(Received for publication, August 15, 1996, and in revised form, October 30, 1996)
From the Institute of Pharmacology and Toxicology, University of Lausanne, 27 Rue du Bugnon, 1005 Lausanne, Switzerland
GLUT2 expression is strongly decreased in
glucose-unresponsive pancreatic cells of diabetic rodents. This
decreased expression is due to circulating factors distinct from
insulin or glucose. Here we evaluated the effect of palmitic acid and
the synthetic glucocorticoid dexamethasone on GLUT2 expression by
in vitro cultured rat pancreatic islets. Palmitic acid
induced a 40% decrease in GLUT2 mRNA levels with, however, no
consistent effect on protein expression. Dexamethasone, in contrast,
had no effect on GLUT2 mRNA, but decreased GLUT2 protein by about
65%. The effect of dexamethasone was more pronounced at high glucose
concentrations and was inhibited by the glucocorticoid antagonist
RU-486. Biosynthetic labeling experiments revealed that GLUT2
translation rate was only minimally affected by dexamethasone, but that
its half-life was decreased by 50%, indicating that glucocorticoids
activated a posttranslational degradation mechanism. This degradation
mechanism was not affecting all membrane proteins, since the
subunit of the Na+/K+-ATPase was unaffected.
Glucose-induced insulin secretion was strongly decreased by treatment
with palmitic acid and/or dexamethasone. The insulin content was
decreased (~55 percent) in the presence of palmitic acid, but
increased (~180%) in the presence of dexamethasone. We conclude that
a combination of elevated fatty acids and glucocorticoids can induce
two common features observed in diabetic
cells, decreased GLUT2
expression, and loss of glucose-induced insulin secretion.
Development of non-insulin-dependent, type II diabetes
mellitus is accompanied by a loss of glucose-stimulated insulin
secretion (GSIS)1 (1, 2). The primary
causes of this secretory defect are not yet completely elucidated.
However, in rodent models of diabetes, the loss of GSIS has been
demonstrated to correlate with a reduced or suppressed expression of
the cell glucose transporter GLUT2 (3-6). Thus, in addition to a
loss of GSIS, a decreased expression of GLUT2 is also a characteristic
of diabetic
cells. In an attempt at identifying the causes of
GLUT2-regulated expression, we previously performed islet
cross-transplantation experiments. When control islets were
transplanted in diabetic mice, GLUT2 expression was suppressed whereas
when GLUT2 nonexpressing islets from diabetic animals were transplanted
into control mice, a complete recovery of transporter expression was
observed. These experiments led to the conclusion that circulating
factors present in the diabetic environment, distinct from glucose and
insulin, were responsible for the loss of GLUT2 expression (6).
Furthermore, the down-expression of GLUT2 in transplanted islets
correlated with a loss of GSIS (7). Identification of the circulating
factors that control GLUT2 expression in
cells is therefore of
critical importance, as they may be responsible for the functional
alterations of
cells in diabetes.
Elevated circulating free fatty acids and triglycerides are part of the
symptoms of both insulin-dependent and
non-insulin-dependent diabetes mellitus (8, 9). Free fatty
acids have been described for many years as being able to induce a
state of insulin resistance in peripheral tissues by a glucose/fatty
acid cycle that prevents a normal uptake of glucose (10). The effects
of free fatty acids on the function of pancreatic islets has been
studied both in in vivo and in vitro experiments.
These studies indicated that short term (1-3 h) exposure of pancreatic
islets to free fatty acids had a stimulatory effect on GSIS (11-13),
whereas longer exposure led to a suppression of insulin secretion
(12-14). This inhibitory effect is also accompanied by a decrease in
insulin biosynthesis, in glucose oxidation, and a reduction in pyruvate dehydrogenase activity with a parallel increase in pyruvate
dehydrogenase kinase activity (15, 16). A major role for free fatty
acids in the development of cell glucose unresponsiveness has thus been proposed. This was further supported by the observation that circulating free fatty acid levels were increased a few weeks before
development of hyperglycemia and loss of GLUT2 expression in male
Zucker diabetic rats, while obese female Zucker rats, which do not
develop hyperglycemia and do not lose GLUT2, did not show this increase
in free fatty acids, even though they develop similar
hypertriglyceridemia (17). Furthermore, incubation of pancreatic islets
in the presence of free fatty acids induced an increase in low
Km glucose usage (18) and an elevated basal insulin
secretion rate (14, 18). However, no apparent regulation of GLUT2
expression by free fatty acids has been observed in islets maintained
in tissue culture (9). Thus, although free fatty acids may lead to a
number of
cells dysfunctions associated with diabetes, they
apparently do not induce the decreased or suppressed expression of
GLUT2. This therefore indicates that additional factors also
participate in the induction of the
cells functional alterations in
diabetes.
Dexamethasone administration in humans and in animals as well as
hypercortisolism in Cushing syndrome are known to induce a state of
insulin resistance. This is also usually accompanied by changes in cell functions, in particular an increase in basal but a decrease in
stimulated insulin secretion, an increase in proinsulin mRNA, a
decrease in islet insulin stores, and an hyperplasia and hypertrophy of
the
cells (19-23). Dexamethasone administration to rats does not,
however, induce a decrease in
cell GLUT2 (24). Only if diabetes is
induced, as for example following repeated injections of high doses of
dexamethasone to Wistar rats or of relatively lower doses to Zucker
fa/fa rats, is a decrease in GLUT2 expression observed (22, 25). The
exact role of dexamethasone on pancreatic
cells is, however,
difficult to evaluate when administered to the intact animal. In
vitro, exposure of RINm5F cells to dexamethasone increased
proinsulin mRNA levels but did not alter the insulin secretion rate
at any glucose concentrations (26). In HIT cells and isolated
cells
dexamethasone induces a decrease in insulin secretion and mRNA
(27). The inhibitory effect on mRNA levels can, however, be
completely prevented by increases in intracellular cAMP (28).
Here we studied the effect of palmitic acid and dexamethasone alone, or in combination on the expression of GLUT2, on GSIS and on insulin mRNA levels in in vitro cultured rat pancreatic islets. We demonstrate that a combination of both substances can reproduce in vitro the decrease in GSIS and GLUT2 expression observed in islets from diabetic rodents.
Male Sprague-Dawley rats were purchased from Biological Research Laboratories Ltd., Ficoll DL-400 and palmitic acid (sodium salt) from Fluka, collagenase (type IV) from Worthington, and bovine serum albumin (BSA fraction V, essentially fatty acid-free) from Sigma. GeneScreen nylon membranes for RNA analysis were from DuPont NEN and random primed labeling kit for cDNA probe labeling from Life Technologies, Inc. Protran nitrocellulose membranes for protein analysis were from Schleicher & Schuell and bicinchoninic acid (BCA) protein assay from Pierce. The enhanced chemiluminescence detection kit for Western blot (ECL) and horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin antibody were from Amersham Corp. Non-esterified fatty acid enzymatic detection kit (NEFA PAP) used for fatty acid concentrations determination was from Biomerieux and antibodies for the insulin radioimmunoassay from Linco Research Inc.
Islet IsolationPancreatic islets were isolated from male Sprague-Dawley rats weighing about 200 g. Islets were isolated following digestion of total pancreas with collagenase and subsequent separation of the total digested pancreas on a discontinuous Ficoll gradient, according to the method of Gotoh et al. (29).
Islet CultureIslets were kept in culture at 37 °C in a
humidified atmosphere containing 5% CO2. On day 0 (just
after the isolation), islets were resuspended at 15-20 islets/ml in
RPMI 1640 medium (11 mM glucose), supplemented with 10%
fetal calf serum, 10 mM Hepes, pH 7.4, 1 mM
sodium pyruvate, and 50 µM -mercaptoethanol. On day 1, medium was changed to RPMI 1640 containing 2.8 mM glucose, and on day 2 islets were placed in RPMI containing either 2.8, 5.6, or
30 mM glucose, in the presence of different concentrations of palmitic acid or dexamethasone, for the indicated periods of time.
Palmitic acid was prepared as a 8 mM solution in Hepes-buffered Krebs-Ringer bicarbonate buffer, pH 7.4 (KRBH) containing 10% bovine serum albumin (essentially fatty acid-free) (final fatty acid/BSA molar ratio: 5.7); fatty acid was equilibrated with BSA overnight at 37 °C and filtered before use. In control conditions, KRBH, 10% BSA was added to a final concentration of 5%. Free fatty acid concentrations in the medium were checked with a non-esterified fatty acid enzymatic detection kit (NEFA PAP).
Dexamethasone and RU-486 stock solutions were prepared in absolute ethanol and added to the medium at final ethanol concentration comprised between 0.1 and 1%.
RNA Extraction and Northern Blot AnalysisTotal RNA was
prepared from 40 islets, in the presence of 20 µg of yeast tRNA as
carrier, according to the acid guanidinium thiocyanate/phenol-chloroform extraction method (30). After separation
by electrophoresis on 1.2% agarose gels containing 2% formaldehyde
and transfer to nylon membranes either overnight by capillary action,
or for 2 h using a vacuum blotter, specific mRNAs were
detected following prehybridization of the filters for 4 h at
42 °C in 50% formamide, 5 × SSC (SSC: 150 mM
NaCl, 17 mM sodium citrate, pH 7.0), 0.1 M
Na2HPO4/NaH2PO4, pH
6.5, 10 mM EDTA, 1% sodium dodecyl sulfate, 5 × Denhardt's (Denhardt's: 0.02% Ficoll 400, 0.02%
polyvinylpyrrolidone, 0.02% bovine serum albumin), and 0.1 mg/ml yeast
tRNA and hybridization overnight in the same buffer with
106 cpm/ml of a 32P-radiolabeled probe.
cDNAs fragments used as probes were a 1.4-kilobase pair
EcoRI fragment from plasmid pLGT-1 for GLUT2 (31), a
0.5-kilobase pair EcoRI fragment from pMSVTKpUChPPI-1 (gift
from K. Docherty, University of Aberdeen) for insulin mRNA, and a
0.8-kilobase pair BamHI-HindIII fragment from
-actin-pGEM for actin mRNA (32). Filters were exposed at
80 °C with an intensifying screen for 2 to 4 days to Kodak X-AR
films.
Islets were lysed in a buffer
containing 80 mM Tris, pH 6.8, 5 mM EDTA, 5%
SDS, 2 mM N-ethylmaleimide, 2 mM
phenylmethylsulfonyl fluoride, and sonicated 1.5 min in the cup of a
sonicator (33). Proteins were quantitated by the BCA assay, using
bovine serum albumin as a standard, and 5 µg (except otherwise
specified) were analyzed on SDS-containing 10% polyacrylamide gels.
The proteins were then transferred to nitrocellulose filters and the
transporter detected with a rabbit antibody raised against a peptide
corresponding to amino acids 513-522 of rat GLUT2 (31) (dilution
1:2,000) and a horseradish peroxidase-coupled donkey anti-rabbit
immunoglobulin antibody (dilution 1:8,000). Detection was with the
enhanced chemiluminescence detection technique. For the subunit of
the Na+/K+-ATPase, an antibody raised against
the purified
subunit of the Bufo marinus
Na+/K+-ATPase was used (34).
For pulse-chase experiments, islets
were first treated for 48 h with or without 1 µM
dexamethasone. After washing twice with PBS, they were incubated 30 min
in RPMI 1640 medium depleted of methionine and complemented with 10%
dialyzed fetal calf serum and labeled with 80 µCi/ml
[35S]methionine for 5 min at 37 °C. Cells were then
washed twice with PBS and lysed in PBS containing 1% Triton X-100 and
5 mM EDTA for 10 min at 4 °C. Nuclei and cells debris
were pelleted by a 15-min centrifugation at 13,000 rpm in a tabletop
centrifuge, and the supernatant was recovered. Incorporated
radioactivity was quantitated by trichloroacetic acid precipitation,
and samples containing equivalent amounts of radioactivity were
immunoprecipitated overnight at 4 °C with 3 µl of each of two
anti-GLUT2 antibodies, raised against peptides corresponding to amino
acids 513-522 and 47-60 of the rat GLUT2, as described (33).
Immunoprecipitates were collected with 30 µl of protein A-Sepharose
beads for 20 min at room temperature. After washings, they were
resuspended in sample buffer and analyzed on SDS-containing 7.5%
polyacrylamide gel, exactly as described (33). Gels were then treated
15 min in glacial acetic acid, 30 min in diphenyloxazol 10% in acetic acid, and washed 30 min in H2O before being dried and
exposed to x-ray films at 70 °C.
For determination of GLUT2 half-life, islets were first treated with or without 1 µM dexamethasone for 24 h. Islets in groups of ~200 were then pulse-labeled as described above except that the pulse was for 3 h in the presence of 200 µCi/ml [35S]methionine. Islets were then washed and either lysed directly or returned to the normal culture medium containing 2 mM cold methionine and chased at 37 °C for 6, 12, or 24 h with or without dexamethasone. Islets were lysed in 100 µl of a buffer consisting of 1% SDS in PBS and protease inhibitors. After further dilution of the lysate in 400 µl of PBS containing 1.25% Triton X-100, GLUT2 was immunoprecipitated from identical amounts of total cellular proteins and analyzed by gel electrophoresis as described above. Quantitation of band intensity was by laser scanning densitometry.
Islets PerifusionAfter incubation of the islets for
48 h in the presence of 0.6 mM palmitic acid and/or 1 µM dexamethasone as mentioned above, batches of 10 islets
were prepared and placed in a perifusion chamber. The perifusion buffer
was a Krebs-Ringer solution containing 0.5% bovine serum albumin, and
the flow rate was adjusted at 1 ml·min1. Perifusion
experiments consisted in a 40-min equilibration period in the presence
of 2.8 mM glucose before switching the glucose concentration to 16.7 mM. Fractions were collected every
minute. Insulin was then quantitated by radioimmunoassay, using rat
insulin as a standard.
Results are presented as mean ± S.E.. Statistical differences were analyzed by the Student's t test.
Pancreatic
islets were kept in tissue culture for 48 h in the presence of 2.8 mM glucose or 30 mM glucose and increasing
concentrations of palmitic acid. Total RNA was isolated from batches of
40 islets, and GLUT2 and actin mRNA levels were evaluated by
Northern blot analysis. Quantitation of GLUT2 was always expressed as
the ratio of GLUT2 to actin mRNA. Fig. 1A
shows that increasing the glucose concentration from 2.8 to 30 mM led to an increase in GLUT2 mRNA, as expected (35,
36), and that addition of 0.6 mM palmitic acid induced a
decrease in GLUT2 mRNA. Fig. 1B shows a quantitation of
the time-dependent modulation of the GLUT2 to actin ratio
over a 6-day period. Maximal reduction was already reached after 1 day.
Fig. 1C shows the dose-dependent effect with a
maximal reduction observed in the presence of 0.6 mM
palmitic acid. Decreases in GLUT2 mRNA were, however, not
correlated with any consistent reduction in GLUT2 protein expression
either in dose response or in time course experiments (not shown).
Dexamethasone Effect on GLUT2 mRNA and Protein
Exposure
of isolated islets to dexamethasone concentrations ranging from 1 nM to 1 µM for 48 h did not
significantly alter GLUT2 mRNA levels (Fig. 2,
A and B) except for an apparently significant increase at 10 nM (142.6 ± 16.2%, mean ± S.E.
of control value (n = 4), p < 0.05).
At the protein level, however, dexamethasone induced a strong
decrease in GLUT2 expression (Fig. 3, A and
B). A significant effect was already observed at 10 nM, and the maximal inhibitory effect was reached at 1 µM dexamethasone (34.9 ± 10.7% of the control
value (n = 5)). Time course experiments showed that
this maximal effect was already observed after 24 h (Fig. 4, A and B).
Dexamethasone is thought to exert its cellular effects by binding to
and activating glucocorticoid receptors. Activation of these receptors
can be inhibited by the antagonist RU-486. We thus assessed whether the
effect of 0.1 µM dexamethasone could be blocked by
increasing concentrations of RU-486. Fig. 5 shows that
the decreased expression of GLUT2 could indeed be completely prevented
by RU-486.
To determine whether the effect of dexamethasone was dependent on the
presence of high glucose concentrations in the medium, we incubated
islets in the presence of 1 µM dexamethasone and different glucose concentrations and measured GLUT2 expression by
Western blot analysis. At 2.8 mM glucose dexamethasone had no effect on GLUT2 protein expression (109.9 ± 21.5% of control value (n = 7)), but at higher glucose concentrations,
dexamethasone induced a decrease in GLUT2 protein: 67.7 ± 9.2%
of control at 5.6 mM glucose (n = 6) and
42.6 ± 9.2% of control at 30 mM glucose (n = 7) (Fig. 6).
Since dexamethasone decreased GLUT2 protein expression without
modifying mRNA levels, we next determined whether the observed effect was at the translational or posttranslational level by performing biosynthetic labeling experiments. Islets previously treated
for 48 h with 1 µM dexamethasone were pulse-labeled
for 5 min with [35S]methionine, lysed, and GLUT2 was
immunoprecipitated and analyzed by gel electrophoresis. In parallel, an
aliquot of the biosynthetically labeled islets was directly lysed in
the electrophoresis sample buffer for quantitative analysis of GLUT2 by
Western blot analysis. These experiments showed that GLUT2 synthesis
rate was only slightly decreased (81.2 ± 5.9% of control
(n = 3)), while at the same time total GLUT2 levels
were decreased to 35.2 ± 5.7% of control (n = 2)
(Fig. 7, A and B). This indicated
that dexamethasone had little effect on GLUT2 translational rate. To
assess whether the half-life of GLUT2 was decreased, batches of 200 islets were first treated with or without 1 µM
dexamethasone for 24 h and then pulse-labeled for 3 h with
[35S]methionine. At the end of the pulse the islets were
washed and either lysed or returned to a normal culture medium
containing an excess of cold methionine and incubated at 37 °C for
6, 12, or 24 h with or without dexamethasone. After
immunoprecipitation and separation by gel electrophoresis, GLUT2 was
quantitated by laser scanning densitometry. Fig. 7C shows
that the half-life of GLUT2 was decreased from 20 to 10 h in the
presence of dexamethasone, indicating a major effect of glucocorticoids
on transporter stability.
Finally, to determine whether the dexamethasone effect was specific for
GLUT2, we evaluated the expression of the subunit of the
Na+/K+-ATPase. Fig. 8 shows that
at the maximal dexamethasone concentration tested, expression of this
protein was not decreased but rather increased by dexamethasone
treatment, suggesting that the effect of dexamethasone was not due to a
general effect on membrane proteins.
Combined Effect of Palmitic Acid and Dexamethasone on GLUT2 mRNA and Protein
The combined effect of dexamethasone and
palmitic acid was tested in 48-h incubations of islets with 0.6 mM palmitate plus increasing dexamethasone concentrations.
At the mRNA level, the presence of dexamethasone at 0.1 µM increased the inhibitory effect of palmitic acid,
leading to a decrease in GLUT2 mRNA levels from 65.6 ± 2.8%
of control levels (n = 5), in the presence of palmitic acid alone, to 41.5 ± 8.1% of control levels (n = 5), in the presence of palmitic acid and dexamethasone
(p < 0.01) (Fig. 9, A and
B). Combination of palmitic acid and dexamethasone led to a
decrease in GLUT2 protein down to 24.6 ± 5.3% of the control at
1 µM dexamethasone (n = 4), which was not
significantly different from the effect of dexamethasone alone (see
Fig. 3).
Insulin Secretion, Islet Insulin Content, and Insulin mRNA
The effect of dexamethasone and/or palmitic acid on
insulin secretion was assessed in islet perifusion experiments. Islets treated for 48 h with 0.6 mM palmitic acid and/or 1 µM dexamethasone showed no change in basal insulin
secretion compared with control islets. However, a 73.1 ± 10.0%
(n = 2) inhibition of the glucose-induced insulin
secretion was observed following dexamethasone treatment, a 72.1 ± 7.7% (n = 2) decrease following palmitic acid
treatment, and an 81.8 ± 2.8% (n = 2) decrease
when both treatments were combined (Fig. 10,
A-C).
The insulin content of islets treated for 48 h with 0.6 mM palmitic acid and/or 1 µM dexamethasone was then measured. Palmitic acid, with or without dexamethasone, induced a decrease in insulin content which reached 46.7 ± 2.8% for palmitic acid alone (n = 2) and 43.3 ± 13.7% for palmitic acid plus dexamethasone (n = 2), compared with control values. On the opposite, dexamethasone treatment increased insulin content to 177.2 ± 17.0% (n = 4) of the control islets.
Islet insulin mRNA levels following palmitic acid and/or dexamethasone treatment were measured by Northern blot analysis. Dexamethasone, present at concentrations from 1 nM to 1 µM, did not induce any change in insulin mRNA, except possibly at 1 µM, where a small increase was observed: 118.4 ± 5.0% of the control (n = 3) (p < 0.05 versus control). On the opposite, islets exposed to palmitic acid exhibited a decrease in insulin mRNA down to 66.4 ± 12.9% of control at 0.6 mM palmitic acid (n = 6). Addition of different concentrations of dexamethasone did not alter palmitic acid effect.
In the present study, we demonstrated that free fatty acids and the synthetic glucocorticoid dexamethasone down-regulate GLUT2 expression in isolated pancreatic islets. Whereas palmitic acid induced a decrease in GLUT2 mRNA levels, it did not induce consistent changes in GLUT2 protein expression. In contrast, dexamethasone induced a strong decrease in GLUT2 protein levels but no change in mRNA levels. Both substances, however, displayed a very strong inhibitory action on glucose-induced insulin secretion.
Free fatty acids are elevated in both type I and type II diabetes and
have been shown to have a number of negative effects on insulin
sensitivity of peripheral tissues and function of pancreatic cells.
The present experiments were undertaken to determine whether palmitic
acid could have a role in the control of GLUT2 expression in addition
to its inhibitory effect on GSIS. The effect of palmitic acid was
detectable only on the regulation of transporter mRNA levels, and
no consistent changes could be observed at the protein level. This
indicates that although free fatty acids are able to induce glucose
unresponsiveness in
cells, in agreement with previously published
work (12-14), they are certainly not the only factor inducing the
dysfunction of these cells in diabetes, since GLUT2 levels are
unaffected.
Dexamethasone effect on islet function as observed in the present
experiment is at least 2-fold: a strong reduction in GLUT2 protein
expression and a severe inhibition of GSIS. The decrease in GLUT2
protein expression is relatively rapid, occurring within 24 h of
exposure to dexamethasone. Strikingly, there is no parallel decrease in
mRNA levels. This, therefore, indicates that the regulation of
transporter expression is at the translational or posttranslational level. Our pulse-labeling experiments demonstrated only a minimal decrease in the rate of transporter translation, suggesting that the
regulation was at a posttranslational level. This was indeed directly
demonstrated in pulse-chase experiments, which showed a 50% decrease
in GLUT2 half-life, induced by glucocorticoid treatment. Since the
effect of dexamethasone could be inhibited by the glucocorticoid receptor antagonist RU-486, transcriptional activation of a gene or a
set of genes is required to increase the rate of GLUT2 degradation. Although we do not know which gene products are responsible for stimulating transporter degradation, possible candidates include components of the ubiquitin-proteasome degradation system. Indeed, in
muscle, dexamethasone has been shown to activate the
energy-dependent protein degradative system and the
expression of ubiquitin (37). Although in this report degradation of
myofibrillar proteins was assessed, it is known that membrane proteins
such as CFTR can also be degraded by the proteasome-ubiquitin system
(38, 39). Whatever degradative system is induced, it must display a
selectivity for the transporter, since another membrane protein, the
subunit of the Na+/K+-ATPase, was not
decreased in the presence of dexamethasone. Another interesting
observation is that degradation of GLUT2 induced by dexamethasone was
more pronounced in the presence of high glucose concentrations. The
effect of dexamethasone cannot simply be explained as an inhibition of
the glucose effect, since it has been demonstrated that the increase in
GLUT2 expression induced by glucose is due to transcriptional
activation of its gene (35, 36), whereas the effect of dexamethasone on
GLUT2 is at the posttranslational level. Activation of the
degradative system is thus both glucose- and
dexamethasone-dependent.
When added to islets in the presence of palmitic acid, dexamethasone increased the inhibitory action of fatty acids on GLUT2 mRNA. It might then be postulated that the effect of fatty acids is increased by dexamethasone by a mechanism involving interaction at the GLUT2 promoter of glucocorticoid receptors and fatty acid-activated transcription factors such as the peroxisome proliferator-activated receptors or stimulation of peroxisome proliferator-activated receptors expression by dexamethasone (40).
Insulin Secretion, Islets Insulin Content, and Insulin mRNADexamethasone and fatty acids, alone or in combination,
had a negative impact on the first and second phases of glucose-induced insulin secretion. This has been demonstrated previously for fatty acids (13, 14, 18). Here, however, we did not observe the increase in
basal secretory activity reported in these preceding studies. We,
however, observed a decrease in proinsulin mRNA levels and in total
insulin content. The inhibitory effect of dexamethasone correlated with
an increase in total insulin content and in the absence of a reduction
in proinsulin mRNA. The mechanism by which fatty acids and
dexamethasone exert their inhibitory effect on GSIS is not known. For
fatty acids, a decrease in pyruvate dehydrogenase and increase in
pyruvate dehydrogenase kinase has been reported which could result in
impaired glucose signaling (15). For dexamethasone, an increase in
glucose-6-phosphatase, which increases glucose cycling in cells,
may reduce the glucose signaling pathway (41, 42). Dexamethasone has
also been demonstrated to increase islets neuropeptide Y content and
secretion (43). This peptide, by binding to specific
Gi-coupled receptors present on
cells, has been shown
to have an inhibitory action on insulin secretion (44). In addition,
the activation of a proteolytic activity, as demonstrated in the
present study, may also lead to the degradation of essential components
of the insulin granules exocytic machinery.
Together, our data show that in addition to fatty acids, dexamethasone
has profound effects on the function of isolated pancreatic cells.
The observed decreased expression of GLUT2 is due to the induction of a
protein degradative system, which is better induced in hyperglycemic
conditions and which shows apparent specificity for the transporter
when compared with the
subunit of the
Na+/K+-ATPase. The strong inhibitory action on
insulin secretion may be due to a combination of different causes,
including alterations in glucose metabolism, increased secretion of
neuropeptide Y, or degradation of key components of the exocytic
machinery. High glucocorticoid levels in the presence of hyperglycemia
may therefore have inhibitory effects on
cells functions that are
different from those reported in dexamethasone-induced insulin
resistance when normoglycemia is prevailing. These effects may explain
the decrease in GLUT2 expression observed for instance in db/db mice (6) which have high circulating levels of glucocorticoids (45).