, immunohistochemical studies revealed that at least MafA and MafB were present within the nuclei of islet beta cells and not within pancreas acinar cells. Because MafA, MafB, and c-Maf were each capable of specifically binding to and activating insulin C1 element-mediated expression, our results suggest that all of these factors play a role in islet beta-cell function.

Upstream stimulatory factor (USF) and neurogenic differentiation/beta-cell E box transactivator 2 (NeuroD/BETA2) contribute to islet-specific glucose-6-phosphatase catalytic-subunit-related protein (IGRP) gene expression.

Islet-specific glucose-6-phosphatase (G6Pase) catalytic-subunit-related protein (IGRP) is a homologue of the catalytic subunit of G6Pase, the enzyme that catalyses the final step of the gluconeogenic pathway. The analysis of IGRP-chloramphenicol acetyltransferase (CAT) fusion-gene expression through transient transfection of islet-derived beta TC-3 cells revealed that multiple promoter regions, located between -306 and -97, are required for maximal IGRP-CAT fusion-gene expression. These regions correlated with trans -acting factor-binding sites in the IGRP promoter that were identified in beta TC-3 cells in situ using the ligation-mediated PCR (LMPCR) footprinting technique. However, the LMPCR data also revealed additional trans -acting factor-binding sites located between -97 and +1 that overlap two E-box motifs, even though this region by itself conferred minimal fusion-gene expression. The data presented here show that these E-box motifs are important for IGRP promoter activity, but that their action is only manifest in the presence of distal promoter elements. Thus mutation of either E-box motif in the context of the -306 to +3 IGRP promoter region reduces fusion-gene expression. These two E-box motifs have distinct sequences and preferentially bind NeuroD/BETA2 (neurogenic differentiation/beta-cell E box transactivator 2) and upstream stimulatory factor (USF) in vitro, consistent with the binding of both factors to the IGRP promoter in situ, as determined using the chromatin-immunoprecipitation (ChIP) assay. Based on experiments using mutated IGRP promoter constructs, we propose a model to explain how the ubiquitously expressed USF could contribute to islet-specific IGRP gene expression.

Cloning and functional analysis of SEL1L promoter region, a pancreas-specific gene.

We examined the promoter activity of SEL1L, the human ortholog of the C. elegans gene sel-1, a negative regulator of LIN-12/NOTCH receptor proteins. To understand the relation in SEL1L transcription pattern observed in different epithelial cells, we determined the transcription start site and sequenced the 5' flanking region. Sequence analysis revealed the presence of consensus promoter elements--GC boxes and a CAAT box--but the absence of a TATA motif. Potential binding sites for transcription factors that are involved in tissue-specific gene expression were identified, including: activator protein-2 (AP-2), hepatocyte nuclear factor-3 (HNF3 beta), homeobox Nkx2-5 and GATA-1. Transcription activity of the TATA-less SEL1L promoter was analyzed by transient transfection using luciferase reporter gene constructs. A core basal promoter of 302 bp was sufficient for constitutive promoter activity in all the cell types studied. This genomic fragment contains a CAAT and several GC boxes. The activity of the SEL1L promoter was considerably higher in mouse pancreatic beta cells (beta TC3) than in several human pancreatic neoplastic cell lines; an even greater reduction of its activity was observed in cells of nonpancreatic origin. These results suggest that SEL1L promoter may be a useful tool in gene therapy applications for pancreatic pathologies.

Inhibition of Fas-mediated apoptosis in mouse insulinoma betaTC-3 cells via an anti-Fas ribozyme.

In this study we have designed and constructed an anti-Fas ribozyme and show that it can specifically cleave the Fas mRNA in vitro. Moreover, to test its efficacy ex vivo, we transfected the anti-Fas ribozyme into betaTC-3 insulinoma cells, using a RNA polymerase III promoter to drive its expression. Like pancreatic beta cells, betaTC-3 cells do not constitutively express Fas, but Fas expression can be induced with IL-1 and IFN-gamma. Transfected cells expressed an average of 5000 copies of anti-Fas ribozyme transcript per cell as assessed by reverse transcriptase-real-time PCR. After IL-1/IFN-gamma treatment, betaTC-3 cells transfected with the anti-Fas ribozyme expressed 80% less Fas compared with mock-transfected cells. In addition, the anti-Fas ribozyme also inhibited Fas expression in NIT-1 insulinoma cells and in primary cultures of dispersed pancreatic islet cells. Inhibition of de novo Fas expression in betaTC-3 cells expressing the anti-Fas ribozyme correlated with resistance to Fas-mediated apoptosis as determined by the number of cells exhibiting caspase 3 proteolytic activity. Hence, we have engineered a ribozyme capable of preventing Fas expression in the betaTC-3 pancreatic insulinoma cell line and conferring resistance to Fas-mediated apoptosis. We suggest that ribozymes may be potentially useful to engineer resistance to apoptosis in transplantable beta cells, a feature that may significantly improve the survival of islet cell grafts.

Assessment of ribozyme cleavage efficiency using reverse transcriptase real-time PCR.

Real-time PCR is a novel technology recently described to perform quantitative analysis of amplified products. Unlike classical quantitative PCR, this method is easy to standardize, does not required extensive manipulation, and is not reagent intensive, so that the risk of contamination is minimized. Therefore, we have chosen reverse transcriptase real-time PCR to quantitate CD95 (Fas) transcripts to test the cleavage efficiency of anti-Fas ribozymes in the mouse insulinoma cell line beta TC-3. Based on the melting-curve analysis of the amplified products, we determined the temperature at which to collect the fluorescent data used for quantification. After constructing a standard curve by plotting the log of the standards' copy number versus their fractional cycle number, the copy numbers of the unknown samples were automatically determined by interpolation of this curve. As we illustrate in this study, it is important, particularly while setting up the technique, to validate the melting-curve profile with standard gel electrophoresis analysis, achieved by matching melting temperature and size of the amplified product. The method is fast and reproducible: Excluding the isolation of RNA and synthesis of cDNA, the results can be obtained in less than 1 hr. The coefficient of variance is 15% in the range of 10(4)-10(6) gene copies. Accordingly, reverse transcriptase (RT) real-time PCR is a technique suitable for screening a large number of ribozymes.

Effects of alginate composition on the metabolic, secretory, and growth characteristics of entrapped βTC3 mouse insulinoma cells

The effects of alginate composition on cell growth as well as the metabolic and secretory profile of transformed beta-cells entrapped in alginate/poly-L-lysine/alginate (APA) solid beads were investigated following entrapment of beta TC3 mouse insulinoma cells in alginate composed of either high mannuronic acid or high guluronic acid residues. Entrapped cultures were maintained in spinner flasks for 40-60 days. The pattern of cell growth and the overall rates of glucose consumption and insulin secretion were investigated. Cultures of beta TC3 cells entrapped in alginate composed predominantly of mannuronic acid units (77%) displayed a linear increase in the rates of glucose consumption and insulin secretion concomitant with an increase in cell population in the periphery of the beads. Conversely, cultures of beta TC3 cells entrapped in alginate composed predominantly of high guluronic acid units (69%) displayed a decrease in the rates of glucose consumption and insulin secretion during the first three weeks of culture, followed by a rapid recovery that surpassed the initial rates by day 40. This biphasic pattern was concomitant to a decrease in viable cells during the first three weeks as ascertained by histology, followed by an increase in cell proliferation. Cell growth in high guluronic acid alginate took place at random locations throughout the solid bead and not in the periphery, as was the case in high mannuronic acid alginate preparations. Possible reasons for these differences and the significance of these findings in the context of a bioartificial pancreas composed of APA entrapped transformed cells are discussed.

In situ electrochemical oxygen generation with an immunoisolation device.

The viability and function of transplanted tissue encapsulated in immunobarrier devices is subject to oxygen transport limitation. In this study, we have designed and used an in situ electrochemical oxygen generator which decomposes water electrolyticaly to provide oxygen to the adjacent planer immunobarrier diffusion chamber. The rate of oxygen generation, which increases linearly with electrical current, was accurately controlled. A theoretical model of oxygen diffusion was also developed and was used to calculate the oxygen profiles in some of the experimental systems. In vitro culture experiments were carried out with beta TC3 cells encapsulated in titanium ring devices. The growth and viability of cells with or without in situ oxygen generation was studied. We found that under otherwise similar culturing conditions, the thickness of the cell layer and the viability of cells was the highest in devices cultured in stirred media with oxygen generation, even though the thickness had not reached the theoretically predicted value, and lowest in those unstirred and without oxygen generation.

Towards the development of a bioartificial pancreas: effects of poly-L-lysine on alginate beads with BTC3 cells.

A bioartificial tissue construct that consists of insulin-secreting cells entrapped in an alginate/poly-L-lysine (PLL) matrix offers a promising approach for the treatment of type I diabetes. Use of transformed cells has been proposed as a solution to the cell availability problem posed by islets. The growth characteristics of transformed cells in their sequestered environment and the effects of PLL on their metabolic and secretory activities have not yet been characterized. Our data demonstrate that mouse insulinoma beta TC3 cells proliferate while they are entrapped in both PLL-free and PLL-coated alginate beads. During this process, cell aggregates develop in the bead periphery, which increase in number and size with time. PLL is crucial for the long-term in vitro structural stability of beads, and it does not appear to affect the metabolic and secretory activities of entrapped beta TC3 cells. The implications of these findings in the development of a bioartificial pancreatic construct based on transformed cells are discussed.

GLUT1 is adequate for glucose uptake in GLUT2-deficient insulin-releasing beta-cells.

GLUT2 may play an important role in pancreatic beta-cell glucose metabolism. A decrease in glucose uptake due to underexpression of GLUT2 has been considered as the cause of beta-cell dysfunction in diabetes with different pathogenesis. However, this view has been challenged by recent studies, in which the underexpression of GLUT2 was not accompanied by a decrease in glucose uptake. Our present aim is to evaluate the presumed importance of GLUT2 in maintaining the efficiency of beta-cell glucose uptake. We studied the kinetic characteristics of 3-O-methylglucose uptake in two beta-cell lines. One of these is the beta TC3 cell line which expresses GLUT1 and the other is the beta HC9 cell line which expresses both GLUT1 and GLUT2. Under equilibrium exchange conditions, 3-O-methylglucose transport in these two cell lines showed similar values of K(m) and V(max). The apparent IC50 of cytochalasin B for inhibiting 3-O-methylglucose transport in beta HC9 cells was nine times as high as in beta TC3 cells, indicating that GLUT1 is the critically important glucose transporter in the beta TC3 cell line and GLUT2 in the beta HC9 cell line. In both cell lines, the rates of glucose uptake were at least three times as fast as that of glucose phosphorylation. Our results suggest that GLUT1 is able to compensate for GLUT2 loss as it occurs in beta TC3 and maintains a commensurately high capacity of glucose uptake to sustain glucose metabolism in pancreatic beta-cells.

IGF-II, IGF-binding proteins and IGF receptors in pancreatic β-cell lines

The IGFs are mitogenic agents which are closely linked to regulatory processes in carbohydrate metabolism. Because limited information is available on the occurrence of the IGF system in the pancreatic beta-cell milieu, we evaluated the presence of IGFs, IGF receptors, and IGF-binding proteins (IGFBPs) in the beta-cell lines beta TC3 and HIT T-15. Serum-free conditioned media (SFCM) from beta TC3 cells contained IGF-II at concentrations greater than 100 ng/ml. High (15 kDa) and low (7.5 kDa) molecular weight IGF-II were detected both by column chromatography followed by RIA and by immunoblotting. GH (10-1000 ng/ml) conditioning of beta TC3 cells stimulated IGF-II secretion in a dose-dependent manner. IGF-II mRNA was detected in beta TC3 cells using Northern blots, and also showed a GH-dependent relationship. IGF-II peptide was detected in SFCM from HIT cells, albeit at lower concentrations. To evaluate the presence of IGF receptors in beta-cell lines, affinity cross-linking studies were performed on beta TC3 cells, demonstrating type I IGF receptors which bound iodinated IGF-II with high affinity, iodinated IGF-I with lesser affinity, and had minimal appreciable binding to iodinated insulin. Type II IGF receptors were not detected. SFCM from beta TC3 and HIT cells was subjected to Western ligand blotting, which disclosed the presence of two major IGFBPs of 29 kDa and 24 kDa, characteristic of IGFBP-2 and IGFBP-4. The identity of the specific IGFBPs was confirmed by immunoprecipitation and Northern blotting. Varying the glucose concentration had no significant effect on the levels of IGFBPs, nor did preconditioning with GH, IGF-I, IGF-II, insulin, or glucagon. Levels of both IGFBPs in beta TC3 cell-conditioned media increased in the presence of dexamethasone at concentrations of 10(-6) M or greater. In summary, we present evidence that beta-cell lines comprise an environment for GH and IGF action. We speculate that IGFs, their receptors and binding proteins function as a complex interactive system which regulates beta-cell growth and function.

A calcium-dependent chloride current in insulin-secreting beta TC-3 cells.

Ca(2+)-dependent conductances have been hypothesized to play a role in the bursting pattern of electrical activity of insulin-secreting beta cells in response to high plasma glucose. A Maxi K+ channel has received the most attention, while a low-conductance Ca(2+)-activated K+ current has also been identified. We used an increasingly popular beta cell model system, the beta TC-3 cell line, and the perforated-patch technique to describe the properties of a novel Ca(2+)-dependent Cl- current [ICl(Ca)] in insulin-secreting pancreatic beta cells. The reported ICl(Ca) could be activated under physiological Ca2+ concentrations and is the first of its kind to be described in pancreatic insulin-secreting cells. We found that long depolarizing steps above -20 mV elicited an outward current which showed slow inward relaxation upon repolarization to negative membrane potentials. Both the outward currents and the inward tails showed dependence on Ca2+ influx: their current/voltage (I/V) relations followed that of the "L-like" Ca2+ current (ICa) present in these cells; they were blocked completely by the removal of external Ca2+ or application of Cd2+ at concentrations sufficient for complete block of ICa; and their magnitude increased with the depolarizing step duration. Moreover, the inward tail decayed monoexponentially with a time constant which at voltages negative to activation of ICa showed a weak linear voltage dependence, while at voltages positive to activation of ICa it followed the voltage dependence of ICa. This Ca(2+)-dependent current reversed at -21.5 mV and when the external Cl- concentration was reduced from 159 mM to 62 mM the reversal potential shifted by approximately +20 mV as predicted by the Nernst relation for a Cl(-)-selective current. Cl- channel blockers such as DIDS (100 microM) and niflumic acid (100 microM) blocked this current. We concluded that this current was a Ca(2+)-dependent Cl- current [ICl(Ca)]. From substitution of the external Cl- with various monovalent anions and from the reversal potentials we obtained the following permeability sequence for ICl(Ca): I- > NO3- > Br- > Cl- > Acetate.

Monitoring single-cell pharmacokinetics by capillary electrophoresis and laser-induced native fluorescence.

The quantification of insulin released from single cells of the insulin-secreting cell line beta TC3 permeabilized by digitonin is demonstrated. A simple method for monitoring the on-column release process by using capillary electrophoresis and laser-induced native fluorescence detection is described. Quantitative measurements of both the amount of insulin released and the amount remaining in the cell can be achieved simultaneously. This protocol provides an alternative approach to the study of cell secretion in the fields of neuroscience and endocrinology.

Regulation of glycosylphosphatidylinositol-specific phospholipase D secretion from beta TC3 cells.

Glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) is abundant in mammalian serum, but the source of the circulating enzyme is unknown. Pancreatic islets have been reported to contain and secrete GPI-PLD. In this report we examined the regulation of GPI-PLD secretion from beta TC3 cells, a mouse insulinoma cell line. In the absence of glucose, phorbol myristic acid (0.1 microM) stimulated insulin secretion by 2.5-fold and GPI-PLD secretion by 2-fold. Carbachol (5 microM), glucagon-like peptide I-(7-36) amide (0.1 microM), and isobutylmethylxanthine (0.1 mM) had no significant effect on insulin or GPI-PLD secretion in the absence of glucose. Glucose (16.7 mM) stimulated both GPI-PLD and insulin secretion from beta TC3 cells by 55% and 235%, respectively. In addition, glucose potentiated the secretagogue effect of isobutylmethylxanthine, phorbol myristic acid, and glucagon-like peptide I on both insulin and GPI-PLD secretion. By immunohistochemistry and confocal microscopy, beta TC3 cells contain both insulin and GPI-PLD, which generally colocalized intracellularly. However, GPI-PLD secretion differed from insulin secretion by a higher rate of basal release (2.8% vs. 0.23%/h), a lower magnitude of response to secretagogues, and a more prolonged period of increased secretion. These results demonstrate that beta TC3 cells secrete GPI-PLD in response to insulin secretagogues and suggest that GPI-PLD may be secreted via the regulated pathway in these cells.

Transcriptional activation of the GLUT2 gene by the IPF-1/STF-1/IDX-1 homeobox factor.

The homeodomain protein PDX-1, referred as IPF-1/STF-1/IDX-1, is a transcriptional factor that plays a critical role in the control of several genes expressed in the pancreatic islet. PDX-1 gene expression has been previously shown to be reduced in cultured beta-cell lines chronically exposed to high glucose concentrations. As the glucose transporter type 2 (GLUT2) gene expression is selectively decreased in the beta-pancreatic cells of experimental models of diabetes, we postulated that the loss of GLUT2 gene expression in the pancreatic islets of diabetic animals may be due to the loss of PDX-1 transacting function on the GLUT2 gene. We, therefore, investigated the potential role of PDX-1 in the transcriptional control of GLUT2. We have identified a repeat of a TAAT motif (5'-TAATA-ATAACA-3') conserved in the sequence of the human and murine GLUT2 promoters. Recombinant PDX-1 binds to this GLUT2TAAT motif in electrophoretic mobility shift experiments. PDX-1 antiserum detects the formation of the complex of PDX-1 with the GLUT2TAAT motif in nuclear extracts from the pancreatic insulin-secreting cell line, beta TC3. The GLUT2TAAT motif was mutated in the murine GLUT2 promoter (-1308/+49 bp) linked to a luciferase reporter gene and transfected into beta TC3 cells. Compared with the transcriptional activity of the wild type promoter, that of the mutated promoter decreases by 41%. Multiple copies of the GLUT2TAAT motif were ligated 5' to a heterologous promoter and transfected into a PDX-1-expressing cell line (beta TC3) and into cell lines lacking the homeobox factor (InR1-G9 and JEG-3). The GLUT2TAAT motif mediates the activation of the heterologous promoter in the PDX-1-expressing cell line but not in InR1-G9 or JEG-3 cell lines. Furthermore, cotransfection in a PDX-1-deficient cell line with the expression vector encoding PDX-1 transactivates specifically the heterologous promoter containing the multimerized GLUT2TAAT motif. These data demonstrate that the murine GLUT2 promoter is controlled by the PDX-1 homeobox factor through the identified GLUT2TAAT motif.

Distribution of glucagon receptors on hormone-specific endocrine cells of rat pancreatic islets.

Glucagon is insulinotropic, but it remains uncertain whether the insulinotropic action is mediated directly by glucagon receptors expressed on beta-cells or by cross-binding to the insulinotropic glucagon-like peptide-1 (GLP-1) receptor known to be expressed on beta-cells. Binding of [125I]glucagon to GLP-1 receptors and not to glucagon receptors has been reported in tumor-derived beta-cells (15). The objectives of the current study were to use receptor-binding techniques and a glucagon receptor-specific antiserum to determine whether glucagon receptors are present on beta-cells. Specific binding (7.2 +/- 0.8%) of [125I]GLP-1 to beta TC-3 cells was displaced equivalently with GLP-1 and exendin-(9-39) )Kd = 0.9 and 0.4 nM. respectively), whereas approximately 700-fold higher concentrations of glucagon were required for equal displacement (Kd = 400 nM). Binding of [125I]glucagon to beta TC-3 cells (approximately 1%) was displaced equivalently with 1 microM glucagon, GLP-1, or exendin-(9-39). These observations support earlier findings that beta TC-3 cells do not express functional glucagon receptors. However, specific binding of [125I]glucagon was observed on INS-1 cells (2.3 +/- 0.2%); this was displaced with glucagon (Kd = 1 nM), but not 1 microM GLP-1 or exendin-(9-39). To examine the distribution of glucagon receptors on native beta-cells, dispersed cultured rat islets were immunostained for glucagon, somatostatin, or insulin in combination with a polyclonal rabbit antiserum raised to an extracellular portion of the glucagon receptor (KD-14). The glucagon receptor antiserum colocalized staining with approximately 97% of immunoreactive insulin cells, 9% of immunoreactive glucagon cells, and 11% of immunoreactive somatostatin cells. Perfusion of the rat pancreas with concentrations of glucagon as low as 10(-12) M resulted in significant insulin release. These results suggest that whereas the tumor-derived beta-cell line beta TC-3 does not express functional glucagon receptors, INS-1 cells and isolated rat pancreatic beta-cells have specific glucagon receptors, as do a subpopulation of alpha- and delta-cells. A model is proposed for the role of glucagon in islet hormone secretion during feeding and fasting.

Glucagon acts through its own receptors in the presence of functional glucagon-like peptide-1 receptors on hamster insulinoma.

The observations that glucagon binds to glucagon-like peptide-1 (tGLP-1) receptors have raised the question of whether glucagon receptors mediate the insulinotropic effect of glucagon. We have investigated the presence and selective activation of glucagon and tGLP-1 receptors on tumor-derived cells. Northern blot analysis detected either glucagon or tGLP-1 receptor messenger RNA in hamster (HIT) and mouse (beta TC3) beta-cell lines, respectively, whereas both receptor messenger RNA were revealed in Syrian hamster insulinoma. Their expression in insulinoma plasma membranes was confirmed by specific covalent labeling with either [125I]glucagon or [125I]tGLP-1. Both glucagon and tGLP-1 receptors showed a single class of high affinity binding sites with respective Kd values of 1.11 +/- 0.11 and 0.82 +/- 0.11 nM. [125I]tGLP binding was dose dependently inhibited with a hierarchy of exendin-4 > tGLP-1 > exendin-(9-39) > oxyntomodulin > glucagon. [125I]Glucagon binding was only inhibited by glucagon and oxyntomodulin. Both glucagon and tGLP-1 increased cAMP formation in insulinoma plasma membranes in a dose-dependent manner, with ED50 values of 170.0 +/- 25.0 and 3.1 +/- 0.4 pM, respectively. Exendin-(9-39), a tGLP-1 receptor antagonist, inhibited tGLP-1-induced, but not glucagon-induced, cAMP formation. Our data demonstrate on hamster insulinoma the presence of high affinity glucagon and tGLP-1 receptors selectively coupled to adenylyl cyclase. The observed low affinity of tGLP-1 receptors for glucagon sustains the idea that each hormone has a direct insulinotropic effect.

Determination of insulin in single pancreatic cells by capillary electrophoresis and laser-induced native fluorescence.

Development of a method for the determination of insulin based on capillary electrophoresis with laser-induced native fluorescence detection is described. Under optimal conditions, insulin as low as 73 amol can be detected with a good signal-to-noise ratio (S/N = 10 peak-to-peak). Application of this method for the determination of insulin content in single cells from the insulin-secreting cell lines RINm5F and beta TC3 is demonstrated. Non-bonded poly(ethylene oxide)-coated and bare capillaries are evaluated for this purpose, with the latter found to be more suitable for single-cell analysis.

CAMP-dependent phosphorylation of the cardiac-type alpha 1 subunit of the voltage-dependent Ca2+ channel in a murine pancreatic beta-cell line.

Two voltage-dependent calcium channels (VDCCs) have been reported in pancreatic islets: the beta-cell/endocrine-brain and cardiac subtypes. The cardiac-type alpha 1 subunit was isolated from cultured beta TC3 cells, a murine pancreatic beta-cell line, by immunoprecipitation with a specific polyclonal antibody. We have examined the effects of 1-isobutyl-3-methylxanthine (IBMX) and forskolin, agonists that elevate cAMP in these cells, on the phosphorylation of this subunit in intact beta TC3 cells using a sensitive back-phosphorylation technique. This technique allows quantitative detection of protein phosphorylation that is specifically stimulated by cAMP. The stimulation of intact beta TC3 cells with forskolin or IBMX resulted in the phosphorylation of the cardiac-type alpha 1 subunit as evidenced by a 40-60% decrease in the ability of the 257-kDa form to serve as a substrate in the in vitro back-phosphorylation reaction with [gamma-32P]ATP and the catalytic subunit of cAMP-dependent protein kinase (PKA). The effects of forskolin were time- and concentration-dependent. The concentration-dependency of forskolin-induced phosphorylation of the cardiac-type alpha 1 subunit and the potentiation of glucose-induced insulin secretion were highly correlated, a finding that is consistent with a role for such phosphorylation in mediating at least some of the effects of cAMP on secretion.

Endogenous tumor necrosis factor-alpha production by a pancreatic beta-cell line: inhibitory effects of hydrocortisone and nicotinamide.

The purpose of this study was to examine regulation of interleukin-1(IL-1)-induced tumor necrosis factor-alpha (TNF-alpha) production by a mouse beta-cell line (beta TC1). Three-hour incubation of beta TC1 cells with 5 U/ml of IL-1 beta results in the expression of TNF-alpha mRNA and intracellular accumulation of TNF-alpha. It has been shown that glucocorticoids and immunosuppressive agents such as cyclosporin and FK-506 inhibit TNF-alpha generation by T-lymphocytes and monocytes. Hydrocortisone of 1 and 10 mumol/l suppressed TNF-alpha mRNA levels and the TNF-alpha content of beta TC1 cells exposed to IL-1 beta, whereas neither cyclosporin nor FK-506 altered the TNF-alpha content. Nicotinamide of 5-10 mmol/l also reduced TNF-alpha mRNA and TNF-alpha protein levels in beta TC1 cells. Addition of exogenous TNF-alpha did not inhibit IL-1-induced transcription of TNF-alpha gene. These observations support the potential therapeutic role of glucocorticoids and nicotinamide in protecting beta-cells against cytokine-mediated damage, although glucocorticoid agonists have hyperglycemic metabolic effects.

Molecular mechanism underlying glucagon-like peptide 1 induced calcium mobilization from internal stores in insulin-secreting beta TC3 cells.

Molecular mechanism underlying glucagon-like peptide 1 induced calcium mobilization from internal stores in insulin-secreting beta TC3 cells.