RAPID COMMUNICATION
Glucose-stimulated insulin secretion is not obligatorily linked to an increase in O2 consumption in beta HC9 cells

Klearchos K. Papas1 and Mary Ann C. Jarema2

1 Core Technologies/Analytics and Bio-Nuclear Magnetic Resonance, Novartis Institute for Biomedical Research, and 2 New Product Marketing, Novartis Pharmaceuticals Corporation, Summit, New Jersey 07901-1398

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

We investigated the effects of glucose on the rates of oxygen consumption (OCR) and insulin secretion (ISR) by beta HC9 cells derived from mouse pancreatic islets with beta -cell hyperplasia. Our results demonstrate that the OCR by beta HC9 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.

fuel hypothesis; nutrient starvation; tissue engineering; bioartificial pancreas

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

THE EFFECTS OF GLUCOSE and various other insulin secretagogues on the rate of oxygen consumption (OCR) by islets have been the subject of intense investigation over the past three decades (5, 7, 10-14, 18, 22, 30-31). This is in part due to the implication of changes in the OCR upon stimulation in the generation of signals (such as ATP related compounds) that may initiate the cascade of events that lead to insulin exocytosis. It is currently widely accepted that elevations in glucose concentration stimulate insulin secretion by increasing the rate of beta -cell metabolism (the "fuel hypothesis") and that glucose metabolism is essential for the generation of one or several intracellular messengers that ultimately lead to granule exocytosis (6, 7, 14, 18-23, 26-28, 35-36). According to the "classical" view of the fuel hypothesis for glucose-stimulated insulin secretion (GSIS), glucose metabolism is linked to granule exocytosis via the closure of the ATP-sensitive potassium channels (K+ATP channels) by metabolically generated coupling factors (Fig. 1). This sequence of events, also known as the "K+ATP channel/Ca2+-dependent pathway," is inherently attractive and has gained considerable attention because it provides a link between metabolic and electrophysiological events in the process of GSIS (7, 18-22). Despite the fact that the importance of the K+ATP channel/Ca2+ dependent pathway was somewhat undermined by the recent discovery of new K+ATP channel- and Ca2+-independent pathways for GSIS (8, 15, 35), the K+ATP channel- and Ca2+-dependent pathways remain the pathways of choice in linking metabolic to ionic events in the process of GSIS (6, 22, 24, 36, 42).


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Fig. 1.   Fuel hypothesis links metabolic to electrophysiological events in the process of glucose-stimulated insulin secretion (GSIS) via the ATP-sensitive K+ (K+ATP) channel/Ca2+-dependent pathway. Proposed sequence of events: extracellular elevation in glucose concentration (1) leads to an intracellular increase in the glucose consumption rate (GCR) (2), an increase in the oxygen consumption rate (OCR) (3), and therefore an increase in the rate of ATP synthesis, which may result in an increase in the intracellular levels of ATP or ATP/ADP (4). Increase in intracellular levels of ATP and/or the ATP/ADP ratio is thought to directly close the K+ATP channels (5). Closure of K+ATP channels results in depolarization of cell membrane and opening of voltage-gated Ca2+ channels. This allows influx of Ca2+ ions into the cell (6), thus establishing a net increase in intracellular Ca2+ concentration (7). Elevated intracellular Ca2+ concentration is thought to be the ultimate messenger for insulin secretion (8).

The large body of literature on islets from a variety of animal species, which without exception demonstrated increases in the OCR on glucose stimulation, solidified the implication of glucose metabolism in the process of GSIS (5-7, 11-14, 18, 22, 30-31). The reported changes in the OCR provided direct evidence for an accelerated rate of beta -cell metabolism upon glucose stimulation, which correlated with changes in insulin secretion and offered substantial support to the fuel hypothesis. Additional support was provided by the demonstration that the increases in the OCR and insulin secretion by islets upon glucose stimulation were blocked by metabolic inhibitors, such as glucosamine and mannoheptulose (10, 13).

Results reported in the literature on a variety of beta -cell lines also demonstrate, without exception, increases in the OCR upon addition of glucose to nutrient-free buffers (2, 16, 22, 39-42). The observed increases in the OCR by beta -cell lines were implicated in the mechanism of GSIS in a manner similar to that of normal islets (2, 16, 22). However, recent results obtained with the beta TC3 cell line demonstrated no change in the OCR upon addition of glucose to glucose-free DMEM (25). These results appeared to be contradictory to reports published at a later time point on the same cell line, which demonstrated an increase in the OCR upon addition of glucose to glucose-free media (16, 22).

An analysis of the experimental protocols used revealed that, without exception, the experiments performed with islets and beta -cell lines that demonstrated increases in the OCR upon glucose stimulation were conducted in nutrient-free buffers (2, 5, 7, 10-14, 16, 18, 22, 30-31, 39-42). We hypothesized that, in the absence of other nutrients, glucose, which is well metabolized by beta -cells, may increase the OCR, but it may not do so when the cells are not starved, i.e., are incubated in glucose-free, nutrient-rich media. The hypothesis that the use of nutrient-free vs. more physiologically relevant nutrient-rich media may influence the outcome of experiments addressing glucose signaling in the process of GSIS was explicitly and elegantly presented in a paper by Ghosh et al. (9) in the early nineties. The concern expressed by Ghosh et al. with this hypothesis did not, however, change the established common practice, i.e., the use of nutrient-free buffers. As a consequence, the vast majority of the studies performed over the past 7 years that investigated changes in metabolic coupling factors and their implications in the mechanism of GSIS were performed in nutrient-free buffers. Furthermore, this hypothesis was never extended to investigations related to the implications of changes in the OCR in the mechanism of GSIS. To further evaluate this hypothesis and to extend it to investigations related to changes in the OCR by beta -cells in the mechanism of GSIS, we performed OCR and insulin secretion rate (ISR) measurements on beta HC9 cells exposed to the following media: glucose-free PBS, PBS containing 25 mM glucose, glucose-free DMEM, and DMEM containing 25 mM glucose. The results obtained, along with their implications in understanding fundamental aspects of the mechanism of GSIS, are discussed.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Cells and cell culture. beta HC9 cells were chosen for our studies because they are closer to normal mouse islets in terms of their glucose metabolism and stimulated insulin secretion (16, 22, 29); they were obtained from Dr. Hanahan (University of California, San Francisco). The cells were cultivated at 37°C in a humidified incubator in an atmosphere of 95% air-5% CO2 and were passaged in T-175-cm2 flasks. Feeding was performed every other day with fresh DMEM, supplemented with 16 mM glucose, 10% (vol/vol) fetal bovine serum, and 1% (vol/vol) penicillin streptomycin (Sigma, no. P-0781). The DMEM used in our experiments (GIBCO BRL, no. 11966-025) was glucose- and pyruvate-free but contained a mixture of 15 amino acids including glutamine. beta HC9 cells were passaged every 10 days at a split ratio of 1:3. Confluent monolayer beta HC9 cells of passages 19-25 were used in all the experiments described here. In all cases in which OCR and ISR measurements were performed, serum-free media [DMEM (GIBCO BRL, no. 11966-025) and PBS (GIBCO BRL, no. 14040-133)] were used.

Entrapment procedure. Cell entrapment in alginate beads was performed on the basis of previously published protocols, with minor modifications (17). beta HC9 monolayers were trypsinized and then pelleted by mild centrifugation (100 g). The supernatant was removed, and the cells were resuspended in 2% wt/vol alginate solution (Keltone LV, product sample, lot no. 37061A, Kelco, San Diego, CA). The homogeneous alginate/cell suspension was then aseptically transferred to a syringe connected to a no. 21G sterile needle. Beads were extruded into a 50-ml centrifuge tube containing sterile CaCl2 (1.1% wt/vol solution). Either one or two T-175-cm2 flasks were used in each experiment. A confluent T-175 flask contained ~7 × 107 beta HC9 cells. beta HC9 cells were suspended in alginate at a density of 7 × 107 cells/3 ml of alginate solution.

OCR measurements. OCR measurements were performed on entrapped beta HC9 cells, because entrapment enabled the measurement of the OCR by the same cells in the different media and under different glucose concentrations. This was achieved because entrapment facilitated the separation of the cells from the incubation media during washes without cell loss. Entrapment also minimized the possibility of cell loss during the injection of glucose to glucose-free media. The apparatus used for the performance of the OCR measurements was designed for batch mode operation and was similar to the one described in detail elsewhere (32). The apparatus consisted of a glass bottle containing an autoclavable floating magnetic stirring bar, and a polarographic dissolved oxygen probe (DOP) with its amplifier (Ingold, Wilmington, MA). A custom-made Delrin cap accommodating the DOP and glucose injection ports was fabricated specifically to fit the glass bottle to ensure an airtight seal. Two ports were provided, both equipped with sterile syringes to accommodate the extra medium volume on injection of glucose in the apparatus and to ensure complete removal of air bubbles potentially trapped during the loading of the beads. The ports were equipped with a Teflon mesh to eliminate the chance of bead loss from the apparatus during glucose injection. Control experiments, performed under the experimental conditions used and with deoxygenated medium, confirmed that the apparatus was airtight (i.e., no oxygen penetrated the system from the surrounding environment) for periods as long as 5 h (data not shown). Any penetration of oxygen within the apparatus during the measurements could potentially have an effect on the rate of observed decline in the oxygen in the system and therefore on the calculated value of the OCR. Data were obtained in the range of 40-100% air saturation. The decline in the dissolved oxygen (DO) concentration in the sealed glass bottle containing the cells was linear with time in this concentration range. The OCR was calculated from the slope of the graph of the DO concentration vs. time (11-12). Additional details of the calculations have been presented elsewhere (25, 32).

Insulin secretion. Insulin concentration in media samples was assayed by ELISA by use of two monoclonal antibodies (Biodesign, Kennebunk, ME) in a sandwich format with the upper antibody being conjugated to biotin. Alkaline phosphatase conjugated to streptavidin was used to quantify binding.

Insulin secretion was measured from confluent beta HC9 monolayers by subtracting the insulin concentration in incubation media at the end of a prespecified time interval from that in the incubation media at the beginning of the time interval. Insulin secretion was averaged over 30-min time intervals. Specific insulin secretion rates were calculated on the basis of viable cell number measured from the monolayers used at the termination of the experiments, as described elsewhere (33). Monolayers, freshly trypsinized, and well-oxygenated entrapped transformed beta -cells have been shown to respond in a similar fashion with regard to insulin secretion and oxygen consumption upon stimulation by glucose (16, 22, 25, 32, 34, 38) and have been used interchangeably for the performance of such studies (16, 22).

Statistics. Results are expressed as means ± SD on the basis of the number of independent measurements (n). Statistical significance was determined with the Student's t-test.

Experimental design. The protocol followed for the measurement of OCR by beta HC9 cells is depicted in Fig. 2. The medium was allowed to equilibrate with the incubator gas phase (95% air-5% CO2) for a defined period. After the stabilization of the probe readings, the probe was calibrated to 100% air saturation and was monitored for stability (A-B). At point B, the entrapped cells were introduced into the chamber and the OCR was measured on the basis of the decline in oxygen concentration with time in the closed system (B-C). At point C, glucose was injected into the chamber through one of the ports with the aid of a sterile syringe. The final glucose concentration in the chamber was 25 mM. The OCR was measured again on the basis of the decline in oxygen concentration with time between points C and D. A change in the slope (an increase) would reflect an increase in the OCR on addition of glucose. At point D, the system was opened, the beads were removed from the chamber, and the chamber was washed and filled with nutrient-free PBS. The apparatus was placed back into the incubator for temperature and gas equilibration. The beads were washed five times with 40-ml volumes of nutrient-free PBS and were then introduced in the system (point E). The decline in oxygen concentration was monitored again between points E and F. Glucose was then added at point F (final concentration of 25 mM), and the decline in oxygen concentration was monitored between points F and G. The experiment was terminated at point G.


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Fig. 2.   Experimental design for OCR measurements. A-B: medium equilibration and probe calibration in glucose-free DMEM; B: addition of cells in chamber; B-C: decline in dissolved oxygen (DO) concentration with time in glucose-free DMEM; C: injection of glucose into chamber (final concentration, 25 mM); C-D: decline in DO with time in DMEM containing 25 mM glucose; D: bead removal and washes with nutrient-free PBS; D-E: temperature and gas equilibration of glucose-free PBS in apparatus; E: introduction of cells into system after repeated washes with glucose-free PBS; E-F: decline in oxygen concentration with time in glucose-free PBS; F: injection of glucose into chamber (final concentration, 25 mM); F-G: decline in oxygen concentration with time in PBS containing 25 mM glucose; G: termination of experiment.

It is important to note that the same cells were used for the measurement of OCR in DMEM-based media and nutrient-free PBS. This excluded the possibility that observed differences in the OCR in the different media were due to differences in the cells used. The possibility that the oxygen consumption in PBS was lower because of cell loss (because of the sequence followed in the experimental design) was eliminated by the fact that the OCR obtained in DMEM was higher than that measured in PBS in experiments performed in which the sequence was reversed, i.e., the cells were exposed first to PBS and then to DMEM (data not shown).

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Oxygen consumption. Figure 3 depicts typical results obtained with a culture of entrapped beta HC9 cells following the experimental protocol just described. Addition of 25 mM glucose to glucose-free DMEM had no significant effect on the OCR by beta HC9 cells (Figs. 3A and 4A), whereas addition of 25 mM glucose to glucose-free PBS increased the OCR by 60% (Figs. 3A and 4A). This increase in OCR was found to be statistically significant at the 99% confidence level. The 60% increase in OCR upon addition of glucose to glucose-free PBS reported here is in good agreement with the 68% increase in OCR reported by Liang et al. (16) for beta HC9 cells when 30 mM glucose was added to glucose-free modified Hanks' solution, and with the 58% increase observed when glucose (16.7 mM) was added to mouse islets incubated in glucose-free Krebs-Ringer phosphate buffer (12). As was the case of PBS, modified Hanks' and Krebs-Ringer phosphate buffers were nutrient free (12, 16).


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Fig. 3.   OCR by beta HC9 in the absence, or in the presence, of 25 mM glucose, in DMEM (A) and PBS (B). Arrows C and F, time points at which glucose was injected into apparatus. Circles, points used for linear curve fits (shown as solid lines in B; not shown in A, for clarity). Data presented in A and B were obtained from the same alginate-entrapped beta HC9 cells as depicted in experimental protocol detailed in Fig. 2.


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Fig. 4.   Oxygen consumption and insulin secretion by beta HC9 cells. A: oxygen consumption rates by beta HC9 cells in DMEM and PBS in the absence or in the presence of 25 mM glucose. Values are means ± SD of measurements performed with 3 independent cultures after experimental protocol depicted in Fig. 2. B: insulin secretion rates by beta HC9 cells in DMEM and PBS in the absence or in the presence of 25 mM glucose. Values are means ± SD of measurements performed with 4 independent cultures. # and * Significant differences (at 99% confidence level) between 0 and 25 mM glucose in PBS and DMEM, respectively. All rates are expressed as percentages of those measured in zero glucose DMEM. Oxygen consumption and insulin secretion by beta HC9 cells incubated in zero glucose DMEM medium were 1.1 ± 0.1 µmol · min-1 · 109 cells-1 and 84.7 ± 13.4 µU · h-1 · 105 cells-1, respectively.

As shown in Fig 4A, the OCR in DMEM-based media was always higher than that measured from the same cells in PBS. This was true even in the case in which the comparison was made between glucose-free DMEM and PBS containing 25 mM glucose. The OCR by beta HC9 cells incubated in glucose-free DMEM (1.1 ± 0.1 µmol · min-1 · 109 cells-1) was 97% higher than the OCR by beta HC9 cells exposed to 25 mM glucose PBS (statistically significant at the 99% confidence level).

Insulin secretion. The ISR increased significantly whether glucose was added to glucose-free DMEM or glucose-free PBS (Fig. 4B). Addition of 25 mM glucose to glucose-free DMEM increased insulin secretion by 88.4% (from 84.7 ± 13.4 to 159.6 ± 11.5 µU · h-1 · 105 cells-1), whereas addition of 25 mM glucose to glucose-free PBS increased insulin secretion by 605.8% (from 12.0 ± 4.3 to 84.7 ± 19.5 µU · h-1 · 105 cells-1). The ISR in glucose-free DMEM was not significantly different from that measured in PBS containing 25 mM glucose. The data shown in Fig. 4 demonstrate that a correlation exists between OCR and ISR when glucose is added to nutrient-free PBS. However, in more physiological nutrient-rich DMEM-based media, no such correlation exists between OCR and ISR.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Effects of glucose on the OCR and ISR by beta -cells. The publication of a report demonstrating substantial changes in the OCR by both beta TC3 and beta HC9 cells upon addition of glucose to glucose-free buffers (16) ruled out the possibility that the reported lack of changes in the OCR by beta TC3 cells upon glucose stimulation (25, 32, 34) was due to differences in glucose metabolism and energy requirements between beta TC3 cells and islets. An alternative hypothesis (34) offered an explanation for the lack of changes in the OCR by beta TC3 cells upon glucose stimulation that was based on the difference in the experimental protocols used (i.e., the use of nutrient-free buffers vs. nutrient-rich DMEM-based media). The data with beta HC9 cells reported in this study support this alternative hypothesis by demonstrating that the published differences on the effects of glucose on the OCR by beta TC3 and beta HC9 cells can be fully explained by the differences in the incubation media used.

The lack of effect of glucose on the OCR by beta TC3 and beta HC9 cells in DMEM may be due to a compensation of the contribution of glucose to the OCR by a reduction in the rate of oxidation of other nutrients, such as amino acids, which are present in DMEM (34). The above hypothesis is supported by the lower rate of ammonia production measured by beta TC3 cells in DMEM containing 16 mM glucose compared with that measured in glucose-free DMEM (32, 34) and by similar effects of glucose on the rate of ammonia production by normal islets, which were attributed to changes in amino acid utilization (19).

OCR, rate of ATP synthesis, and ISR by beta -cells incubated in PBS and DMEM. The reported stimulation in the OCR by beta HC9 and beta TC3 cells upon addition of glucose to nutrient-free buffers suggested an increase in the rate of ATP synthesis under stimulated conditions of secretion (16, 22). The calculated glucose-induced increase in the rate of ATP synthesis correlated very well with glucose-induced insulin secretion under the experimental conditions used (16, 22). The plot of the rate of ATP production vs. insulin secretion generated an energy-secretion diagram on which data from both beta TC3 and beta HC9 cells were projected on a single sigmoidal curve (16, 22). On the basis of these data, it was concluded that the rate of ATP synthesis may be an important parameter that controls insulin release and other glucose-dependent functions of beta -cells (16, 22).

Our observations that 1) switching the incubation media from PBS containing 25 mM glucose to glucose-free DMEM has no effect on insulin secretion but almost doubles the OCR by beta HC9 cells and 2) addition of 25 mM glucose to glucose-free DMEM almost doubles the ISR without affecting the OCR suggest a dissociation between the rate of ATP synthesis and ISR. Because these results demonstrate two cases in which the ISR and the OCR [and presumably (16, 22) the rate of ATP synthesis] are not correlated, they argue against an intimate role for the higher rate of ATP synthesis in the stimulus-secretion coupling in these cells.

Media effects on the OCR by beta -cells: implications in tissue engineering. In addition to their implications for understanding fundamental aspects of the mechanism of GSIS, the findings reported here are important in other areas, such as the area of tissue engineering. The values of OCR by islets are useful and have been incorporated into mathematical models directed toward the design and optimization of islet-based bioartificial tissue constructs to be used for the long-term treatment of type I diabetes (3-4). Because to our knowledge all of the published values on the OCR by islets have been obtained in nutrient-free buffers and because of the observed higher values, as well as the reported invariance in the OCR upon glucose stimulation by transformed beta -cells incubated in DMEM-based media, we believe that there is a need for the performance of OCR measurements in physiologically relevant media that are more representative of the in vivo environment.

Speculation. The lack of an effect of glucose on the OCR by beta TC3 and beta HC9 cells in DMEM and the recent findings demonstrating an energetic requirement for GSIS distal to calcium influx (37) raise the following questions with regard to glucose metabolism and GSIS. 1) Is an increase in the rate of glucose metabolism essential for the generation of the signals or coupling factors that ultimately lead to insulin exocytosis? 2) Is glucose metabolism in the absence of other nutrients merely permissive and necessary to support the energy requirements for exocytosis?

We believe that the use of inhibitors of glucose metabolism, but not oxidative phosphorylation, in the presence of other nutrients that can directly enter the tricarboxylic acid cycle, is critical in addressing the questions we have raised. The performance of such experiments is warranted because, to our knowledge, the vast majority of experiments investigating the effects of inhibitors of glycolysis on GSIS by islets and beta -cells have been performed in buffers lacking exogenous nutrients other than glucose. Therefore, we believe that the possibility that inhibitors of glucose metabolism in the absence of other nutrients block GSIS by maintaining the cellular energy stores below a permissive threshold level (1, 37), and not by blocking signals generated from glucose metabolism, cannot be safely ruled out. This suggestion is supported by recent findings (37) demonstrating that several inhibitors of oxidative phosphorylation block GSIS but not glucose-stimulated Ca2+ influx. We believe that the observations described in this study, combined with the findings that in nutrient-rich media glucose stimulates insulin secretion without affecting the OCR (25, 32, 34), the intracellular AMP (9), and ATP levels (9, 22, 32, 34), or the intracellular ATP-to-(ADP)(Pi) ratio (9) in beta -cells, warrant the performance of experiments to reevaluate the implications and the importance of glucose metabolism and metabolically generated coupling factors, in the K+ATP channel/Ca2+-dependent pathway of GSIS, under more physiologically relevant conditions.

In conclusion, the results presented in this study demonstrate that for the beta HC9 cell line, which closely resembles normal mouse islets with regard to glucose metabolism and stimulated insulin secretion (16, 22, 29), 1) the OCR in DMEM is always higher than that measured in PBS, and it is dissociated from the rate of insulin secretion; 2) glucose stimulates insulin secretion in both PBS and DMEM media; and 3) an increase in the rate of insulin secretion is not obligatorily linked to an increase in the OCR.

On the basis of these findings, we suggest that the reported increases in OCR upon addition of glucose to nutrient-free buffers may be unrelated to the process of GSIS and instead related to nutrient starvation. Increases in the OCR upon glucose stimulation may not be observed in more physiologically relevant media and, hence, in the in vivo environment. We believe that our findings, which were extended to a so-far-undisputed metabolic coupling event (the increase in OCR) in the mechanism of GSIS, reinforce the concerns of Ghosh et al. (9) with regard to the use of nutrient-free buffers vs. nutrient-rich media. We hope that these findings will stimulate further experimentation in more physiologically relevant media and a reassessment of the implications of changes in the OCR and other metabolic coupling factors in the mechanism of GSIS.

    NOTE ADDED IN PROOF

Since the submission of this paper we became aware of an article (Carlsson, P. O., P. Liss, A. Andersson, and L. Jansson. Measurements of oxygen tension in native and transplanted rat pancreatic islets. Diabetes 47: 1027-1032, 1998) in which measurements of oxygen tension (PO2) were performed on islets within the pancreas and on islets transplanted beneath the renal capsule. It was demonstrated that acute hyperglycemia did not affect the oxygen tension in any of the investigated tissues. The authors' explanation for their findings was that an increase in islet blood flow induced by hyperglycemia compensated for the increased consumption of oxygen by the islet beta -cells. An alternative explanation, which is consistent with the results presented in the current study, is that glucose did affect the oxygen consumption by beta -cells in vivo where islets were exposed to a nutrient-rich environment. This possibility can be directly evaluated in future experiments.

    ACKNOWLEDGEMENTS

We thank Joann Olejarczyk-Gurkan for the insulin assays and Gregory Hywel and Paul Ameye [Sandoz Research Institute (SRI) machine shop], Don Haenichen (SRI electronics shop), and John Procreva and Theo Dunn (Arotec Corporation) for the computer interfacing. We also thank Drs. Beth Dunning, Tom Hughes, Brian Boettcher, Bork Balkan, Mark Prentki, Athanassios Sambanis, Ioannis Constantinidis, Robert C. Long, Jr., Sid Topiol, Anthony Mancuso, and Michael Shapiro, and Evangelos Tziampazis, for suggestions and for critical review of the manuscript.

    FOOTNOTES

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests: K. K. Papas, Core Technologies/Analytics & Bio-NMR, Novartis Institute for Biomedical Research, 556 Morris Ave., Summit, NJ 07901-1398.

Received 23 June 1998; accepted in final form 25 August 1998.

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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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Am J Physiol Endocrinol Metab 275(6):E1100-E1106
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