From the Touchstone Center for Diabetes Research and Departments of
Biochemistry, § Internal Medicine, and ** Human
Nutrition, University of Texas Southwestern Medical Center, Dallas,
Texas 75390 and the
Department of Biochemistry and Medicine,
Dartmouth Medical School, Hanover, New Hampshire 03755
Received for publication, November 15, 2000
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The long-chain acyl-CoA (LC-CoA) model of
glucose-stimulated insulin secretion (GSIS) holds that secretion is
linked to a glucose-induced increase in malonyl-CoA level and
accumulation of LC-CoA in the cytosol. We have previously tested the
validity of this proposal by overexpressing goose malonyl-CoA
decarboxylase (MCD) in INS-1 cells, but these studies have been
criticized due to: 1) the small insulin secretion response (2-4 The regulation of insulin secretion by glucose is mediated by
metabolism of the sugar in pancreatic islet Our laboratory has recently addressed the LC-CoA model using
adenovirus-mediated overexpression of goose malonyl-CoA decarboxylase (MCD) in INS-1 cells (8). In these studies, we were able to partially
block the glucose-induced increase in malonyl-CoA levels and lessen
glucose-mediated inhibition of fatty acid oxidation. Despite this
perturbation of the link between glucose and lipid metabolism, GSIS
remained unchanged. However, the findings and conclusions of this paper
have been called into question at several levels (9, 10). Concerns
raised include: 1) the INS-1 cell line used in our studies exhibited
only a 2-4 The current study was undertaken to address these outstanding concerns.
This has been facilitated by our recent development of INS-1-derived
cell lines (e.g. 832/13) with robust
KATP-channel-dependent and
-independent GSIS (12). Under normal ionic conditions, 832/13 cells
exhibit a 10-fold increase in insulin secretion when glucose is raised
from 3 to 15 mM, and are also responsive to glucose when
the KATP channel is by-passed by application of
depolarizing K+ and diazoxide. Thus, these cells appear to
be an improved model for evaluation of secretory mechanisms relative to
parental INS-1 cells, which comprised a mixture of glucose-responsive
and -unresponsive cells (12). In addition, since publication of our
prior study, the human MCD cDNA has been cloned (13), and mutant
forms of the cDNA lacking the mitochondrial and peroxisomal
localization sequences have been inserted into adenovirus vectors. With
this cadre of improved reagents, we have re-examined the effect of MCD
overexpression on GSIS, and find no impairment of robust GSIS in 832/13
cells, despite complete blockade of the glucose-induced rise in
malonyl-CoA level. These findings extend our previous observations (8)
and are not consistent with the LC-CoA hypothesis as originally set forth.
All materials were from Sigma, unless otherwise stated.
Cell Culture--
The INS-1-derived cell line 832/13 was used
throughout this study (12). These cells were cultured in RPMI 1640 with
11.1 mM D-glucose, supplemented with 10% fetal
bovine serum, 100 units/ml penicillin, 100 µg/ml streptomycin, 10 mM HEPES, 2 mM glutamine, 1 mM
sodium pyruvate, and 50 µM Preparation and Use of Recombinant Adenoviruses--
Fragments
of the cDNA encoding human MCD lacking the mitochondrial
localization sequence (amino acids 1-39) or the mitochondrial and
peroxisomal localization sequences (amino acids 1-39 and 490-493; see
Ref. 13 for complete sequence of human MCD) were cloned into the
adenovirus vector pACCMV.pLpA (14) and used to prepare recombinant
adenoviruses (AdCMV-MCD6 and AdCMV-MCD Malonyl-CoA Decarboxylase Activity Assay--
MCD activity was
determined as the rate of decarboxylation of malonyl-CoA to acetyl-CoA
as previously described (8). In brief, the rate of acetyl-CoA formation
was monitored by cleavage of its thioester bond by carnitine
acetyltransferase. The thiol group of CoA was colorimetrically measured
at 412 nm over 5-10 min, a time period over which the rate of product
accumulation was linear. Activity was measured either in crude cell
extracts or in cytosol-enriched fractions. Crude extracts were prepared by collection of cells in 50 mM HEPES buffer, pH 7.2, containing protease inhibitors (2 mM phenylmethylsulfonyl
fluoride, 5 µg/ml leupeptin, 50 mM benzamidine, and 50 µg/ml trypsin inhibitor), and homogenization by 10 strokes in a
Dounce homogenizer. A portion of these extracts was used to prepare
cytosol-enriched fractions by centrifugation at 12,000 rpm for 20 min,
transfer of the supernatant into a new tube, and further centrifugation
at 100,000 × g for 20 min. The resultant supernatant
was collected for assay of MCD activity, and values were normalized to
total protein.
Malonyl-CoA Assay--
832/13 cells were cultured in RPMI
containing 3 mM glucose for 24 h following viral
treatment. Cells were washed with HEPES-balanced salt solution (HBSS:
114 mM NaCl, 4.7 mmol KCl, 1.2 mM
KH2PO4, 1.16 mM MgSO4,
20 mM HEPES, 2.5 mM CaCl2, 25.5 mM NaHCO3, pH 7.2) containing 3 mM
glucose and incubated in the same buffer for 2 h. Following this
preincubation, cells were switched to HBSS containing 3 or 15 mM glucose for 30 min. Cells were collected into 2-ml isopropylene tubes by adding 1.5 ml of ice-cold 3.5% (v/v)
HClO4 to each plate and centrifugation of the collected
extract at 12,000 rpm for 5 min at 4 °C. The supernatant containing
acid-soluble metabolites was neutralized to pH 2-3 with 5 N KOH and to pH 6 with 2.5 M KHCO3.
After centrifugation at 2000 rpm for 5 min, the supernatant volume was
measured, prior to its transfer to a 15-ml tube. For assay, each sample
was divided into two 0.2-ml aliquots. 50 pmol of malonyl-CoA was added
to one of the two samples, and malonyl-CoA levels were assayed in both
samples according to the method of McGarry et al. (17),
using purified fatty acid synthase.
[1-14C]Palmitate Oxidation--
832/13 cells in
6-well plates were harvested by light trypsinization and kept in HBSS
containing 3 mM glucose for 30 min at 37 °C. The cells
were contained in center wells (Kontes, Vineland, NJ) suspended from a
rubber sleeve stopper (Fisher, Pittsburgh, PA) inserted into a glass
scintillation vial. A reaction mixture consisting of 0.5 mM
palmitic acid complexed to 1% bovine serum albumin (essentially fatty
acid free), with ~1 × 107 cpm/µmol
[1-14C]-palmitic acid (PerkinElmer Life Sciences, Boston,
MA) as tracer, 0.8 mM L-carnitine, and glucose,
at a final concentration of 3 or 15 mM, was added and the
vials sealed. After 2 h the reaction was terminated by injection
of 100 µl of 7% perchloric acid into the center well. The rate of
[1-14C]palmitate oxidation was measured as released
14CO2, which was trapped by adding 300 µl of
benzethonium hydroxide to the bottom of the sealed vials, followed by
an additional 2-h incubation at 37 °C. Then, the center wells and
rubber stoppers were discarded, scintillation mixture was added, and
14CO2 counted.
Insulin Secretion Studies--
For assay of insulin secretion,
832/13 cells were grown to confluence in 12-well plates and treated
with the various recombinant adenoviruses. After 24 h, cells were
washed in HBSS with 0.2% bovine serum albumin and 3 mM
D-glucose followed by preincubation in 3 ml of the same
buffer for 2 h. Insulin secretion was then measured by static
incubation of the cells for 2 h in 1.5 ml of HBSS containing
glucose, triacsin C, and oleate/palmitate (2:1 molar ratio)
concentrations indicated in figure legends. When ATP-sensitive
K+ channel-independent GSIS was examined, the
K+ concentration in the HBSS during the static incubation
was increased to 35 mM, while the Na+
concentration was lowered to 89.8 mM, and 250 µM diazoxide was added. Because the human proinsulin gene
is stably expressed in the 832/13 cell line (12), insulin was measured
using the Coat-a-Count kit (DPC, Los Angeles, CA). The antibody in this
assay recognizes human insulin and cross-reacts ~20% with rat insulin.
Statistical Analysis--
Data represent mean ± S.E. and
different experimental groups were compared either with a one-tailed
Student's t test or a one-way ANOVA followed by
Newman-Keul's test for comparisons post-hoc. A probability
level of p < 0.05 was considered to be statistically significant.
MCD Activities of the Different Recombinant Adenoviruses in 832/13
Cells--
In our previous work, a recombinant adenovirus containing
the cDNA encoding malonyl-CoA decarboxylase from the goose
uropygial gland (AdCMV-MCDgoose) was used to partially
attenuate increases in malonyl-CoA levels during glucose stimulation of
INS-1 cells (8). The cDNA used in this virus encoded a protein that
lacked its N-terminal mitochondrial, but not the C-terminal peroxisomal localization sequence. These features, coupled with its non-mammalian origin, may have limited the ability of the modified goose MCD to
degrade malonyl-CoA formed in the cytosol of rat cells, which is the
critical pool for modulation of carnitine palmitoyltransferase I enzyme
activity. To deal with this concern, we have constructed three new
adenoviruses containing modified human MCD cDNAs. The first of
these is analagous to the goose MCD construct in that it lacks its
N-terminal mitochondrial localization sequence (AdCMV-MCD6), and the
second lacks both the mitochondrial and SKL targeting sequences
(AdCMV-MCD
To assess the capacity of the different recombinant adenoviruses to
increase MCD activity, 832/13 cells were treated with increasing doses
of the viruses. Twenty-four h after viral treatment, total MCD activity
was determined in unfractionated cell extracts. As is shown in Fig.
1, each of the viruses encoding
functional enzymes (AdCMV-MCDgoose, AdCMV-MCD6, or
AdCMV-MCD
More relevant than total MCD activity in whole cell extracts is the
amount of activity residing in the cytosolic compartment of cells. To
estimate this, different batches of 832/13 cells were treated with the
same titers of AdCMV-MCDgoose, AdCMV-MCD6, or AdCMV-MCD Effect of MCD Overexpression on Malonyl-CoA Levels--
In our
previous study in which goose MCD was overexpressed in INS-1 cells,
cellular malonyl-CoA levels were increased in response to changes in
external glucose concentrations, although total malonyl-CoA levels were
significantly lower in MCD overexpressing cells than in controls (8).
The incomplete blockade in malonyl-CoA formation could have led to an
erroneous conclusion about its role in regulation of insulin secretion.
We therefore investigated the ability of our new MCD adenoviruses to
lower malonyl-CoA levels in 832/13 cells. As shown in Fig.
3, malonyl-CoA levels were increased by
4.8-fold in AdCMV- Impact of Recombinant MCD Adenoviruses on Fatty Acid Oxidation in
832/13 Cells--
We next proceeded to evaluate the metabolic effects
of the AdCMV-MCD
There is a possibility that the most pronounced metabolic effects of
MCD overexpression occur at higher viral titers than used in the
experiment of Fig. 4. Alternatively, high viral titers could have
compromised certain aspects of metabolic function, so that the effects
on fatty acid oxidation may have been more pronounced at lower viral
titers. To address this, we determined the titer dependence of the
effect of AdCMV-MCD Glucose-stimulated Insulin Secretion in AdCMV-MCD
Insulin secretion was also studied in the presence of 35 mM
K+ and 250 µM diazoxide, which allows
measurement of the KATP channel-independent pathway of glucose sensing (2). It has been suggested that his pathway
is responsible for the LC-CoA-mediated potentiation of GSIS (10, 11).
However, in the presence of the high K+ concentration and
diazoxide, 15 mM glucose had the same stimulatory effect
(2.3-fold) on insulin secretion in AdCMV-
We have previously demonstrated that treatment of INS-1 cells with
triacsin C, an inhibitor of LC-CoA synthetase, results in a 50%
decrease in LC-CoA levels, with no effect on GSIS (8). We therefore
examined the effects of combined treatment of 832/13 cells with the
AdCMV-MCD Preincubation of AdCMV-MCD The idea that malonyl-CoA could be a coupling factor in In contrast to these results, we have previously demonstrated that
adenovirus-mediated overexpression of goose MCD in INS-1 cells reduced
malonyl-CoA levels and partially alleviated inhibition of fatty acid
oxidation by glucose (8). Triacsin C, an inhibitor of LC-CoA synthetase
was also shown to block glucose incorporation into lipids and lower
LC-CoA levels, but neither the molecular nor the pharmacologic
approaches to impairing LC-CoA formation had any effect on GSIS (8).
The current study has attempted to address perceived limitations of the
prior work that have come to light (9, 10), and to resolve the apparent
discrepancy between our work and that of others. Key improvements in
our experimental approach reported herein include. 1) The use of a new
INS-1-derived cell line with robust KATP
channel-dependent or -independent pathways of glucose
sensing (12). 2) The construction of new recombinant adenoviruses
containing the cDNAs encoding human forms of MCD, including one in
which both the mitochondrial and peroxisomal localization sequences
were removed to enhance expression of the enzyme in the cytosolic
compartment. 3) New studies involving preincubation of cells in a
mixture of oleate and palmitate to boost cellular lipid stores. 4)
Co-application of the molecular (AdCMV-MCD How can our findings be reconciled with those supporting the
malonyl-CoA/LC-CoA model of glucose sensing? First, the rise in
malonyl-CoA levels that occurs prior to insulin release is not proof of
a causal relationship between the two phenomena. Also, the recently
reported effect of palmitoyl-CoA on insulin secretion is modest, and
the use of permeabilized cells in these studies raises questions about
the physiological relevance of the experiment (7). Moreover, in view of
the proposed critical role of mitochondrial metabolism in GSIS (22),
abrogation of GSIS conferred by hydroxycitrate may be due in part to
perturbed mitochondrial metabolism. It should also be noted that the
inhibitory effect of hydroxycitrate on GSIS is not observed in isolated
rat islets (23). The enhanced GSIS that accompanies inhibition of fatty
acid oxidation by carnitine palmitoyltransferase I inhibitors may be
explained by the fact that these compounds, e.g.
2-bromopalmitate or etomoxir, are modified fatty acids, which may act
like native lipids to potentiate GSIS independent of their effects on
fatty acid oxidation (18). Finally, chronic lowering of malonyl-CoA levels in INS-1 cells with a stable transfection strategy may cause
profound alterations in fold)
of the INS-1 cells used; 2) unknown contribution of the ATP-sensitive K+ (KATP) channel-independent
pathway of GSIS in INS-1 cells, which has been implicated as the site
at which lipids regulate insulin granule exocytosis; and 3) deletion of
the N-terminal mitochondrial targeting sequence, but not the C-terminal
peroxisomal targeting sequence in the goose MCD construct, raising the
possibility that a significant fraction of the overexpressed enzyme was
localized to peroxisomes. To address these outstanding concerns,
INS-1-derived 832/13 cells, which exhibit robust
KATP channel-dependent and -independent pathways of GSIS, were treated with a new adenovirus encoding human MCD lacking both its mitochondrial and peroxisomal targeting sequences (AdCMV-MCD
5), resulting in large increases in
cytosolic MCD activity. Treatment of 832/13 cells with AdCMV-MCD
5 completely blocked the glucose-induced rise in malonyl-CoA and attenuated the inhibitory effect of glucose on fatty acid oxidation. However, MCD overexpression had no effect on
KATP channel-dependent or
-independent GSIS in 832/13 cells. Furthermore, combined treatment of
832/13 cells with AdCMV-MCD
5 and triacsin C, an inhibitor of long
chain acyl-CoA synthetase that reduces LC-CoA levels, did not impair
GSIS. These findings extend our previous observations and are not
consistent with the LC-CoA hypothesis as originally set forth.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-cells, resulting in an
increase in the ATP:ADP ratio and closure of ATP-dependent K+
(KATP)1
channels. Subsequently, voltage-gated Ca2+ channels open,
intracellular Ca2+ rises, and insulin exocytosis is
initiated (1). However, it has become clear that this is a minimal
model of glucose sensing, because glucose-stimulated insulin secretion
(GSIS) still occurs when closure of the KATP
channel or the rise in intracellular Ca2+ are prevented (2,
3). It has been suggested that factors that complement changes in
ATP:ADP ratios in regulation of insulin secretion may arise from
post-mitochondrial metabolism of glucose, and interaction of glucose
and lipid metabolism. The long-chain acyl-CoA (LC-CoA) model of GSIS
holds that malonyl-CoA levels increase in response to increasing
glucose concentrations, resulting in inhibition of carnitine
palmitoyltransferase I and fatty acid oxidation (4-6). This
could lead to accumulation of LC-CoA in the cytosol, which may act as a
coupling factor in stimulation of insulin secretion (7).
fold stimulation of insulin secretion as glucose was
raised from 3 to 15 mM, as opposed to freshly isolated rat
islets, which can exhibit a 10-15-fold response. 2) It is unclear if
the so-called KATP channel-independent pathway
of GSIS is operative in INS-1 cells. This pathway, which is revealed
when
-cells are exposed to depolarizing K+, has been
implicated as the site at which lipids regulate insulin granule
exocytosis (10, 11). 3) The goose MCD cDNA used in our previous
study had its N-terminal mitochondrial targeting sequence deleted, but
contained an intact C-terminal SKL peroxisomal targeting motif, raising
the possibility that a significant fraction of the overexpressed enzyme
failed to localize to the cytosol.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-mercaptoethanol, at
37 °C in a humidified atmosphere containing 5% CO2.
5, respectively) as described
previously (15). The resulting viruses were plaque-purified (15) and
used to treat confluent 832/13 cells at varying titers as described in
the figure legends. A virus containing the bacterial
-galactosidase
gene (AdCMV-
GAL) was used as a control (16). After a 1-h incubation
with viruses, cells were washed once in phosphate-buffered saline,
culture medium was added, and assays and analyses were undertaken
24 h later.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
5). Finally, a control virus was constructed containing
the MCD cDNA of a patient with two point mutations in the coding
sequence that render the enzyme catalytically inactive (AdCMV-MCDmut) (13).
5) increased enzyme activity in a
dose-dependent manner, with maximal values ~20-fold
higher than in either of the control groups (AdCMV-
GAL-treated or
AdCMV-MCDmut-treated cells). Interestingly, the maximal MCD activity achieved was 10-fold higher in this study than in our previous
report (8). The reasons for this increased efficiency of expression of
MCD in the current study are not known, but could relate to the fact
that we treated the cells with a higher viral titer over a shorter time
period than in our previous study. Alternatively, expression of the
construct may have been more efficient in the novel 832/13 cells than
in the parental INS-1 cells used previously (8).
View larger version (27K):
[in a new window]
Fig. 1.
Titering of adenoviruses expressing MCD.
832/13 cells were treated with the indicated multiplicities of
infection (MOI) of various recombinant adenoviruses,
abbreviated as described under "Materials and Methods." MCD
enzymatic activity was measured in total cell extracts 24 h after
viral treatment. Results are mean ± S.E. of three independent
experiments performed in duplicate.
5
adenoviruses, and used for preparation of cytosolic cell fractions
24 h after viral treatment. Fig. 2
shows that the MCD activity in the cytosolic fraction of AdCMV-MCD
5
was increased by 120 and 50% compared with cells treated with
AdCMV-MCDgoose or AdCMV-MCD6, respectively. Thus, while the
three MCD viruses caused similar increases in MCD activity in whole
cell extracts, AdCMV-MCD
5 containing the cDNA that lacks both
its mitochondrial and peroxisomal localization sequences caused a
larger increase in cytosolic activity than the other two constructs. In
theory, AdCMV-MCD
5 may therefore be a more specific tool for
perturbing the link between metabolism of glucose and lipids, because
the pool of malonyl-CoA that is implicated in
-cell
stimulus-secretion coupling is formed in the cytosol (9).
View larger version (20K):
[in a new window]
Fig. 2.
Cytoplasmic MCD activities in
MCD-overexpressing 832/13 cells. 832/13 cells were treated with
the indicated recombinant adenoviruses at an MOI of 25 for each virus.
Total cell extracts were prepared, and a portion was used for
measurement of MCD enzymatic activity (white bars, left).
Cytoplasm-enriched fractions were then prepared with the remaining
total cell extract as described under "Materials and Methods," and
MCD activity was measured in this fraction (black bars,
right). Data represents the mean ± S.E. for three
independent experiments; *, p < 0.05, and **,
p < 0.01.
GAL-treated 832/13 cells as glucose was raised
from 3 to 15 mM. This glucose-induced rise in malonyl-CoA levels was completely blocked in 832/13 cells treated with any of the
three MCD viruses, AdCMV-MCDgoose, AdCMV-MCD6, or
AdCMV-MCD
5. The more effective blockade of malonyl-CoA production is
consistent with the higher levels of enzyme activity achieved in this
study compared with the previous one (8). Given the similar performance of the three viral constructs, all subsequent experiments were carried
out exclusively with AdCMV-MCD
5.
View larger version (27K):
[in a new window]
Fig. 3.
Malonyl-CoA levels in MCD-overexpressing
832/13 cells. 832/13 cells were treated with the indicated
recombinant adenoviruses (see "Materials and Methods" for
description of abbreviations). After viral treatment, cells were
cultured in 3 mM glucose for 24 h, at which point
cells were washed by HBSS containing 3 mM glucose, and
further incubated in the same buffer for 2 h. Cells were then
switched to HBSS containing either 3 or 15 mM glucose, and
after an additional 30 min, malonyl-CoA was extracted and measured as
described under "Materials and Methods." Data represent the
mean ± S.E. for three independent measurements. The
asterisk symbol (*) indicates that the malonyl-CoA level at
15 mM glucose was significantly higher than at 3 mM glucose in the AdCMV- GAL-treated control cells, with
p < 0.001
5 virus. [1-14C]Palmitate oxidation
was measured in 832/13 cells treated with a titer of AdCMV-MCD
5 that
raised MCD activity to ~1.8 µmol/mg/min. As shown in Fig.
4, incubation of AdCMV-
GAL-treated
832/13 cells at 15 mM glucose reduced the rate of
[1-14C]palmitate oxidation to 31% of that in cells
incubated in 3 mM glucose. Thus, glucose regulates fatty
acid oxidation in 832/13 cells in a fashion similar to what has been
reported for rat islets (18) and INS-1 cells (8). In contrast, the high
glucose concentration was much less effective at suppressing fatty acid
oxidation in cells treated with AdCMV-MCD
5, such that the rate
remained at 63% of that in cells incubated in 3 mM
glucose.
View larger version (22K):
[in a new window]
Fig. 4.
[1-14C]Palmitate oxidation in
832/13 cells. 832/13 cells were treated with AdCMV-MCD 5 or
AdCMV-
GAL. 24 h after viral treatment,
[1-14C]palmitate oxidation was measured as described
under "Materials and Methods." Results represent the mean ± S.E. of four independent experiments. The double asterisk
symbol (**) (p < 0.01) indicates that at 15 mM glucose, cells treated with AdCMV-MCD
5 oxidized fatty
acids at a significantly higher rate than cells treated with
AdCMV-
GAL.
5 on fatty acid oxidation. At one-fourth the
viral titer used in the foregoing experiments (5 × 105 pfu/well corresponding to an MCD activity of 0.85 µmol/mg/min) the effect of MCD overexpression to impair
glucose-mediated inhibition of fatty acid oxidation was already
maximal. Moreover, it was neither increased nor decreased when the
titer was increased stepwise over a 4-fold range (data not shown). This
incomplete reversal of glucose suppression of fatty acid oxidation in
AdCMV-MCD
5 expressing cells, despite the complete blockade of
glucose-induced malonyl-CoA accumulation, suggests that factors other
than the malonyl-CoA level are involved in regulation of fatty acid
oxidation by high glucose.
5-transduced
and/or Triacsin-treated 832/13 Cells--
Having established that
AdCMV-MCD
5 blocks glucose-induced malonyl-CoA production and
attenuates the inhibitory effect of glucose on fatty acid oxidation, we
proceeded to evaluate the effects of this new reagent on insulin
secretion. In 832/13 cells treated with the control virus AdMV-
GAL,
15 mM glucose caused a 10-fold increase in insulin
secretion relative to secretion at 3 mM glucose (Fig.
5A). Cells treated with
AdCMV-MCD
5 exhibited the same robust response to stimulatory
glucose, providing clear evidence that a rise in malonyl-CoA and
complete blockade of fatty acid oxidation are not required events in
GSIS. This lack of effect of MCD was not specific to AdCMV-MCD
5, as
cells treated with AdCMV-MCD6 exhibited the same potent response to
stimulatory glucose (data not shown).
View larger version (27K):
[in a new window]
Fig. 5.
Glucose-stimulated insulin secretion in
832/13 cells. 832/13 cells were treated with the indicated
adenoviruses and cultured for 24 h in medium containing 3 mM glucose. Insulin secretion was then measured by
incubating cells in HBSS containing either 3 or 15 mM
glucose for 2 h. Panel A, experiments conducted in the
presence of normal K+ concentrations. Panel B,
experiments conducted in the presence of depolarizing K+
(35 mM) and 250 µM diazoxide to measure
KATP channel-independent glucose sensing.
Results represent the mean ± S.E. for eight independent
experiments for panel A and four independent experiments for
panel B. Insulin secretion was compared with basal secretion
measured at 3 mM glucose for cells treated with each virus,
using a one-way ANOVA followed by Newman-Keul's test
post-hoc; *, p < 0.05; **,
p < 0.01; ***, p < 0.001.
GAL or
AdCMV-MCD
5-treated 832/13 cells (Fig. 5B), demonstrating
that blockade of malonyl-CoA production has no effect on glucose
sensing via the KATP channel-independent pathway.
5 virus, which blocks malonyl-CoA accumulation, and
triacsin C, which lowers LC-CoA levels. The combined addition of
AdCMV-MCD
5 and triacsin C to 832/13 cells had no effect on the
10-fold stimulation of insulin secretion by 15 mM glucose under normal culture conditions (Fig. 5A) or on the
stimulation by glucose of insulin secretion via the
KATP channel-independent pathway (Fig.
5B). Thus, complete blockade of malonyl-CoA accumulation and
simultaneous lowering of the cellular LC-CoA pool has no effect on
GSIS.
5-treated 832/13 Cells with Free Fatty
Acids--
Finally, we examined the possibility that limited
endogenous lipid stores in insulinoma-derived clonal
-cells could
impact our ability to demonstrate a link between glucose and lipid
metabolism for regulation of GSIS. To test this possibility, 832/13
cells were preincubated for 48 h in 0.5 mM
oleate/palmitate, prior to treatment with AdCMV-MCD
5. GSIS was then
assayed after an additional 24 h of culture in 0.5 mM
oleate/palmitate. These maneuvers were designed to increase cellular
storage of lipids. As seen in Fig. 6,
culture of 832/13 for 3 days in 0.5 mM oleate/palmitate
resulted in attenuation of GSIS, such that these cells exhibited a
4-fold response to 15 mM glucose, compared with the 10-fold
response of cells grown in normal medium. This blunting effect of
chronic culture in fatty acids is similar to that previously described in isolated rat and human islets (19, 20). Nevertheless, glucose responsiveness was not different in AdCMV-
GAL versus
AdCMV-MCD
5-treated cells that had been cultured in the presence of
0.5 mM oleate/palmitate. The impact of acute stimulation of
832/13 cells with free fatty acid was also tested in cells
cultured both in regular RPMI and in RPMI supplemented with 0.5 mM oleate/palmitate. Again, GSIS was similarly potentiated
by 0.5 mM oleate/palmitate in AdCMV-
GAL and
AdCMV-MCD
5-treated cells, regardless of whether they were preincubated with the fatty acid mixture. Thus, neither an increment in
cellular lipids nor acute exogenous administration of lipids uncovers a
requirement for malonyl-CoA in regulation of insulin secretion.
View larger version (24K):
[in a new window]
Fig. 6.
Effect of preincubation of 832/13 cells in
fatty acids. 832/13 cells were treated with either AdCMV-MCD 5
or AdCMV-
GAL after 48 h of culture in medium lacking
(left bars) or containing (right bars) 0.5 mM oleate/palmitate. After viral treatment, medium
containing 0.5 mM oleate/palmitate was again added to the
cells, and after a further 24 h GSIS was assayed in a 2-h static
incubation with or without co-stimulation by 1 mM
oleate/palmitate. Results represent mean ± S.E. for five
independent experiments for each condition. Insulin secretion was
compared with basal secretion measured at 3 mM glucose for
cells treated with each virus, using a one-way ANOVA followed by
Newman-Keul's test post-hoc. In addition, comparisons
between cells stimulated at 15 mM glucose with or without
the addition of 1 mM oleate/palmitate are shown as
bars; *, p < 0.05; **, p < 0.01; ***, p < 0.001.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-cell
stimulus-secretion originated from the observation that the levels of
this intermediate rise prior to insulin secretion when HIT-T15 hamster
insulinoma cells are exposed to glucose (4-6). Furthermore, the
glucose-induced rise in malonyl-CoA was coincident with inhibition of
fatty acid oxidation and was assumed to result in an increase in
cytosolic levels of LC-CoA. That LC-CoA could be an effector molecule
in
-cell stimulus-secretion coupling was further suggested by the
fact that perfusion of permeabilized HIT-T15 cells with LC-CoA-esters
induced insulin secretion (7). Additional supporting evidence for the
LC-CoA model comes from the use of hydroxycitrate, which inhibits
ATP-citrate lyase and consequently malonyl-CoA formation. Use of this
agent to disrupt the link between glucose and malonyl-CoA formation was
shown to inhibit GSIS from the perfused rat pancreas (18). Conversely, inhibition of carnitine palmitoyltransferase I and fatty acid oxidation
with agents such as etomoxir-stimulated GSIS from the perfused pancreas
(18). Finally, stable expression of an acetyl-CoA carboxylase-antisense
construct in INS-1 cells resulted in lowering of malonyl-CoA levels,
increased fatty acid oxidation, and inhibition of GSIS (21).
5) and pharmacologic
(triacsin C) reagents to maximally perturb malonyl-CoA and LC-CoA
synthesis. These improvements have allowed us to demonstrate in a clear
and unequivocal fashion that complete blockade of malonyl-CoA
accumulation has no impact on the KATP
channel-dependent or independent pathways of GSIS. The co-application of AdCMV-MCD
5 and triacsin C also has no effect on
insulin secretion, suggesting that robust glucose sensing is not linked
to changes in total LC-CoA levels. Furthermore, preincubation of 832/13
cells for 3 days in 0.5 mM oleate/palmitate impaired glucose sensing, as has been described extensively in islets (19, 20).
However, culture of cells in fatty acids does not uncover an effect of
AdCMV-MCD
5 on GSIS. Finally, treatment of 832/13 cells with
AdCMV-MCD
5 does not affect fatty acid potentiation of GSIS,
supporting the view that this action of fatty acids is not related to
their oxidation (18).
-cell metabolism and function (21). Of
particular concern here is that chronic lowering of malonyl-CoA levels
could result in depletion of stored lipids. It has been shown that
depletion of
-cell lipids in nicotinamide-treated animals or humans
(24, 25) or in hyperleptinemic rats (26) blocks insulin secretion in
response to glucose and many other secretagogues. Interestingly, in
both cases, GSIS is immediately restored by provision of free fatty
acids. These findings clearly support an essential role for lipids in
regulation of insulin secretion, possibly at the level of membrane
lipid turnover or acylation of regulatory proteins. The latter
possibility is also supported by recent studies in which insulin
secretion was inhibited by addition of cerulenin, an inhibitor of
protein acylation (11). However, while a minimal pool of lipids may be
essential for normal regulation of insulin secretion, the current study
provides strong evidence that glucose sensing can occur in the absence
of a rise in malonyl-CoA and despite acute perturbation of lipid metabolism.
![]() |
FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grants DK-42582 (to C. B. N.) and DK-18573 (to J. D. M) and a grant from the Reynold's Foundation (to C. B. N. and J. C.).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. Section 1734 solely to indicate this fact.
¶ Recipient of a Postdoctoral Fellowship from the Juvenile Diabetes Foundation International.
To whom correspondence should be addressed: Touchstone Center
for Diabetes Research, Rm. Y8.212, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390. Fax: 214-648-9191; E-mail: newgard@utsw.swmed.edu.
Published, JBC Papers in Press, December 11, 2000, DOI 10.1074/jbc.M010364200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: KATP, ATP-sensitive K+; GSIS, glucose-stimulated insulin secretion; LC-CoA, long-chain acyl-CoA; MCD, malonyl-CoA decarboxylase; HBSS, Hepes balanced salt solution.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Newgard, C. B., and McGarry, J. D. (1995) Annu. Rev. Biochem. 64, 689-719[CrossRef][Medline] [Order article via Infotrieve] |
2. | Gembal, M., Gilon, P., and Henquin, J. C. (1992) J. Clin. Invest. 89, 1288-1295[Medline] [Order article via Infotrieve] |
3. | Komatsu, M., Schermerhorn, T., Aizawa, T., and Sharp, G. W. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 10728-10732[Abstract] |
4. |
Corkey, B. E.,
Glennon, M. C.,
Chen, K. S.,
Deeney, J. T.,
Matschinsky, F. M.,
and Prentki, M.
(1989)
J. Biol. Chem.
264,
21608-21612 |
5. |
Prentki, M.,
Vischer, S.,
Glennon, M.,
Regazzi, R.,
Deeney, J.,
and Corkey, B. E.
(1992)
J. Biol. Chem.
267,
5802-5810 |
6. | Prentki, M., and Corkey, B. E. (1996) Diabetes 45, 273-283[Abstract] |
7. |
Deeney, J. T.,
Gromada, J.,
Hoy, M.,
Olsen, H. L.,
Rhodes, C. J.,
Prentki, M.,
Berggren, P. O.,
and Corkey, B. E.
(2000)
J. Biol. Chem.
275,
9363-9368 |
8. |
Antinozzi, P. A.,
Segall, L.,
Prentki, M.,
McGarry, J. D.,
and Newgard, C. B.
(1998)
J. Biol. Chem.
273,
16146-16154 |
9. | Corkey, B. E., Deeney, J. T., Yaney, G. C., Tornheim, K., and Prentki, M. (2000) J. Nutr. 130 Suppl. 2S, 299S-304S[Medline] [Order article via Infotrieve] |
10. | Komatsu, M., Yajima, H., Yamada, S., Kaneko, T., Sato, Y., Yamauchi, K., Hashizume, K., and Aizawa, T. (1999) Diabetes 48, 1543-1549[Abstract] |
11. | Yajima, H., Komatsu, M., Yamada, S., Straub, S. G., Kaneko, T., Sato, Y., Yamauchi, K., Hashizume, K., Sharp, G. W. G., and Aizawa, T. (2000) Diabetes 49, 712-717[Abstract] |
12. | Hohmeier, H. E., Mulder, H., Chen, G., Henkel-Rieger, R., Prentki, M., and Newgard, C. B. (2000) Diabetes 49, 424-430[Abstract] |
13. |
Gao, J.,
Waber, L.,
Bennett, M. J.,
Gibson, K. M.,
and Cohen, J. C.
(1999)
J. Lipid Res.
40,
178-182 |
14. |
Gomez-Foix, A. M.,
Coats, W. S.,
Baque, S.,
Alam, T.,
Gerard, R. D.,
and Newgard, C. B.
(1992)
J. Biol. Chem.
267,
25129-25134 |
15. | Becker, T. C., Noel, R. J., Coats, W. S., Gomez-Foix, A. M., Alam, T., Gerard, R. D., and Newgard, C. B. (1994) Methods Cell Biol. 43, 161-189[Medline] [Order article via Infotrieve] |
16. | Herz, J., and Gerard, R. D. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 2812-2816[Abstract] |
17. | McGarry, J. D., Stark, M. J., and Foster, D. W. (1978) J. Biol. Chem. 253, 8291-8293[Abstract] |
18. | Chen, S., Ogawa, A., Ohneda, M., Unger, R. H., Foster, D. W., and McGarry, J. D. (1994) Diabetes 43, 878-883[Abstract] |
19. |
Lee, Y.,
Hirose, H.,
Ohneda, M.,
Johnson, J. H.,
McGarry, J. D.,
and Unger, R. H.
(1994)
Proc. Natl. Acad. Sci. U. S. A.
91,
10878-10882 |
20. | Zhou, Y. P., and Grill, V. (1995) J. Clin. Endocrin. Metab. 80, 1584-1590[Abstract] |
21. | Zhang, S., and Kim, K. H. (1998) Cell. Signal. 10, 35-42[CrossRef][Medline] [Order article via Infotrieve] |
22. |
Maechler, P.,
Kennedy, E. D.,
Pozzan, T.,
and Wollheim, C. B.
(1997)
EMBO J.
16,
3833-3841 |
23. | Sener, A., and Malaisse, W. J. (1991) Biochimie (Paris) 73, 1287-1290[Medline] [Order article via Infotrieve] |
24. |
Stein, D. T.,
Esser, V.,
Stevenson, B. E.,
Lane, K. E.,
Whiteside, J. H.,
Daniels, M. B.,
Chen, S.,
and McGarry, J. D.
(1996)
J. Clin. Invest.
97,
2728-2735 |
25. | Dobbins, R. L., Chester, M. W., Daniels, M. B., McGarry, J. D., and Stein, D. T. (1998) Diabetes 47, 1613-1618[Abstract] |
26. | Koyama, K., Chen, G., Wang, M. Y., Lee, Y., Shimabukuro, M., Newgard, C. B., and Unger, R. H. (1997) Diabetes 46, 1276-1280[Abstract] |