1Department of Internal Medicine, Karolinska Institutet, Stockholm South Hospital, SE-118 83 Stockholm; 2Department of Medical Cell Biology, University of Uppsala, SE-751 23 Uppsala; 3Department of Molecular Medicine, The Rolf Luft Center for Diabetes Research, Karolinska Institutet, Karolinska Hospital, SE-171 76 Stockholm, Sweden; and 4Department of Pharmacology, College of Medicine, University of Tennessee, Memphis, Tennessee 38163
Submitted 10 February 2003 ; accepted in final form 6 April 2003
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ABSTRACT |
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diabetes mellitus; pancreatic islet; insulin secretion; sulfonylurea; protein kinase C; ATP-dependent K+ channels
Experiments have unveiled that glucose retains the ability to release
insulin even in the presence of maximally depolarizing concentrations of
K+ and diazoxide, an opener of ATP-regulated K+
(KATP) channels
(18,
25). Thus signaling molecules
other than ATP and Ca2+ may be involved in glucose
sensing in the -cell, although the precise nature of such signals has
remained elusive. Additionally, sulfonylureas promote insulin exocytosis from
permeabilized cells (14),
suggesting KATP-independent actions also of these drugs. Although
poorly defined, this occurs by effects exerted on the secretory machinery
itself not involving closure of KATP and initiation of
Ca2+-dependent electrical activity
(14). Moreover, sulfonylureas
stimulate insulin exocytosis in
-cells from sulfonylurea
receptor-deficient mice, an effect particularly pronounced at high
concentrations of glucose (Berggren P-O, unpublished observations).
Furthermore, reports show that >90% of glibenclamide-binding sites are
localized intracellularly in the -cell
(7,
31). Interestingly, under
chronic treatment, glibenclamide specifically and progressively accumulates in
islets in association with secretory granules and mitochondria and causes
long-lasting stimulation of insulin secretion
(21).
In this study, we set out to determine whether glibenclamide also, in part,
acts independently of the KATP-dependent pathway in the -cell
and may directly affect specific intracellular targets, controlling fuel
partitioning, to stimulate insulin exocytosis. In doing so, we focused on a
pivotal enzyme implicated in fuel partitioning, carnitine palmitoyltransferase
1 (CPT-1; palmitoyl-CoA:L-carnitine
O-palmitoyltransferase; EC 2.3.1.21
[EC]
), located in the outer
mitochondrial membrane (9).
CPT-1 is an important determinant of cellular fatty acid oxidative flux
(29,
32). The enzyme catalyzes
transfer of long-chain fatty acyl groups from coenzyme A to carnitine and is
inhibited by sulfonylureas in hepatocytes
(9,
29,
32). The clinical significance
of CPT is illustrated by the fact that CPT deficiency in humans causes fasting
hypoglycemia (6), although this
may in part be due to hepatic effects. Pioneering studies by McGarry
(29) in liver have elucidated
the role of CPT-1 in the regulation of hepatic fatty acid oxidation and
ketogenesis. His studies also identified malonyl-CoA as an important
physiological inhibitor of CPT-1 and an important element in the
carbohydrate-induced sparing of fatty acid oxidation
(29).
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MATERIALS AND METHODS |
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Islet cell preparation and culture. Pregnant Wistar rats,
purchased from B & K Universal (Sollentuna, Sweden), were killed by
cervical dislocation on day 21 of gestation and the fetuses rapidly
removed. Islets were prepared from pancreatic glands as previously described
(20,
39). Briefly, the pancreata
were finely chopped and digested for a short time with collagenase. The
carefully washed digest was plated in culture dishes allowing cell attachment
(Nunc, Roskilde, Denmark) and cultured for 5 days at 37°C in a humidified
atmosphere of 5% CO2 in ambient air in medium RPMI 1640 containing
11.1 mM glucose, 10% fetal calf serum, 2 mM L-glutamine, 100 U/ml
benzylpenicillin, and 0.1 mg/ml streptomycin. At the end of the culture
period, groups of islets were transferred to fresh medium containing 1% FCS
and cultured free-floating overnight, a procedure that minimizes fibroblast
proliferation. Spherical islets, free of connective tissue, were then selected
under a stereomicroscope and used for the different analyses listed below. In
each experiment, all test groups received the same amount of solvent used to
dissolve various drugs. We chose to use fetal islets to obtain islet tissue
enriched in -cells; previous studies have shown that fetal islets
contain 9095%
-cells, which display a full secretory response to
glucose after a 5-day culture period similar to that of the adult
-cell
(20,
39).
Analysis of -cell CPT-1 mRNA and enzyme activity.
Islet cell expression of CPT-1 mRNA was assessed by Northern blot analysis
essentially as described (4,
17).
Activity of CPT in islets was assayed by measuring the incorporation of tritium-labeled carnitine into acylcarnitine by using a modification of the procedure previously used for liver mitochondria (17). The low protein content of islet preparations in culture necessitated using high specific radioactivity and high concentration of carnitine. Islet samples of 0.20.3 mg of protein each were homogenized in 125 µl of medium containing 0.25 M sucrose, 1 mM EDTA, and 3 mM Tris (pH 7.2) by use of a micro dounce homogenizer. Whole homogenates were assayed for CPT activity. Each assay contained, in a total volume of 500 µl, 10 µg of islet protein, 82 mM sucrose, 70 mM KCl, 70 mM imidazole (pH 7.0), 1 mg of BSA, 2 mM L-carnitine (2 µCi/µmol L-[methyl-3H]carnitine), 0.5 µg of antimycin A, 100 µM myristoyl-CoA, 2 mM ATP, and 2 mM MgCl2. Reactions were carried out at 37°C for 20 min. Assays were linear with respect to time up to 35 min and were also linear with respect to protein in the range assayed.
Analysis of -cell fatty acid oxidation. Duplicate
groups of 25 islets, labeled overnight with [14C]palmitate (10
µCi/ml) to achieve sufficient uptake and steady state, were incubated in
small glass vials at 37°Cfor2hin100 µl of a Krebs-Ringer bicarbonate
buffer (26) supplemented with
10 mM HEPES (KRBH), 3 or 20 mM D-glucose, glibenclamide, or
etomoxir. For measurements of fatty acid oxidation, reactions were terminated
by the addition of 100 µl of 0.05 mM antimycin A in ethanol. The
14CO2 formed was released from incubation medium by the
addition of 100 µl of 0.4 M Na2HPO4 (pH 6.0) and
trapped in 250 µl of hyamine. After overnight postincubation, the
radioactivity was measured by liquid scintillation counting.
Lipid extraction and quantification of diacylglycerol. Groups of
250300 islets were cultured free-floating overnight in RPMI 1640 medium
supplemented with 1% fetal calf serum. Islets were preincubated for 45 min at
37°C in KRBH buffer. They were then swiftly transferred to Eppendorf tubes
containing 1 ml KRBH buffer (prewarmed to 37°C) supplemented with 40 µM
glibenclamide. Islets were then incubated for the indicated time period,
rapidly pelleted, and quickly rinsed once in ice-cold PBS. Tubes were then
immediately plunged into liquid nitrogen and kept frozen at -80°C pending
further analysis of their diacylglycerol (DAG) content. After samples on ice
were thawed, islets were sonicated in a 500-µl extraction solution
consisting of chloroform-methanol-HCl (100:100:1, vol/vol/vol) and 100 µl
of PBS with 10 mM EDTA. After centrifugation (5 min, 12,000 g), the
aqueous phase was removed and reextracted with 100 µl of chloroform, which
were added to the organic phase. The combined chloroform phases were
evaporated under a stream of liquid nitrogen and resolubilized in 50 µl of
the chloroform solution. This solution was reextracted with 10 µl of PBS
with 10 mM EDTA and then reevaporated. Samples were then stored at -80°C
under nitrogen until analyzed for DAG. 1,2-DAGs were quantified as described
(39). Briefly, dried lipids
were solubilized in 20 µl of an
octyl--D-glucoside-cardiolipin solution (7.5%
octyl-
-D-glucoside, 5 mM cardiolipin in 1 mM
diethylenetriaminepentaacetic acid) by sonication in a bath sonicator. The
reaction was then carried out in 100 µl containing 20 µl of sample
solution, 50 mM imidazole HCl (pH 6.6), 50 mM NaCl, 12.5 mM MgCl2,
1 mM EGTA, 2 mM DTT, 6.6 µg DAG kinase, and 1 mM
[
-32P]ATP for 30 min at room temperature. Lipids were
extracted and evaporated as above. Samples were then run on Kieselgel 60
plates activated by preheating at 120°C. Plates were developed with
chloroform-methanol-acetic acid (65:15:5, vol/vol/vol) and subjected to
autoradiography. Standard samples of D-1,2-dipalmitine were run in
parallel. The intensities of the spots corresponding to phosphatidic acid were
quantified using densitometry and are expressed as arbitrary units (optical
density).
PKC translocation assay. Groups of 100 cultured islets were incubated for 30 min in RPMI 1640 medium plus 10% FCS with the various additions indicated in Fig. 5. The islets were quickly washed with cold PBS and used directly for preparation of membrane and cytosol fractions as described by Alcázar et al. (1). Membrane and cytosol proteins were separated on a 9% SDS-PAGE gel and transferred to a nitrocellulose membrane. The membranes were incubated with mouse anti-PKC monoclonal antibody (MC5, Santa Cruz Biotechnology, Santa Cruz, CA) followed by incubation with a horseradish peroxidase-linked secondary antibody. Antibody binding was visualized using the enhanced chemiluminescence immunoblotting detection system (Amersham International). Band intensities were quantified by densitometry, and the results are expressed as percentage of the membrane-to-cytosol ratio of control islets.
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Insulin exocytosis determinations. Duplicate groups of 10 islets were preincubated for 45 min in KRBH buffer (26) containing 3 mM glucose at 37°C. They were then incubated for another 30 min in fresh medium, supplemented as indicated in Fig. 6. To circumvent any influence of KATP channels on membrane potential and cytosolic Ca2+ concentration, cells were incubated under Ca2+-clamped conditions, i.e., in the presence of 20 mM glucose, 25 mM KCl, and 400 µM diazoxide (18). The insulin concentration in incubation medium was analyzed radioimmunologically (22). Dextrancoated charcoal in 0.2 M glycine buffer was used to separate bound and free insulin. Interassay coefficient of variation (CV) was 2.8% at 21 µU/ml insulin and 2.3% at 104 µU/ml (n = 28, duplicates). Intra-assay CV was 1.2% at 20 µU/ml insulin and 4.0% at 123 µU/ml (n = 16, duplicates). Standard curve range was 3.9250 µU/ml insulin.
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Statistical analysis. Results presented are derived from independent experiments performed on different days. Means ± SE were calculated and groups of data compared using Student's t-test.
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RESULTS |
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Glibenclamide dose-dependently inhibits CPT enzymatic activity. As
shown in Fig. 2A,
glibenclamide and malonyl-CoA dose-dependently inhibit CPT enzymatic activity
in islet homogenates, the apparent IC50 occurring at 50 µM
for glibenclamide and at 25 µM for malonyl-CoA. It should be noted that the
enzyme assays were performed in the presence of albumin (2 mg/ml), which
tightly binds glibenclamide. This was done because CPT enzyme activity
measurements require the addition of protein because of the minute amount of
tissue available. Hence, the plots grossly underestimate the inhibitory
potency of glibenclamide (
10-fold on the basis of assays of recombinant
CPT in the presence and absence of BSA;
Fig. 2B). Another
important implication of this study is that glibenclamide inhibits both CPT-1
and CPT-2 in islets and that each enzyme is identically sensitive because it
binds at the active site, not the malonyl-CoA site. Therefore, when 5 µM
free glibenclamide inhibits CPT-1 by 50% and CPT-2 by 50%, the total effect on
the pathway is inhibition of >50%, because two enzymes in the same pathway
are being inhibited. This can be seen from the plot
(Fig. 2C) in which
extrapolation of inhibition to infinite concentration leads to 100% inhibition
of CPT activity (CPT-1 and CPT-2 inhibited) by glibenclamide, whereas
extrapolation of inhibition by malonyl-CoA to infinitely high concentration
leads to only
50% inhibition (only CPT-1 inhibited). Another
KATP channel-blocking insulin secretagogue, repaglinide, was also
tested and was found not to affect CPT enzymatic activity at 10 or 50 µM
(data not shown). Repaglinide possesses many of the physicochemical properties
of glibenclamide but does not affect KATP-independent insulin
secretion.
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Glibenclamide inhibits islet fatty acid oxidation. As shown in
Fig. 3, exposure to
glibenclamide (at 3 mM glucose) for 90 min causes a substantial suppression of
the oxidation of endogenous fatty acids in islets prelabeled with
[14C]palmitate, amounting to an 40% reduction of control
values. Such a reduction of mitochondrial
-oxidation is expected from
the finding of CPT inactivation by glibenclamide. This inhibitory effect is
comparable in magnitude to that caused by high glucose. Additionally, the
specific CPT-1 inhibitor etomoxir (in 3 mM glucose), serving as a positive
control, causes an
70% reduction in the rate of palmitate oxidation.
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Rapid DAG formation by glibenclamide exposure. Reducing CPT-1 activity, and thereby fatty acid oxidation, with glibenclamide should lead to a shunting of acyl-CoA esters to lipid esterification products. That this indeed occurs in the sulfonylurea-exposed islets is amply illustrated in Fig. 4. Thus glibenclamide stimulation of intact islets results in a robust and transient accumulation of DAG. The effect is significant already at 10 min, being maximally sixfold enhanced at 30 min and leveling off by 60 min of glibenclamide exposure.
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Glibenclamide translocates PKC to membranes. To assess whether
accelerated DAG formation by glibenclamide is associated with a biological
action, we measured PKC activation (as estimated by its translocation from
cytosol to membranes) because DAG activates the classical isoforms of this
enzyme. Entirely consistent with the findings above, stimulating islets with
glibenclamide causes a dose-dependent activation of PKC, shown as
translocation of the enzyme from the cytosol to membranes
(Fig. 5, A and
B). At 5 µM, glibenclamide enhances PKC activity
approximately twofold, a figure that is further increased to threefold at 40
µM of the drug (Fig. 5, A and
B). In comparison, 10 nM of the phorbol ester TPA elicits
a 2.6-fold translocation of PKC. The PKC antibody used detects the classical
enzyme isoforms expressed in islets, i.e., ,
, and
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Glibenclamide-stimulated insulin exocytosis occurs partly through KATP-independent and PKC-dependent pathways. Figure 6 reports short-term experiments in which insulin exocytosis is measured in Ca2+-clamped conditions (i.e., presence of elevated glucose and K+) with the KATP channels bypassed (presence of diazoxide). Even under these Ca2+-clamped conditions, glibenclamide causes a robust 50% stimulation of insulin secretion at 20 mM glucose. A similar 150% increase is noted in response to the PKC-activating phorbol ester TPA (10 nM). Additionally, no additive effects between glibenclamide and TPA occur, arguing indirectly in favor of a common mechanism of action of these two agents. Etomoxir (50 µM), a specific CPT-1 inhibitor, stimulates insulin secretion to the same extent as glibenclamide (Fig. 6). Again, no additive effects between etomoxir- and glibenclamide-stimulated insulin release are detected. Finally, the coaddition of the PKC inhibitor H-7 (10 µM) completely prevents the insulinotropic actions of both TPA and glibenclamide (Fig. 6). Similar findings were obtained under Ca2+-clamped conditions at 3 mM glucose. Thus, in a separate series of experiments in 3 mM glucose, etomoxir (50 µM) stimulates insulin secretion (46 ± 7% over basal; n = 6, P < 0.05) to the same extent as glibenclamide (48 ± 6% over basal; n = 6, P < 0.05). Again, no additive effects between etomoxir- and glibenclamide-stimulated insulin release are detected (not shown). In their entirety, these findings indicate that glibenclamide can promote insulin secretion through KATP channel-independent mechanisms and that this stimulation is in part dependent on the activation of PKC elicited by the sulfonylurea after fat oxidation inhibition.
In an attempt to assess the quantitative importance of this novel
PKC-dependent and KATP-independent pathway in
glibenclamide-stimulated insulin exocytosis at different glucose
concentrations, we compared the extent to which glibenclamide promotes
exocytosis under normal vs. Ca2+-clamped conditions at
low and high glucose in a separate series of nine observations. Thus, at 3 mM
glucose, glibenclamide (40 µM) stimulated insulin exocytosis 602 ±
116%, a figure that was reduced to 120 ± 39% in the additional presence
of 25 mM KCl and 400 µM diazoxide. At 20 mM glucose, glibenclamide
stimulated insulin exocytosis 46 ± 7%, a figure that was reduced to 21
± 3% in the additional presence of 25 mM KCl and 400 µM diazoxide.
Hence, the results indicate that the KATP channel-independent
pathway accounts for 20% (120 vs. 602%) of the maximal insulin-releasing
capacity of glibenclamide at 3 mM glucose; an effect that increases to
45% (21 vs. 46%) in 20 mM glucose, figures that are in fair agreement
with capacitance measurements in mouse islets in a previous report
(14).
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DISCUSSION |
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We also noted a suppressed rate of fatty acid oxidation by both glucose and
glibenclamide, as expected from their inhibitory effect on CPT activity. When
fatty acid oxidation is decreased, the acyl-CoA esters are diverted to the
biosynthesis of esterified lipid products. Entirely consistent with this
scenario is our observation of a rapid and marked accumulation of DAG in
response to glibenclamide and the resultant PKC activation. With respect to
PKC action in the -cell, its activation by glucose-derived DAG or
phorbol esters may promote insulin exocytosis by controlling the
phosphorylation of several key proteins, e.g., voltage-dependent
Ca2+ channels
(2,
3). Previous reports indicate
that islet DAG mass is not affected by variations in
Ca2+
(12,
30,
40). Likewise, islet inositol
1,4,5-trisphosphate levels are not altered by artificially stimulating
Ca2+ influx by ionomycin or K+ or by blocking
it with EGTA (5,
42). Furthermore, islet PKC
activity is only minimally affected by changing Ca2+
from subnanomolar to submicromolar concentrations
(34). All these findings make
it unlikely that the observed increase in DAG levels and PKC activity by
glibenclamide would be secondary to Ca2+ influx
stimulated by the sulfonylurea. Additionally, any Ca2+
influence on DAG levels and PKC activity obviously has no functional
significance for insulin release, since glibenclamide (and TPA) evoked a
robust increase in insulin secretion even under
Ca2+-clamped conditions. Also, the secretory response to
glibenclamide under Ca2+-clamped conditions was
nonadditive with TPA and blocked by the PKC inhibitor H-7, thus clearly
indicating that the KATP-independent effect of glibenclamide on
insulin exocytosis is PKC mediated and not affected by
Ca2+.
The clinical significance of CPT is illustrated by the fact that CPT
deficiency in humans causes fasting hypoglycemia
(6). Additional evidence
supporting the view that CPT inhibition is linked to -cell stimulation
by glucose can be derived from the fact that, when insulin secretion is
suppressed, such as during chronic hyperlipidemia, the CPT-1 gene is
upregulated in association with enhanced fat oxidation in the
-cell
(4). The importance of CPT-1 in
-cell glucose signaling is further underscored by the finding that
blocking the penultimate step (ATP-citrate lyase) in malonyl-CoA synthesis
curtails glucose-sensitive insulin release
(8,
15). Conversely, CPT-1
overexpression in INS-1 insulinoma cells not only results in exaggerated fatty
acid oxidation rates but also impaired glucose-stimulated insulin secretion
(37).
Our findings are compatible with the view that glibenclamide, through its intracellular inactivation of CPT-1, in part promotes insulin exocytosis via KATP-independent and PKC-dependent pathways. Also consistent with this view is the observation that the specific CPT-1 inhibitor etomoxir caused similar effects to glibenclamide on insulin secretion and fat oxidation and that their effects are not additive. This new KATP-independent effect of glibenclamide is quantitatively significant and glucose dependent. Differences in lipophilicity and uptake rates may affect the timing by which the different agents activate PKC. Thus, if TPA (the more lipophilic drug) activated PKC faster than glibenclamide (as one would expect), it would trigger insulin exocytosis more rapidly and give rise to a larger amount of insulin being released over 30 min, as was observed in this case. Glibenclamide seems to be exceptional among the sulfonylureas in that it specifi-cally and progressively accumulates in islets and associates with secretory granules and mitochondria, causing long-lasting stimulation of insulin secretion (21). It is conceivable that the inactivation of CPT-1 may explain the KATP-independent and PKC-dependent insulin secretion by sulfonylurea reported previously (14, 35). This possibility is also in accord with previous reports indicating a hypoglycemic effect of fatty acid oxidation inhibitors in vivo (16). It was recently shown that acyl-CoA activates atypical forms of PKC (43), which may be consistent with our present findings of PKC involvement in glibenclamide-stimulated secretion. Nonetheless, a direct stimulatory effect of long-chain acyl-CoA esters on the exocytotic process (11) may also be involved in the effects of glibenclamide noted herein. Additionally, a direct effect of sulfonylurea on PKC activity was ruled out in a previous study (41).
In hepatocytes, the sensitivity of CPT-1 to malonyl-CoA inhibition is
increased by insulin and decreased in diabetes
(9,
17). Whether a reduced
sensitivity of CPT-1 to malonyl-CoA inhibition in the -cell, resulting
in decreased DAG production and deficient PKC activation, may contribute to
the impaired glucose-stimulated insulin secretion characterizing human type 2
diabetes mellitus is an obvious possibility that remains to be tested.
The physiological significance of our results remains to be determined. At a first glance, it would seem that the submicromolar therapeutical concentrations of glibenclamide maximally achieved in the postabsorptive state (23, 24) would not be sufficient to affect the islet CPT system in diabetic patients (Fig. 2). Interestingly, however, under chronic treatment, glibenclamide progressively and selectively accumulates in islets and is slowly cleared from islets on drug withdrawal (21). Additionally, there are large interindividual variations in the pharmacodynamics and pharmacokinetics of glibenclamide, which are furthermore greatly influenced by genetic polymorphisms of the cytochrome P-450 2C9 system (24). Hence, carriers of certain CYP2C9 genotype variants show markedly reduced clearance rates of glibenclamide, resulting in severalfold-elevated serum concentrations of the drug (24). There are also several important drug interactions that may additionally elevate glibenclamide serum levels, which may be significant in elderly diabetic patients who are often on multiple medications and have impaired drug metabolism (23, 27). Add to this the high levels normally achieved of biologically active and long-acting glibenclamide metabolites that may also impact the CPT system (23). Thus it cannot be excluded that the inhibitory effect of glibenclamide on islet CPT activity described here may contribute to the sustained hypoglycemic effect of the drug observed in some diabetic patients, even after drug removal for 2448 h, to which no explanation has been given to date. Repaglinide, a KATP channel-blocking insulin secretagogue possessing many of the physicochemical properties of glibenclamide but without affecting KATP-independent insulin secretion, did not affect CPT activity. We went on to test all the sulfonylureas that we could obtain and found that, for each drug, the potency of inhibition of CPT corresponded to that drug's potency as an effective antidiabetic agent (not shown). In Ref. 9, we showed inhibition by glibenclamide and tolbutamide with the most potent acting sulfonylurea on insulin secretion, glibenclamide, being the more potent CPT inhibitor. The inactive metabolite of tolbutamide carboxytolbutamide is not a CPT inhibitor (9).
Furthermore, the specific CPT-1 inhibitor etomoxir stimulates insulin secretion nonadditively with glibenclamide both in our hands (Fig. 6) and in previous reports from other groups (8, 44). Moreover, etomoxir elevates DAG contents (19), adding further credence to our concept. Glibenclamide also inhibits CPT-1 enzymatic activity in the liver (9). Whether liver output of lipids is stimulated by glibenclamide in vivo is difficult to address conclusively, because such an effect may be counteracted systemically by the portal delivery of insulin elicited by glibenclamide-induced insulin release from the pancreas occurring when the drug is administered in vivo.
In conclusion, we suggest a model in which islet -cell CPT-1 activity
is reduced by glibenclamide, thereby diverting fatty acid metabolism from
mitochondrial oxidation to the biosynthesis of DAG, which causes
KATP-independent and PKC-dependent exocytosis of insulin. We
suggest that chronic CPT inhibition, through the progressive islet
accumulation of glibenclamide, may explain the prolonged stimulation of
insulin secretion in some diabetic patients, even after drug removal, that
contributes to the sustained hypoglycemia of the sulfonylurea. Whether this
mechanism also results in lipid overload in the
-cell, causing
-cell lipoapoptosis (13,
32) explaining the clinical
phenomenon of "secondary failure" to sulfonylureas
(27), is currently being
evaluated in our laboratory.
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DISCLOSURES |
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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. Section 1734 solely to indicate this fact.
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REFERENCES |
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