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Anaplerotic flux into the Kreb's cycle is crucial for glucose-stimulated insulin secretion from pancreatic beta-cells. However, the regulation of flux through various anaplerotic pathways in response to combinations of physiologically relevant substrates and its impact on glucose-stimulated insulin secretion is unclear. Because different pathways of anaplerosis generate distinct products, they may differentially modulate the insulin secretory response. To examine this question, we applied 13C-isotopomer analysis to quantify flux through three anaplerotic pathways: 1) pyruvate carboxylase of pyruvate derived from glycolytic sources; 2) pyruvate carboxylase of pyruvate derived from nonglycolytic sources; and 3) glutamate dehydrogenase (GDH). At substimulatory glucose, anaplerotic flux rate in the clonal INS-1 832/13 cells was approximately 40% of Kreb's cycle flux, with similar contributions from each pathway. Increasing glucose to 15 mm stimulated insulin secretion approximately 4-fold, and was associated with a approximately 4-fold increase in anaplerotic flux that could mostly be attributed to an increase in PC flux. In contrast, the addition of glutamine to the perfusion media stimulated GDH flux approximately 6-fold at both glucose concentrations without affecting insulin secretion rates. In conclusion, these data support the hypothesis that a signal generated by anaplerosis from increased pyruvate carboxylase flux is essential for glucose-stimulated insulin secretion in beta-cells and that anaplerosis through GDH does not play a major role in this process.

Islet transplantation is a promising method for restoring normoglycemia and alleviating the long term complications of diabetes. Widespread application of islet transplantation is hindered by the limited supply of human islets and requires a large increase in the availability of suitable insulin secreting tissue as well as robust quality assessment methodologies that can ensure safety and in vivo efficacy. We explore the application of nuclear magnetic resonance (NMR) spectroscopy in two areas relevant to beta cell engineering and islet transplantation: (1) the effect of genetic alterations on glucose metabolism, and (2) quality assessment of islet preparations prior to transplantation. Results obtained utilizing a variety of NMR techniques demonstrate the following: (1) Transfection of Rat1 cells with the c-myc oncogene (which may be involved in cell proliferation and cell cycle regulation) and overexpression of Bcl-2 (which may protect cells from stresses such as hypoxia and exposure to cytokines) introduce a wide array of alterations in cellular biochemistry, including changes in anaerobic and oxidative glucose metabolism, as assessed by 13C and 31P NMR spectroscopy. (2) Overnight incubation of islets and beta cells in the bottom of centrifuge tubes filled with medium at room temperature, as is sometimes done in islet transportation, exposes them to severe oxygen limitations that may cause cell damage. Such exposure, leading to reversible or irreversible damage, can be observed with NMR-detectable markers using conventional 13C and 31P NMR spectroscopy of extracts. In addition, markers of irreversible damage (as well as markers of hypoxia) can be detected and quantified without cell extraction using high-resolution magic angle spinning 1H NMR spectroscopy. Finally, acute ischemia in a bed of perfused beta cells leads to completely reversible changes that can be followed in real time with 31P NMR spectroscopy.

This investigation implements specifically designed solvent-suppressed adiabatic pulses whose properties make possible the long-term monitoring of (1)H NMR detectable metabolites from alginate/poly-l-lysine/alginate (APA)-encapsulated betaTC3 cells. Our encapsulated preparations were maintained in a perfusion bioreactor for periods exceeding 30 days. During this prolonged cultivation period, the cells were exposed to repetitive hypoxic episodes of 4 and 24 h. The ratio of the total choline signal (3.20 ppm) to the reference signal (observed at 0.94 ppm assigned to isoleucine, leucine, and valine) decreased by 8-10% for the 4-h and by 20-32% for the 24-h episodes and returned to its prehypoxic level upon reoxygenation. The decrease in the mean value of total choline to reference signal ratio for three 4-h and two 24-h episodes in two different cultures was highly significant (P<0.01). The rate of recovery by this ratio was slower than the rates of recovery by oxygen consumption, lactate production, or glucose consumption. A step-up in oxygen level led to a new, higher value for the total choline to reference ratio. From spectra of extracts at 400 MHz, it was determined that 63.6% of the total choline signal is due to intracellular phosphorylcholine. Therefore, it is inferred that the observed changes in total choline signal are linked to an oxygen level dependence of the intracellular phosphorylcholine. Several possible mechanisms in which oxygen may influence phosphorylcholine metabolism are suggested. In addition, the implications of these findings to the development of a noninvasive monitoring method for tissue-engineered constructs composed of encapsulated cells are discussed., (Copyright 2000 Academic Press.)

Confluent monolayer cultures of betaTC3 cells were exposed for 4 h to acidic, neutral, or alkaline pH media. Studies determined the impact of pH on viability, insulin secretion rate, glucose consumption rate, lactate production rate, and ATP content. Cell viability was not affected by exposure to media of different pH (>95% for all groups). Insulin release from cells exposed to acidic media (pH of 6.4) was approximately 75% higher than that from cells exposed to either neutral (pH of 7.1) or alkaline (pH of 7.8) conditions. Conversely, ATP content was significantly reduced in cultures exposed to acidic conditions, although there was no statistical difference between neutral and alkaline conditions. Glucose consumption and lactate production rates increased linearly with increasing pH., (Copyright 2000 Academic Press.)

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.

As a result of Myc-dependent transcription of the LDH-A gene, Myc-transformed cells (Rat1-Myc) exhibit increased lactate production rates (LPR) even under aerobic conditions (the Warburg effect). Recently, the increased susceptibility to stress-induced apoptosis associated with Myc transfection has been linked to the overexpression of the LDH-A gene. In this report we demonstrate that the overexpression of the anti-apoptotic protein Bcl-2 in Rat1-Myc cells (Rat1-Myc-Bcl-2) reduces the molar ratio of lactate production to glucose consumption (Y(L/G)). The Bcl-2 induced reduction in Y(L/G) may be associated with reduced expression of the LDH-A gene, or a decrease in LDH-A activity. Stimulation of apoptosis by staurosporine, a protein kinase C inhibitor, reduces the LPR in Rat1-Myc cells in a dose-dependent manner. The staurosporine effect on the LPR is rapid and precedes the execution phase of apoptosis as defined by caspase activation and PARP cleavage. This effect on LPR is completely blocked by Bcl-2 overexpression. Serum starvation alone does not affect the LPR of Rat1-Myc or Rat1-Myc-Bcl-2 cells; however, the effect of staurosporine on the LPR of Rat1-Myc cells is potentiated by serum starvation. These data demonstrate that Bcl-2 overexpression reduces the Y(L/G) in Rat1-Myc cells, perhaps via a reduction in the activity or expression of the LDH-A gene, and this reduction may desensitize cells to some pro-apoptotic stimuli. The reduction in LPR in response to staurosporine may be an early step in the induction of apoptosis in Rat1-Myc cells. By abolishing the reduction in LPR, Bcl-2 may protect Rat1-Myc cells from staurosporine-induced apoptosis. Moreover, the lack of effect by serum starvation on the LPR supports a model in which serum starvation induces apoptosis through a pathway distinct from that of the staurosporine and glucose-dependent apoptotic pathway(s) in Myc-transformed cells.

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., (Copyright 1999 John Wiley & Sons, Inc.)

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., (Copyright 1999 John Wiley & Sons, Inc.)

We investigated the effects of glucose on the rates of oxygen consumption (OCR) and insulin secretion (ISR) by betaHC9 cells derived from mouse pancreatic islets with beta-cell hyperplasia. Our results demonstrate that the OCR by betaHC9 cells incubated in nutrient-rich DMEM is unaffected by glucose (0-25 mM), is dissociated from the ISR (which increases with the addition of glucose), and is always higher than that measured in PBS. Glucose (25 mM) increases both the OCR and ISR when added to nutrient-free PBS. On the basis of results presented here, we suggest that, contrary to the current consensus, the observed increases in the OCR by beta-cells upon addition of glucose to nutrient-free buffers may be unrelated to the process of glucose-stimulated insulin secretion (GSIS) and, instead, related to nutrient starvation. We believe that a reevaluation of the implication of changes in OCR upon glucose stimulation in the process of GSIS is warranted and that OCR and ISR measurements should be performed in more physiological media to avoid nutrient starvation artifacts.

Understanding the biochemical events associated with glucose-stimulated insulin secretion by pancreatic beta cells is of importance in gaining insight into both the pathophysiology of diabetes and the development of tissue-engineered bioartificial pancreatic substitutes. We have investigated the effects of glucose concentration on the bioenergetic status and on the metabolic and secretory functions exhibited by mouse insulinoma betaTC3 cells entrapped in calcium alginate/poly-L-lysine/alginate (APA) beads. Cells entrapped in APA beads constitute a possible implantable bioartificial pancreas for the long-term treatment of insulin-dependent diabetes mellitus. Our results show that, in entrapped betaTC3 cells, the oxygen consumption rate and the intracellular nucleotide triphosphate levels are unaffected by a step change in glucose concentration from 16 mM to 0 mM for 4.5 h and then back to 16 mM. The intracellular Pi level and the ammonia production rate were doubled, while insulin secretion was decreased 10-fold, upon switching from 16 mM to 0 mM glucose. The implications of these findings in the context of pancreatic beta cell biochemistry and the mechanism of the 'Fuel Hypothesis' are discussed.

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.

We have investigated the rates of glucose consumption, lactate production and insulin secretion by mouse insulinoma beta TC3 cells exposed to high glucose and oxygen concentrations in the range of 132 mmHg (normoxia) to 0 mmHg (anoxia). The rates of glucose consumption and lactate production, and the yield of lactate on glucose were 6.4 +/- 0.2 nmol/h - 10(5) cells, 7.7 +/- 0.5 nmol/h - 10(5) cells, and 1.2 +/- 0.1 respectively, at oxygen concentrations between 132-25 mmHg. These values increased gradually as the oxygen concentration was reduced below 25 mmHg, reaching a maximum value of 12.8 +/- 0.4, 23.8 +/- 1.1, 1.9 +/- 0.1 respectively, at complete anoxia. Insulin secretion remained constant at 360 +/- 24 pmol/h - 10(8) cells at oxygen concentrations between 132-7 mmHg, but was inhibited at lower oxygen concentrations, dropping to 96 +/- 24 pmol/h - 10(8) cells at 0 mmHg. The rate of insulin secretion in the presence of high glucose under anoxia was significantly higher than the rate of basal secretion (28.2 +/- 3.0 pmol/h - 10(8) cells) at normoxia. The secretory properties of beta TC3 cells at low oxygen concentrations may have implications in the development of a diffusion-based bioartificial tissue constructs for the long-term treatment of Insulin Dependent Diabetes Mellitus.

Oxygen consumption rates of beta TC3 mouse insulinoma cells were measured in three different cell culture systems: monolayer, freshly trypsinized cell suspension, and trypsinized cells entrapped in alginate-poly-L-lysine-alginate (APA) polymer beads. The oxygen consumption rate for cells in the APA beads was similar to the rate for freshly trypsinized cells, indicating that cells can be entrapped without affecting their oxygen consumption rate. The cells in monolayers consumed oxygen at a higher rate than freshly trypsinized cells or cells in APA beads, suggesting that trypsinization may lower oxygen consumption capability. In addition, the oxygen consumption of beta TC3s during glucose-induced insulin secretion was not significantly different from the basal rate, in contrast to the increased oxygen demand of actively secreting islets. Our results suggest that beta TC3s may be better suited than islets for use in a bioartificial pancreas due to their stable oxygen requirements.

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.

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 same 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.