Development of a bioartificial pancreas: II. Effects of oxygen on long-term entrapped betaTC3 cell cultures

Tissue-engineered pancreatic constructs based on immunoisolated, insulin-secreting cells are promising in providing an effective, relatively inexpensive, long-term treatment for type I (insulin-dependent) diabetes. An in vitro characterization of construct function under conditions mimicking the in vivo environment is essential prior to any extensive animal experimentation. Encapsulated cells may experience hypoxic conditions postimplantation as a result of one or more of the following: the design of the construct; the environment at the implantation site; or the development of fibrosis around the construct. In this work, we studied the effects of 3- and 4-day-long hypoxic episodes on the metabolic and secretory activities and on the levels of intracellular metabolites detectable by phosphorus-31 nuclear magnetic resonance ((31)P NMR) of alginate/poly-L-lysine/alginate entrapped betaTC3 mouse insulinomas continuously perfused with culture medium. Results show that, upon decreasing the oxygen concentration in the surrounding medium, the encapsulated cell system reached a new, lower metabolic and secretory state. Hypoxia drove the cells to a more anaerobic glycolytic metabolism, increased the rates of glucose consumption (GCR) and lactate production (LPR), and reduced the rates of oxygen consumption (OCR) and insulin secretion (ISR). Furthermore, hypoxia reduced the levels of intracellular nucleotide triphosphates (NTP) and phosphorylcholine (PC) and caused a rapid transient increase in inorganic phosphate (P(i)). Upon restoration of the oxygen concentration in the perfusion medium, all parameters returned to their prehypoxic levels within 2 to 3 days following either gradual unidirectional changes (ISR, NTP, PC) or more complicated dynamic patterns (OCR, GCR, LPR). A further increase in oxygen concentration in the perfusion medium drove OCR, ISR, NTP, PC, and P(i) to new, higher levels. It is concluded that (31)P NMR spectroscopy can be used for the prolonged noninvasive monitoring of the bioenergetic changes of encapsulated betaTC3 cells occurring with changes in oxygen tension. The data also indicate that the oxygen-dependent states might be related to the total number of viable, metabolically active cells supported by the particular oxygen level to which the system is exposed. These findings have significant implications in developing and non-invasively monitoring a tissue-engineered bioartificial pancreas based on transformed beta cells, as well as in understanding the biochemical events pertaining to insulin secretion from betaTC3 insulinomas.     

Cultivation of recombinant,insulin-secreting AtT-20 cells as free and entrapped spheroids

Animal cells from endocrine glands have potential applications in bioprocessing, for the production of hormones, enzymes, possibly also recombinant proteins, and in tissue engineering, for the development of immunoisolated, implantable devices for long-term treatment of endocrine disorders. Immunoisolation can be achieved by surrounding cells with a biocompatible polymer which allows diffusion of nutrients and metabolites, including hormones, but excludes higher molecular weight antibodies and cytotoxic cells. Primary hormone-secreting cells cannot be effectively amplified in culture, so the large-scale application of implantable systems based on such cells is limited by cell availability. In this study, we conducted an initial assessment of the feasibility of using transformed, continuous cell lines in immunoisolated devices. The model system employed consisted of mouse pituitary tumor AtT-20 cells which secrete recombinant proinsulin and an insulin-like peptide and exhibit a high growth potential. Cells were cultivated as spheroids in spinner flasks and entrapped as such in alginate/polylysine/alginate beads. Free and entrapped spheroids were propagated in fed-batch, suspension cultures. Entrapment did not significantly affect spheroid metabolism or basal secretion. Entrapped spheroids did not increase in size or number and maintained roughly constant metabolic and basal secretory activities over a 15-day period. Free spheroids in suspension increased in size during the sami period, but also maintained constant metabolism and basal secretion, apparently because of a concomitant increase in hypoxic and/or necrotic cells. The potential of using continuous cell lines in the development of bioartificial endocrine organs is discussed.     

Magnetically labeled insulin-secreting cells

Iron oxide nanoparticles have been shown to magnetically label cells in order to visualize them in vivo via MR imaging. This technology has yet to be implemented in insulin secreting cells, thus it is not known whether the presence of these nanoparticles in the cytoplasm of the cells affects insulin secretion. This study investigates the effectiveness and consequence of labeling mouse insulinoma betaTC3 and betaTC-tet cells with monocrystalline iron oxide nanoparticles (MION). Our data show that MION can be internalized in both betaTC3 and betaTC-tet cells following a 24h exposure to 0.02mg/ml MION solution. The metabolic and secretory activities of both MION-labeled cell lines were statistically indistinguishable from sham treatment. Furthermore, cell viability and apoptosis remained constant throughout the cell's exposure to MION. Finally, MR images demonstrated significant contrast between labeled and sham-treated cells. Thus, labeling murine insulinoma cell lines with magnetic iron oxide nanoparticles does not hinder their insulin secretion, while it provides MR imaging contrast.     

The insulin secretion of a minced neonatal rat pancreas cultured in a pancreatic chamber, in response to various insulin secretagogues

The minced pancreas of the neonatal rat was cultured for 35 days in a pancreatic chamber which was constructed of a plastic tube and an ultrafiltration membrane. Insulin and amylase secreted from this pancreatic chamber into the culture medium were measured. During the experiment, the concentration of glucose in the culture medium was changed between 5.5 and 16.5 mM at 2-3 day intervals in order to determine the insulin secretory response of the pancreatic tissue. Insulin secretion was markedly increased in response to 16.5 mM glucose. The ratio of insulin secretion to amylase secretion in the culture medium increased with the advance of culture days although secretions of both insulin and amylase decreased individually. On the 7th culture day, short term incubations were performed to test with various insulin secretagogues; obvious insulin release into the incubation medium was observed. These results show that the pancreatic chamber also in vitro secretes insulin rapidly and significantly in response to various stimuli; that by longer culture of a neonatal rat pancreas in this device, insulin secretory cells without exocrine tissue would be obtained without using digestive enzymes; that application of a pancreatic chamber for a pancreatic transplantation may be feasible.     

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 βTC3 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 βTC-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.     

Hybrid pancreatic tissue substitute consisting of recombinant insulin-secreting cells and glucose-responsive material

Insulin-dependent diabetes is a serious pathological condition, currently treated by blood glucose monitoring and daily insulin injections, which, however, do not prevent long-term complications. A tissue-engineered pancreatic substitute has the potential to provide a more physiologic, less invasive, and potentially less costly treatment of the disease. A major issue in developing such a substitute is the cells being used. Nonpancreatic cells, retrieved from the same patient and genetically engineered to secrete insulin constitutively or with some glucose responsiveness, offer the significant advantages of being immune-acceptable and relaxing the tissue availability limitations, which exist with islets from cadaveric donors. These cells, however, do not have insulin secretion dynamics appropriate for restoration of euglycemia in higher animals and, eventually, humans. In this study, we present the concept of a hybrid pancreatic substitute consisting of such cells sequestered in a material exhibiting glucose-dependent changes of its permeability to insulin. A Concanavalin A-glycogen material sandwiched between two polycarbonate membranes and exhibiting glucose-dependent sol-gel transformations was used. Rates of insulin transport through this material in gel and sol forms were characterized for both FITC-labeled insulin in solution and insulin secreted by betaTC3 mouse insulinoma cells. Effective diffusivities through sol were found to be up to 3.5-fold higher than through the gel state of the material. A mathematical model of a hybrid construct was formulated and analyzed to simulate the secretory behavior in response to step ups and downs in the surrounding glucose concentration. The experimental and modeling studies indicate that a hybrid pancreatic substitute consisting of constitutively secreting cells and glucose-responsive material has the potential to provide a more physiologic regulation of insulin release than the cells by themselves or in an inert material.     

Insulin release from BK virus-induced and in vitro maintained insulinoma cells of Syrian golden hamster

The pancreatic tumor cells (In 111) derived from BK virus-induced insulinoma of Syrian golden hamsters were maintained in culture for several passages and were studied for their insulin secretory ability under various stimulatory conditions. Insulin release was not increased by D-glucose stimulation (27.8 mM), while dibutyryl cyclic AMP (1 mM), theophylline (1 mM), 3-isobutyl-l-methylxanthine (0.1 mM) and elevation of medium calcium from 0.5 to 2.7 mM stimulated insulin release 2.5- to 4-fold. There was a concomitant increase of medium cyclic AMP with addition of theophylline. Streptozotocin (2 mM) treatment for 48 hours significantly reduced insulin release, while alloxan (2 mM), had no inhibitory effect on insulin release. The results indicate that while in vitro-maintained islet tumor cells, In 111, have a cyclic AMP-mediated process involved in insulin secretion analogous to normal beta cells, these cells lack the ability to recognize glucose as an insulin secretagogue probably due to a defect in the cell membrane, though the possibility of alteration in glucose metabolism cannot be fully excluded.     

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 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 to¹H and³¹P, 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 acquisition of¹H images and³¹P 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.     

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.     

Inhibitory effect of rat amylin on the insulin responses to glucose and arginine in the perfused rat pancreas

Amylin, a 37-amino acid polypeptide, is the main component of amyloid deposits in the islets of Langerhans, and has been identified in the B-cell secretory granules. We have investigated the effect of rat amylin on the insulin and glucagon release by the isolated, perfused rat pancreas. Amylin infusion at 750 nM, markedly reduced unstimulated insulin release (ca. 50%, P less than 0.025), whereas it did not modify glucagon output. At the same concentration, amylin also blocked the insulin response to 9 mM glucose (ca. 80%, P less than 0.025) without affecting the suppressor effect of glucose on glucagon release. The inhibitory effect of amylin on glucose-induced insulin secretion was confirmed by lowering the amylin concentration (500 nM) and increasing the glucose stimulus (11 mM); again, no effect of amylin on glucagon release was observed. Finally, amylin, at 500 nM, reduced the insulin response to 3.5 mM arginine (ca. 40%, P less than 0.025) without modifying the secretion of glucagon elicited by this amino acid. It can be concluded that, in the rat pancreas, the inhibitory effect of homologous amylin on unstimulated insulin secretion, as well as on the insulin responses to metabolic substrates (glucose and arginine), favours the concept of this novel peptide as a potential diabetogenic agent.     

Functional examination of microencapsulated bioengineered insulin-secreting beta-cells.

Clonal insulin-secreting BRIN-BD11 cells engineered by electrofusion were encapsulated inside natrium alginate beads and cultured in RPMI 1640 culture media. Acute insulin secretory responses to glucose and amino acids were compared between microencapsulated cells and non-encapsulated cells maintained in monolayer culture. Encapsulated cells exhibited a 1.5-fold, 2.9-fold and 4.2-fold increase (P< 0.001) in insulin release in response to 16.7 mmol/l glucose, 10 mmol/l L-arginine and 10 mmol/l L-alanine respectively. Insulin output by non-encapsulated cells was approximately 30% greater but the relative magnitudes of responses were similar. This is the first study to demonstrate the stability of cellular engineered insulin-secreting cells encapsulated in alginate beads, illustrating the utility of this approach for cellular engineering and potential transplantation in diabetes.     

Maintenance of pancreatic endocrine cells of the neonatal rat: VII. The effect of 3-amino-3-deoxyglucose on insulin secretion from perifused B cells

Monolayer cultures of the pancreas of the neonatal rat were maintained in TCM 199 medium, supplemented with 5.5 mM glucose, with or without 5 mM 3-amino-3-deoxyglucose, and perifused to examine the changes which occurred in the insulin secretory response during culture. On day 0, B cells showed a monophasic insulin secretion in response to 16.7 mM glucose, whereas in the presence of 200 nM 12-o-tetradecanoyl phorbol-13-acetate, 40 microM lysophosphatidylcholine, 10 microM forskolin or 1 mM 3-isobutyl-1-methylxanthine, the same dose of glucose stimulated insulin secretion in a biphasic fashion. Under culture conditions without 3-amino-3-deoxyglucose, the response to glucose totally disappeared after 7 days, and that to 10 mM of either leucine or 2-ketoisocaproate was as low as that of day 0. In contrast, B cells that had been cultured for 7 days in medium with 3-amino-3-deoxyglucose showed an adult-like biphasic pattern in response to glucose. When stimulated by glucose at a linear gradient concentration running from 0 to 20 mM, the B cells responded to increasing concentrations of glucose in a dose-dependent fashion. Further, the response of cAMP to glucose was increased by adding forskolin or 3-isobutyl-1-methylxanthine, which also enhanced the secretion of insulin under either a step-wise or slow-rise stimulation with glucose. The effect of 12-o-tetradecanoyl phorbol-13-acetate was also outstanding. Likewise, the addition of either leucine or 2-keptoisocaproate induced a striking increase in the secondary phase secretion as well as promoting the rates of glutamine oxidation within the cells. In conclusion, it is suggested that the high response to a wider variety of stimuli may represent the reaction of neonatal B cells to the cultural milieu rather than a process of physiological development, and these effects exhibited by 3-amino-3-deoxyglucose would be related to a change in the constituents of glycoproteins in the cells.     

Biosynthesis of insulin secretory granule membrane proteins. Control by glucose.

The biosynthesis of a component SGM 110, specifically localized to the membrane of insulin secretory granules, was studied in rat insulinoma cells and in normal islets of Langerhans. Cells or islets were labelled with [35S]methionine or [3H]mannose and SGM 110 was immunoprecipitated by using a monoclonal antibody. Pulse-chase experiments demonstrated that the nascent polypeptide was cotranslationally glycosylated to form a 97,000 Da peptide which in turn was processed to the mature 110,000 Da form. A 50,000 Da form detected by immunoblotting with the same antibody was not conspicuously labelled even after a 20 h chase incubation, suggesting that it represented late processing of SGM 110 in lysosomes. With insulinoma cells, an increase in medium glucose concentration from 3 mM to 20 mM was without effect on the secretion of insulin or on the biosynthesis of (pro)insulin or SGM 110. In normal islets, however, 20 mM-glucose produced a 17-fold increase in (pro)insulin biosynthesis and a 13-fold increase in SGM 110 biosynthesis, compared with only a 2-fold increase in total protein synthesis, as judged by incorporation of [35S]methionine during a 1 h incubation. The effect of glucose on both (pro)insulin and SGM 110 biosynthesis was blocked by the addition of mannoheptulose, but not by the removal of extracellular calcium, both of which conditions inhibit insulin secretion. In contrast tolbutamide, an agent which stimulates insulin secretion, did not enhance the biosynthesis of (pro)insulin or SGM 110. It is concluded that at least one protein component of the insulin secretory granule membrane is synthesized co-ordinately with proinsulin and is subject to similar regulatory mechanisms. Factors which acutely control insulin secretion may also control granule biogenesis, although the two processes are not coupled in an obligatory fashion.     

Theoretical analysis of the effect of convective flow on solute transport and insulin release in a hollow fiber bioartificial pancreas

The bioartificial pancreas, in which transplanted pancreatic tissue or isolated cells are cultured on a hollow fiber membrane, is an attractive approach to restore physiologic insulin delivery in the treatment of diabetes. Insulin response in prototype devices has been unacceptable due to the large mass transport limitations associated with the membrane and the surrounding shell region. Although available theoretical analyses provide some insight into the combined effects of transport and reaction in the bioartificial pancreas, they cannot quantitatively account for the effects of convective recirculation flow, complex intrinsic insulin secretory kinetics, and non-uniform distribution of pancreatic cells. We have developed a detailed model for glucose and insulin transport and insulin secretion in the hollow fiber bioartificial pancreas based on the solution of the mass and momentum conservation equations describing flow and transport in the lumen, matrix, and shell. Model predictions are in good agreement with literature data obtained in a hollow fiber device with minimal radial convective flow. Although no quantitative data are available for a device with significant radial convection, model simulations demonstrate that convective recirculation flow can dramatically improve insulin response, allowing the device to accurately capture the bi-phasic insulin secretion characteristic of the normal physiologic response. Results provide fundamental insights into the coupling between kinetics and transport in the hollow fiber system and a rational basis for the design of clinical devices.     

In vitro study of using calcium phosphate cement as immunoisolative device to enclose insulinoma/agarose microspheres as bioartificial pancreas

In this study, the feasibility of using calcium phosphate cement (CPC) as immunoisolative device to enclose insulinoma/agarose microspheres as bioartificial pancreas was evaluated. We fabricated a chamber by CPC and utilized X-ray diffraction, Scanning electron microscope and Mercury intrusion porosimetry to identify the characters of the CPC chamber. The nominal molecular weight cut-off and cytotoxicity of CPC chamber were also evaluated. An insulinoma cell line (RIN-m5F) was chosen as insulin source and encapsulated in agarose microspheres and then enclosed in preformed CPC chamber. Insulin secretion was analyzed by Enzyme-linked immunosorbant assay to evaluate the function of insulinoma enclosed in CPC chamber. Results showed that the CPC chamber was non-cytotoxicity to insulinoma and can block the penetration of molecules which molecular weight larger than 12.4 kDa. Insulinoma inside the CPC chamber can secrete insulin in stable level for 30 days. This study indicated that we may use CPC as immunoisolative material to enclose insulinoma/agarose microspheres as bioartificial pancreas.     

Towards the development of artificial endocrine tissues: (31)P NMR spectroscopic studies of immunoisolated, insulin-secreting AtT-20 cells

Transformed, insulin-secreting endocrine cells have been proposed as an alternative to islets for the development of a bioartificiat pancreas. With appropriate immunoprotection, such cells may be implanted without the need for patient immunosuppression. Use of continuous cell lines alleviates the cell availability limitation, but poses questions regarding thestability and biochemical and secretory function of the preparation, especially in the longterm. We have developed a bioreactor/perfusion system, compatible with a horizontal-bore NMR instrument, that can maintain immunoprotected endocrine cells for prolonged periods of time. (31)P NMR spectroscopy was used to study the bioenergetics of recombinant, insulin-secreting mouse pituitary AtT-20 cells entrapped as spheroids in calcium alginate/poly-L-lysine/alginate beads. NMR provided data verifying the macroscopic homogeneity within the bioreactor and allowing the evaluation of changes in cellular bioenergetics for a period of 70 days under different culture conditions. Levels of high-energy phosphates changed slightly during the first 40 days of the experiment, then decreased considerably as cell death occurred. Rates of glucose consumption and insulin-related peptide secretion also remained constant for 40 days and decreased rapidly thereafter. This study constitutes the beginning of an extensive quantitative analysis of the biochemistry of transformed endocrine cell lines in a sequestered, artificial tissue environment. (c) 1995 John Wiley & Sons Inc.     

Entrapment of dispersed pancreatic islet cells in CultiSpher-S macroporous gelatin microcarriers: Preparation, in vitro characterization, and microencapsulation.

Immunoprotection of pancreatic islets for successful allo- or xenotransplantation without chronic immunosuppression is an attractive, but still elusive, approach for curing type 1 diabetes. It was recently shown that, even in the absence of fibrotic overgrowth, other factors, mainly insufficient nutrition to the core of the islets, represent a major barrier for long-term survival of intraperitoneal microencapsulated islet grafts. The use of dispersed cells might contribute to solve this problem due to the conceivably easier nutritional support to the cells. In the present study, purified bovine islets, prepared by collagenase digestion and density gradient purification, and dispersed bovine islet cells, obtained by trypsin and DNAsi (viability > 90%), were entrapped into either 2% (w/v) sodium alginate (commonly used for encapsulation purposes) or (dispersed islet cells only) macroporous gelatin microcarriers (CulthiSpher-S, commonly used for the production of biologicals by animal cells). Insulin release studies in response to glucose were performed within 1 week and after 1 month from preparation of the varying systems and showed no capability of dispersed bovine islet cells within sodium alginate microcapsules to sense glucose concentration changes. On the contrary, bovine islet cells entrapped in CulthiSpher-S microcarriers showed maintained capacity of increasing insulin secretion upon enhanced glucose concentration challenge. In this case, insulin release was approximately 60% of that from intact bovine islets within sodium alginate microcapsules. MTT and hematoxylineosin staining of islet cell-containing microcarriers showed the presence of viable and metabolically active cells throughout the study period. This encouraging functional data prompted us to test whether the microcarriers could be immunoisolated for potential use in transplantation. The microcarriers were embedded within 3% sodium alginate, which was then covered with a poly-L-lysine layer and a final outer alginate layer. Maintained insulin secretion function of this system was observed, which raises the possibility of using microencapsulated CulthiSpher-S microcarriers, containing dispersed pancreatic islet cells, in experimental transplantation studies.     

Metabolic concomitants in pure, pancreatic beta cells during glucose-stimulated insulin secretion

The role of the redox potential in insulin secretion by beta cells stimulated with high glucose was investigated using an in vitro pancreas perfusion system. To assess glycolytic flux the sum of fructose-1,6-P2 + triose-P was determined in pure beta cells microdissected from lyophilized sections of the isolated perfused pancreas quick frozen during the early insulin secretory response. L-Glycerol 3-phosphate and dihydroxyacetone phosphate were measured as indicators of the free cytosolic [NAD+]/[NADH] ratio and NADH and NADPH were also measured. Fructose-1,6-P2 + triose-P was increased in beta cells simultaneously with the onset of insulin secretion indicating an increase in glucose metabolism had occurred. The ratio of [dihydroxyacetone phosphate]/[L-glycerol 3-phosphate] increased simultaneously with the onset of insulin secretion. NADH content increased only after initiation of insulin secretion and NADPH levels remained unchanged during the early secretory response to high glucose. These data contradict the hypothesis that insulin secretion is triggered by a more reduced cytosolic redox state and instead indicate that insulin secretion is initiated by other metabolic coupling factor(s) generated in beta cells stimulated by high glucose.     

Cellular interaction between mouse pancreatic alpha-cell and beta-cell lines: possible contact-dependent inhibition of insulin secretion

The endocrine cells in the pancreatic islet have cellular communication between the heterotypic cells as well as the homotypic cells. The present study was conducted to elucidate the cellular interaction between pancreatic alpha cells and beta cells utilizing differentiated mouse cell lines (i.e., alphaTC clone 6 and betaTC cells). Co-culture of these two cell lines on a gyratory shaker generated numerous cellular aggregates of homogenous size within 48 h. Immunohistochemical staining for insulin and glucagon demonstrated that betaTC cells were located in the central core of each aggregate, while alphaTC cells formed a mantle layer surrounding the betaTC cells. This segregation was observed regardless of the ratios of the two cell types employed. Although glucagon at concentrations of 10(-8) M or higher stimulated insulin secretion from betaTC cells in both monolayer and aggregates, an increase in the ratio of alphaTC/betaTC cells in aggregate cultures was accompanied by a decrease in secreted insulin and a rise in intracellular insulin content of the betaTC component. The inhibitory effect of alphaTC cells on betaTC insulin secretion was not limited to aggregate culture, since insulin secretion from betaTC cells was also suppressed, and intracellular insulin content increased, by co-culture of alphaTC with betaTC cells in monolayer. On the other hand, the secreted and intracellular insulin of betaTC cells was not affected by alphaTC cells in a Transwell co-culture system in which direct cell-to-cell contacts were prevented by a semipermeable membrane that permitted chemical communication via medium metabolites. These data suggest that the insulin secretion from betaTC cells may be inhibited possibly as a result of the contact with alphaTC cells.