Insulinoma betaTC3 Cells Research Articles

Microfabrication of scaffold-free tissue strands for three-dimensional tissue engineering

In this note, we report a practical and efficient method based on a coaxial extrusion and microinjection technique for biofabrication of scaffold-free tissue strands. Tissue strands were obtained using tubular alginate conduits as mini-capsules with well-defined permeability and mechanical properties, where their removal by ionic decrosslinking allowed the formation of scaffold-free cell aggregates in the form of cylindrical strands with well-defined morphology and geometry. Rat dermal fibroblasts and mouse insulinoma beta TC3 cells were used to fabricate both single-cellular and heterocellular tissue strands with high cell viability, self-assembling capability and the ability to express cell-specific functional markers. By taking advantage of tissue self-assembly, we succeeded in guiding the fusion of tissue strands to fabricate larger tissue patches. The presented approach enables fabrication of cell aggregates with controlled dimensions allowing highly long strands, which can be used for various applications, including fabrication of scale-up complex tissues and of tissue models for drug screening and cancer studies.

Glucose Induces MafA Expression in Pancreatic Beta Cell Lines via the Hexosamine Biosynthetic Pathway

MafA is a basic leucine zipper transcription factor that regulates gene expression in both the neuroretina and pancreas. Within the pancreas, MafA is exclusively expressed in the beta cells and is involved in insulin gene transcription, insulin secretion, and beta cell survival. The expression of the mafA gene within beta cells is known to increase in response to high glucose levels by an unknown mechanism. In this study, we demonstrate that pyruvate, which is produced by glycolysis from glucose, is not sufficient to induce mafA gene expression compared with high glucose. This suggests that the signal for MafA induction is independent of ATP levels and that a metabolic event occurring upstream of pyruvate production leads to the induction of MafA. Furthermore, insulin secretion mediated by high glucose is not important for MafA expression. However, the addition of glucosamine to beta cell lines stimulates MafA expression in the absence of high glucose, and inhibition of the hexosamine biosynthetic pathway in the presence of high glucose abolishes MafA induction. Moreover, we demonstrate that the expression of UDP-N-acetylglucosaminyl transferase, the enzyme mediating O-linked glycosylation of cytosolic and nuclear proteins, is essential for glucose-dependent MafA expression. Consistent with this observation, inhibition of N-acetylglucosaminidase, the enzyme involved in the removal of the O-GlcNAc modification from proteins, with O-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-N-phenylcarbamate stimulates MafA expression under low glucose conditions. The presented data suggest that MafA expression mediated by high glucose requires flux through the hexosamine biosynthetic pathway and the O-linked glycosylation of an unknown protein(s) by UDP-N-acetylglucosaminyl transferase.

Insulin secretion dynamics of free and alginate-encapsulated insulinoma cells

This study investigates the effect of alginate/poly-L: -lysine/alginate (APA) encapsulation on the insulin secretion dynamics exhibited by an encapsulated cell system. Experiments were performed with the aid of a home-built perfusion apparatus providing a 1 min temporal resolution. Insulin profiles were measured from: (i) murine insulinoma betaTC3 cells encapsulated in calcium alginate/poly-L: -lysine/alginate (APA) beads generated with high guluronic (G) or high mannuoric (M) content alginate, and (ii) murine insulinoma betaTC-tet cells encapsulated in high M APA beads and propagated in the presence and absence of tetracycline. Results show that encapsulation in APA beads did not affect the insulin secretion profile shortly post-encapsulation. However, remodeling of the beads due to cell proliferation affected the insulin secretion profiles; and inhibiting remodeling by suppressing cell growth preserved the secretion profile. The implications of these findings regarding the in vivo function of encapsulated insulin secreting cells are discussed.

Use of glucose‐responsive material to regulate insulin release from constitutively secreting cells

Genetically-engineered cells offer a solution to the cell availability problem in tissue engineering a pancreatic substitute for the treatment of insulin-dependent diabetes. These cells can be non-beta cells, such as hepatocytes or myoblasts, retrieved as a biopsy from the same patient and genetically engineered to secrete recombinant insulin constitutively or under transcriptional regulation. However, the continuous or slowly responsive insulin secretion dynamics from these cells cannot provide physiologic glucose regulation in patients. Our objective consists of using such cells as an insulin source and of regulating insulin release by incorporating a glucose-responsive material, which acts as a control barrier for insulin in a cell-material hybrid device. Experiments were performed with insulinoma betaTC3 cells, HepG2 hepatomas, and C2C12 myoblasts, the latter two genetically-modified to constitutively secrete insulin. The control barrier consisted of concanavalin A (con A)-based glucose-responsive material, which forms a gel at low and a sol at high glucose concentrations. Results demonstrated that the device released insulin at a higher rate in response to glucose challenges. In contrast, a device containing an inert hydrogel instead of glucose-responsive material released insulin at an essentially constant rate, irrespective of the surrounding glucose concentration. Necessary material improvements include increased sensitivity to glucose, so that the material responds to physiologically relevant glucose concentrations, and increased stability. The prospects of developing a properly functional, implantable substitute based on engineered non-beta cells and glucose-responsive material, and the material and device improvements that need to be made prior to in vivo experiments, are discussed.

Glucose Stimulates the Tyrosine Phosphorylation of Crk-associated Substrate in Pancreatic β-Cells

Several years ago, we demonstrated that glucose induced tyrosine phosphorylation of a 125-kDa protein (p125) in pancreatic beta-cells (Konrad, R. J., Dean, R. M., Young, R. A., Bilings, P. C., and Wolf, B. A. (1996) J. Biol. Chem. 271, 24179-24186). Glucose induced p125 tyrosine phosphorylation in beta-TC3 insulinoma cells, beta-HC9 cells, and in freshly isolated rat islets, whereas increased tyrosine phosphorylation was not observed with other fuel secretagogues. Initial efforts to identify p125 were unsuccessful, so a new approach was taken. The protein was purified from betaTC6,F7 cells via an immunodepletion method. After electrophoresis and colloidal Coomassie Blue staining, the area of the gel corresponding to p125 was excised and subjected to tryptic digestion. Afterward, mass spectrometry was performed and the presence of Crk-associated substrate (Cas) was detected. Commercially available antibodies against Cas were obtained and tested directly in beta-cells, confirming glucose-induced tyrosine phosphorylation of Cas. Further experiments demonstrated that in beta-cells the glucose-induced increase in Cas tyrosine phosphorylation occurs immediately and is not accompanied by increased focal adhesion kinase tyrosine phosphorylation. Finally, it is also demonstrated via Western blotting that Cas is present in normal isolated rat islets. Together, these results show that the identity of the previously described p125 beta-cell protein is Cas and that Cas undergoes rapid glucose-induced tyrosine phosphorylation in beta-cells.

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.

Role of glucose in chronic desensitization of isolated rat islets and mouse insulinoma (betaTC-3) cells to glucose-dependent insulinotropic polypeptide.

It is well documented that the release of insulin from isolated perifused islets attenuates over time, despite a continued glucose stimulation. In the current study we have shown that potentiation of insulin release by the intestinal hormone glucose-dependent insulinotropic polypeptide (GIP) is also attenuated after its continuous application. In less than 20 h of maintained stimulus with either hyperglycaemia (11.0 mM glucose) or GIP (10 nM) under hyperglycaemic conditions, insulin release returned to basal values. This was not due to loss of islet viability or reduction in the releasable pool of insulin granules, as 1 mM isobutylmethylxanthine was able to stimulate equivalent insulin release under both conditions. Further examination of chronic GIP desensitization was examined in cultured mouse insulinoma (betaTC-3) cells. GIP-stimulated cAMP production was not greatly affected by the prevailing glucose conditions, suggesting that the glucose dependence of GIP-stimulated insulin release occurs distally to the increase in intracellular cAMP in betaTC-3 cells. The GIP-stimulated cAMP response curve after desensitization was of similar magnitude at all glucose concentrations, but GIP pretreatment did not affect forskolin-stimulated cAMP production. Desensitization of the cAMP response in betaTC-3 cells was shown not to involve induction of dipeptidyl peptidase IV or pertussis toxin-sensitive G-proteins, activation of protein kinase C or protein kinase A, or modulation of phosphodiesterase activity. Homologous desensitization of the insulin-potentiating activity of GIP was found to affect both GIP-stimulated and forskolin-stimulated insulin release, indicating desensitization of distal steps in the stimulus-exocytosis cascade.

Effects of short-term hypoxia on a transformed cell-based bioartificial pancreatic construct.

Hypoxia is an adverse condition that can jeopardize the function of a bioartificial pancreatic construct. In this study we have investigated the effects of short-term hypoxic exposure (up to 24 h) on the bioenergetic status, metabolism, and insulin secretion of perfused pancreatic constructs composed of alginate/poly-L-lysine/alginate (APA) encapsulated mouse insulinoma betaTC3 cells. The bioenergetic status of the encapsulated cells was monitored noninvasively with the aid of 31P NMR spectroscopy, while glucose, lactate, and insulin concentrations were measured with off-line assays from media samples removed from the perfusion loop. Our results demonstrate that in freshly prepared constructs insulin secretion was not affected by the hypoxic conditions, although intracellular ATP concentration decreased and glucose consumption increased. Alternatively, in constructs that were maintained in our perfusion system for at least 10 days, identical hypoxic conditions resulted in a decreased insulin secretion concomitant to a decreased intracellular ATP concentration and increased glucose consumption. These results suggest that the effects of hypoxia on a transformed cell-based pancreatic construct are not constant throughout the duration of an in vitro culture. The observed differences are attributed to the significant cell growth and rearrangement that occurs with time during an in vitro culture of the constructs.

The Calcium/Calmodulin-dependent Phosphodiesterase PDE1C Down-regulates Glucose-induced Insulin Secretion

To understand the role cAMP phosphodiesterases (PDEs) play in the regulation of insulin secretion, we analyzed cyclic nucleotide PDEs of a pancreatic beta-cell line and used family and isozyme-specific PDE inhibitors to identify the PDEs that counteract glucose-stimulated insulin secretion. We demonstrate the presence of soluble PDE1C, PDE4A and 4D, a cGMP-specific PDE, and of particulate PDE3, activities in betaTC3 insulinoma cells. Selective inhibition of PDE1C, but not of PDE4, augmented glucose-stimulated insulin secretion in a dose-dependent fashion thus demonstrating that PDE1C is the major PDE counteracting glucose-dependent insulin secretion from betaTC3 cells. In pancreatic islets, inhibition of both PDE1C and PDE3 augmented glucose-dependent insulin secretion. The PDE1C of betaTC3 cells is a novel isozyme possessing a K(m) of 0.47 microM for cAMP and 0.25 microM for cGMP. The PDE1C isozyme of betaTC3 cells is sensitive to 8-methoxymethyl isobutylmethylxanthine and zaprinast (IC(50) = 7.5 and 4.5 microM, respectively) and resistant to vinpocetine (IC(50) > 100 microM). Increased responsiveness of PDE1C activity to calcium/calmodulin is evident upon exposure of cells to glucose. Enhanced cAMP degradation by PDE1C, due to increases in its responsiveness to calcium/calmodulin and in intracellular calcium, constitutes a glucose-dependent feedback mechanism for the control of insulin secretion.

Development of a bioartificial pancreas: I. Long-term propagation and basal and induced secretion from entrapped ?TC3 cell cultures

Bioartificial pancreatic constructs based on immunoisolated, insulin-secreting cells have the potential for providing effective, long-term treatment of type I (insulin-dependent) diabetes. Use of insulinoma cells, which can be amplified in culture, relaxes the tissue availability limitation that exists with normal pancreatic islet transplantations. We have adopted mouse insulinoma betaTC3 cells entrapped in calcium alginate/poly-L-lysine/alginate (APA) beads as our model system for a bioartificial pancreas, and we have characterized the effects of long-term propagation and of glucose concentration step changes on the bioenergetic status and on the metabolic and secretory activities of the entrapped cells. Cell bioenergetics were evaluated nonivasively by phosphorus-31 nuclear magnetic resonance ((31)P NMR) spectroscopy, and metabolic and secretory parameters by assaying cell culture medium. Data indicate that net cell growth occurred between days 3 and 10 of the experiment, resulting in an approximate doubling of the overall metabolic and secretory rates and of the intracellular metabolite levels. Concurrently, a reorganization of cell distribution within the beads was observed. Following this growth period, the measured metabolic and secretory parameters remained constant with time. During glucose step changes in the perfusion medium from a high concentration of 12 to 15 mM to 0 mM for 4.5 h to the same high glucose concentration, the oxygen consumption rate was not affected, whereas insulin secretion was always glucose-responsive. Intracellular nucleotide triphosphates did not change during 0 mM glucose episodes performed early in culture history, but they declined by 20% during episodes performed later in the experiment. It is concluded that the system of APA-entrapped betaTC3 cells exhibits several of the desirable characteristics of a bioartificial pancreas device, and that a correlation between ATP and the rate of insulin secretion from betaTC3 cells exists for only a domain of culture conditions. These findings have significant implications in tissue engineering a long-term functional bioartificial endocrine pancreas, in developing noninvasive methods for assessing construct function postimplantation, and in the biochemical processes associated with insulin secretion.

Characterization of the promoter of the mouse prohormone convertase PC2 gene.

Prohormone convertase 2 (PC2) is a member of a family of mammalian subtilisin-like endoproteases that are involved in the processing of prohormones, neuropeptides and many other precursor derived proteins. The expression of PC2 is restricted to neuroendocrine tissues such as pancreatic islets, the pituitary and the brain. To understand the regulation of the PC2 gene, we cloned and characterized the promoter region of the mouse PC2 gene. The transcriptional start site of the mouse PC2 gene is identical to that of the human. There is 79% identity in the sequences of the promoter regions between the mouse and human PC2 genes. The mouse PC2 gene, like the human, does not have a TATA-like motif in the region just upstream of the start of the transcription. Studies with promoter-reporter gene, chloramphenicol acetyltransferase (CAT), constructs showed that the region from -400 to -170 bp was necessary for high level expression of the mouse PC2 gene in the betaTC-3 insulinoma cells.

Syntaxin, but not soluble NSF attachment protein (SNAP), biosynthesis by rat pancreatic islets is regulated by glucose in parallel with proinsulin biosynthesis.

Recent studies have revealed that soluble N-ethylmaleimide sensitive factor attachment receptor (SNARE)-related proteins, originally identified in neural tissues, are also expressed in pancreatic beta cells. In this study, we investigated the effect of glucose on syntaxin 1 and alpha/beta SNAP biosynthesis in pancreatic beta cells and we demonstrated that syntaxin 1, but not alpha/beta SNAP biosynthesis by rat isolated pancreatic islets was stimulated specifically by glucose nearly in parallel with proinsulin biosynthesis. Stimulation of syntaxin 1 and proinsulin biosynthesis by glucose was dose-dependent (Km = approximately 8 mmol/l) and reached the maximum (about 8-12 fold) at concentrations over 11 mmol/l. In contrast, 22 mmol/l glucose increased alpha/beta SNAP biosynthesis about 2-fold only, similar to the increase in total protein synthesis. Stimulation of syntaxin 1 biosynthesis by glucose was also time-dependent, taking around 3 h to reach the maximum, and was not affected by actinomycin-D, suggesting regulation at the translational level. On the other hand, glucose had a similar stimulating effect on both syntaxin 1 and alpha/beta SNAP biosynthesis by mouse insulinoma betaTC3 cells as it did on proinsulin biosynthesis. The evidence showing coordinated stimulation of syntaxin 1 and proinsulin biosynthesis by glucose in rat islets suggested the critical functional role of syntaxin 1 in the insulin exocytotic mechanism.

Calcium-stimulated Phosphorylation of MAP-2 in Pancreatic ॆTC3-cells Is Mediated by Ca2+/Calmodulin-dependent Kinase II

An understanding of the role of CaM kinase II in the pancreatic beta-cell is dependent on the identification of its cellular targets. One of the best substrates of CaM kinase II in vitro that could function in secretory events is the microtubule-associated protein, MAP-2. By immunoblot analysis, a high molecular weight protein with electrophoretic properties characteristic of MAP-2, was identified in rat insulinoma betaTC3 cells and isolated rat islets. In immunoprecipitation experiments employing alpha-toxin-permeabilized betaTC3 cells, elevation of intracellular Ca2+ or addition of forskolin, an adenylate cyclase activator, induced significant phosphorylation of MAP-2 in situ. The effect of Ca2+ was rapid, concentration-dependent and closely correlated with activation of CaM kinase II under similar experimental conditions. H-89, a specific and potent inhibitor of cAMP-dependent protein kinase (PKA), prevented forskolin-induced MAP-2 phosphorylation but had little effect on MAP-2 phosphorylation stimulated by elevated Ca2+. Phosphopeptide mapping revealed that the phosphorylation pattern observed in situ upon incubation of the betaTC3 cells with increased free Ca2+, was strikingly similar to that generated in vitro by CaM kinase II, most notably with regard to the increased phosphate incorporated into one prominent site. These data provide evidence that MAP-2 is phosphorylated by CaM kinase II in the pancreatic beta-cell in situ, and that this event may provide an important link in the mediation of Ca2+-dependent insulin secretion.

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.

Mouse insulinoma beta TC3 cells express prodynorphin messenger ribonucleic acid and derived peptides: a unique cellular model for the study of prodynorphin biosynthesis and processing

The tumor cell line beta TC3 has been established from insulinomas derived from transgenic mice carrying a hybrid insulin promoter-simian virus-40 tumor antigen gene. The beta TC3 cells express high steady state levels of proinsulin messenger RNA (mRNA). In this same cell line, we describe in the present study high expression levels of prodynorphin (pro-Dyn) mRNA and its derived peptides. By Northern blot analysis, the screening of 23 cell lines of endocrine (n = 10) and of nonendocrine (n = 13) origin revealed the presence of high levels of the 2.6-kilobase pro-Dyn transcript only in beta TC3 cells. The beta TC3 cells expressed levels of pro-Dyn mRNA comparable to those in rat tissues expressing pro-Dyn. Chromatographic and radioimmunological studies showed that pro-Dyn mRNA was translated and fully processed into opioid peptides with leucine-enkephalin (Leu-Enk)-extended sequences [dynorphin-A-(1-8), dynorphin-B-(1-13), and alpha-neo-endorphin]. The expression of the prohormone convertases was also examined in beta TC3 cells by Northern blot analysis. In addition to the ubiquitously expressed furin, beta TC3 cells have abundant levels of prohormone convertase-1 (PC1) and PC2 mRNAs, but undetectable levels of PACE4 or PC5 mRNAs. Incubation of beta TC3 cells with 8-bromo-cAMP for 24 h stimulated 3-fold both the pro-Dyn mRNA levels and the secretion of opioid peptides. In contrast to pro-Dyn mRNA, furin, PC1, and PC2 mRNA levels were not affected by 8-bromo-cAMP. The beta TC3 cells constitute a unique model to elucidate the biosynthetic pathway of pro-Dyn processing, to identify the proteolytic enzymes responsible for the production of pro-Dyn end products, and to assess the potential role of opioid peptides in the regulation of pancreatic function.

Mouse insulinoma beta TC3 cells express prodynorphin messenger ribonucleic acid and derived peptides: a unique cellular model for the study of prodynorphin biosynthesis and processing.

The tumor cell line beta TC3 has been established from insulinomas derived from transgenic mice carrying a hybrid insulin promoter-simian virus-40 tumor antigen gene. The beta TC3 cells express high steady state levels of proinsulin messenger RNA (mRNA). In this same cell line, we describe in the present study high expression levels of prodynorphin (pro-Dyn) mRNA and its derived peptides. By Northern blot analysis, the screening of 23 cell lines of endocrine (n = 10) and of nonendocrine (n = 13) origin revealed the presence of high levels of the 2.6-kilobase pro-Dyn transcript only in beta TC3 cells. The beta TC3 cells expressed levels of pro-Dyn mRNA comparable to those in rat tissues expressing pro-Dyn. Chromatographic and radioimmunological studies showed that pro-Dyn mRNA was translated and fully processed into opioid peptides with leucine-enkephalin (Leu-Enk)-extended sequences [dynorphin-A-(1-8), dynorphin-B-(1-13), and alpha-neo-endorphin]. The expression of the prohormone convertases was also examined in beta TC3 cells by Northern blot analysis. In addition to the ubiquitously expressed furin, beta TC3 cells have abundant levels of prohormone convertase-1 (PC1) and PC2 mRNAs, but undetectable levels of PACE4 or PC5 mRNAs. Incubation of beta TC3 cells with 8-bromo-cAMP for 24 h stimulated 3-fold both the pro-Dyn mRNA levels and the secretion of opioid peptides. In contrast to pro-Dyn mRNA, furin, PC1, and PC2 mRNA levels were not affected by 8-bromo-cAMP. The beta TC3 cells constitute a unique model to elucidate the biosynthetic pathway of pro-Dyn processing, to identify the proteolytic enzymes responsible for the production of pro-Dyn end products, and to assess the potential role of opioid peptides in the regulation of pancreatic function.

Tissue engineering of a bioartificial pancreas: modeling the cell environment and device function.

Cell-based implantable artificial tissues are most promising for the long-term treatment of endocrine diseases, such as diabetes. One type of a bioartificial pancreas device consists of calcium alginate microbeads containing insulin-secreting cells and is surrounded by a poly(L-lysine) (PLL) membrane. The membrane is semipermeable, allowing cellular nutrients and metabolites to diffuse through but excluding the antibodies and cytotoxic cells of the host, thus immunoprotecting the cells. The device can be modeled by writing the equations for diffusion of nutrients and metabolites through the polymer and for consumption of the former and production of the latter by the cells. In this paper, we describe the construction and analysis of such a model for alginate/PLL microbeads with insulin-secreting recombinant mouse pituitary AtT-20 and mouse insulinoma beta TC3 cells. Entrapped AtT-20 cells are a simplified model system, whereas microbeads with beta TC3 cells constitute a realistic artificial pancreatic device. Effective diffusivities of key compounds through the polymer with entrapped, inactivated AtT-20 spheroids were measured first. The kinetics of glucose and oxygen consumption and insulin secretion were modeled next, and the equations for diffusion and reaction were then combined to describe the entire system. The model was used to compute nutrient and metabolite concentration profiles in beads and the bead secretory response for different bead sizes and cell loadings. The size and loading necessary for the cells to be well nourished and for the beads to be rapidly responsive to step-ups and step-downs of secretion stimuli were evaluated. It was shown that if the cells are hypersensitive to glucose, i.e., they do not shut off secretion at the physiological glucose threshold but at a lower one, so are the microbeads. This work demonstrates the usefulness of mechanistic models with representative parameter values in optimizing the design of artificial tissues and in characterizing aspects of their behavior that are of importance for restoring in vivo function.

Towards the development of a bioartificial pancreas: immunoisolation and NMR monitoring of mouse insulinomas.

A promising method for diabetes treatment is the implantation of immunoisolated cells secreting insulin in response to glucose. Cell availability limits the application of this approach at a medically-relevant scale. We explore the use of transformed cells that can be grown to large homogeneous populations in developing artificial pancreatic tissues. We also investigate the use of NMR in evaluating, non-invasively, cellular bioenergetics in the tissue environment. The system employed in this study consisted of mouse insulinoma beta TC3 cells entrapped in calcium alginate/poly-L-lysine (PPL)/alginate beads. The PPL layer imposed a molecular weight cutoff of approximately 60 kDa, allowing nutrients and insulin to diffuse through but excluding high molecular weight antibodies and cytotoxic cells of the host. We fabricated a radiofrequency coil that can be double-tuned to 1H and 31P, and an NMR-compatible perfusion bioreactor and support circuit that can maintain cells viable during prolonged studies. The bioreactor operated differentially, was macroscopically homogeneous and allowed the acquisitions of 1H images and 31P NMR spectra in reasonable time intervals. Results indicated that entrapment had little effect on cell viability; that insulin secretion from beads was responsive to glucose; and that the bioenergetics of perfused, entrapped cells were not grossly different from those of cells never subjected to the immobilization procedure. These findings offer promise for developing an artificial pancreatic tissue for diabetes treatment based on continuous cell lines.

Biosynthesis of islet amyloid polypeptide. Elevated expression in mouse beta TC3 cells

Islet amyloid polypeptide (IAPP) messenger RNA levels, biosynthesis, processing, and secretion were studied in cultured mouse beta TC3 insulinoma cells. Northern blot analysis revealed that the size of IAPP mRNA (0.9 kb) in beta TC3 cells was the same as that in normal mouse islets; IAPP mRNA was approximately 60% of the level of insulin mRNA in beta TC3 cells. However, the ratio of synthesis of insulin to IAPP was approximately 6:1, suggesting that IAPP mRNA is not translated efficiently in these cells. Metabolic labeling of beta TC3 cells with [3H]leucine revealed the synthesis of both a precursor form of IAPP (pro-IAPP) of apparent Mr 7400 and a mature form (IAPP) of apparent Mr 3900. In pulse-chase experiments, pro-IAPP could be shown to be processed to IAPP in a manner similar to proinsulin. The t1/2 for conversion of pro-IAPP to IAPP was about 25 min, faster than the t1/2 for proinsulin to insulin of 70 min. A significant proportion of newly synthesized IAPP and insulin precursors were secreted via a constitutive pathway from beta TC3 cells. Possible effects of dexamethasone and forskolin on IAPP mRNA levels and biosynthesis were examined but no effects were observed. In conclusion, the IAPP gene is strongly expressed in beta TC3 cells leading to the biosynthesis, proteolytic processing, and secretion of IAPP, a putative islet hormone.