1 Core Technologies/Analytics
and Bio-Nuclear Magnetic Resonance, We investigated the effects of glucose on the
rates of oxygen consumption (OCR) and insulin secretion (ISR) by
fuel hypothesis; nutrient starvation; tissue engineering; bioartificial pancreas
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
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
HC9
cells derived from mouse pancreatic islets with
-cell hyperplasia. Our results demonstrate that the OCR by
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
-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.
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
-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).
View larger version (24K):
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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 -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 -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
-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
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 -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
-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
-cells in the mechanism of GSIS, we performed
OCR and insulin secretion rate (ISR) measurements on
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.
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MATERIALS AND METHODS |
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Cells and cell culture.
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.
HC9 cells were passaged every 10 days at a split ratio of
1:3. Confluent monolayer
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). 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
HC9 cells.
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 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 confluentStatistics. 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 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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RESULTS |
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Oxygen consumption.
Figure 3 depicts typical results obtained
with a culture of entrapped 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
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
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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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 · h1 · 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.
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DISCUSSION |
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Effects of glucose on the OCR and ISR by -cells.
The publication of a report demonstrating substantial changes in the
OCR by both
TC3 and
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
TC3 cells upon glucose stimulation
(25, 32, 34) was due to differences in glucose metabolism and energy
requirements between
TC3 cells and islets. An alternative hypothesis
(34) offered an explanation for the lack of changes in the OCR by
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
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
TC3 and
HC9 cells can be fully explained by the
differences in the incubation media used.
OCR, rate of ATP synthesis, and ISR by -cells
incubated in PBS and DMEM.
The reported stimulation in the OCR by
HC9 and
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
TC3 and
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
-cells (16, 22).
Media effects on the OCR by -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
-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 TC3 and
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?
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NOTE ADDED IN PROOF |
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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 -cells. An alternative explanation, which is consistent
with the results presented in the current study, is that glucose did
affect the oxygen consumption by
-cells in vivo where islets were
exposed to a nutrient-rich environment. This possibility can be
directly evaluated in future experiments.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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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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