Glucagon-Like Peptide 1 Stimulates Lipolysis in Clonal Pancreatic ß-Cells (HIT)
Gordon C. Yaney,
Vildan N. Civelek,
Ann-Marie Richard,
Joseph S. Dillon,
Jude T. Deeney,
James A. Hamilton,
Helen M. Korchak,
Keith Tornheim,
Barbara E. Corkey, and
Aubrey E. Boyd, III
From the Obesity Research Center (G.C.Y., V.N.C., A.-M.R, J.T.D., J.A.H.,
K.T., B.E.C.), Evans Department of Medicine, and the Departments of
Biochemistry (B.E.C., K.T.) and Biophysics (J.A.H.), Boston Medical Center,
Boston, Massachusetts; the Division of Endocrinology (J.S.D.), University of
Iowa School of Medicine, Iowa City, Iowa; and the Immunology Division
(H.M.K.), Children's Hospital of Philadelphia, Philadelphia,
Pennsylvania.
Address correspondence and reprint requests to Dr. Barbara E. Corkey, Obesity
Research Center, EBRC-808, Boston Medical Center, 650 Albany St., Boston, MA
02118. E-mail:
bcorkey{at}med-med1.bu.edu
.
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ABSTRACT
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Glucagon-like peptide 1 (GLP-1) is the most potent physiological incretin
for insulin secretion from the pancreatic ß-cell, but its mechanism of
action has not been established. It interacts with specific cell-surface
receptors, generates cAMP, and thereby activates protein kinase A (PKA). Many
changes in pancreatic ß-cell function have been attributed to PKA
activation, but the contribution of each one to the secretory response is
unknown. We show here for the first time that GLP-1 rapidly released free
fatty acids (FFAs) from cellular stores, thereby lowering intracellular pH
(pHi) and stimulating FFA oxidation in clonal ß-cells (HIT).
Similar changes were observed with forskolin, suggesting that stimulation of
lipolysis was a function of PKA activation in ß-cells. Triacsin C, which
inhibits the conversion of FFAs to long-chain acyl CoA (LC-CoA), enhanced
basal FFA efflux as well as GLP-1-induced acidification and efflux of FFAs
from the cell. Increasing the concentration of the lipase inhibitor orlistat
progressively and largely diminished the increment in secretion caused by
forskolin. However, glucose-stimulated secretion was less inhibited by
orlistat and only at the highest concentration tested. Because the acute
addition of FFAs also increases glucose-stimulated insulin secretion, these
data suggest that the incretin function of GLP-1 may involve a major role for
lipolysis in cAMP-mediated potentiation of secretion.
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INTRODUCTION
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Glucagon-like peptide 1 (GLP-1) is the most potent potentiator of
glucose-induced insulin secretion that has been described
(1,2).
This peptide causes the elevation of cAMP and the activation of protein kinase
A (PKA) (3); however, it
releases insulin only in the presence of stimulatory glucose
(4) and thus serves as an
incretin rather than a secretagogue
(5). Activation of PKA leads to
phosphorylation of multiple ß-cell proteins, many of which have been
hypothesized to play a role in insulin secretion
(6,7,8).
The nature of the endogenous substrates for PKA that may potentiate insulin
secretion is unknown. Because the islet contains large stores of triglycerides
(9), particularly in diabetes
(10), another possible role of
the normal rise in cAMP could be to stimulate lipolysis (via lipase
activation), thereby providing the cell with free fatty acids (FFAs). Recent
research on hormone-sensitive lipase (HSL) in ß-cells yielded results
consistent with that notion
(11). The released FFAs may
directly effect secretion, or they may do so indirectly via generation of
other lipids, including the putative long-chain acyl CoA (LC-CoA) signal,
diacylglycerol (DAG), and phosphatidic acid (PA)
(12). The acute addition of
exogenous FFAs is also known to enhance glucose-stimulated secretion
(9,13,14,15).
We have shown in previous studies that added FFAs cause acidification in
ß-cells (16) and fat
cells (17) as a consequence of
the flip-flop mechanism of diffusion across the plasma membrane
(16,17).
Furthermore, we have shown in adipocytes that the elevation of cAMP stimulates
lipolysis, with a resulting decrease in the intracellular pH (pHi)
caused by the release of FFAs, which become partially ionized
(17). Therefore, in this study
we assessed whether GLP-1 has a similar effect on lipolysis in ß-cells.
Our data show a decrease in pHi and a release of FFAs by agents
that increase cAMP, presumably via activation of HSL. Furthermore, the
incretin effect was largely diminished by a lipase inhibitor, whereas
glucose-stimulated secretion was less affected. These findings indicate that
cAMP-mediated lipolysis may play an important role in ß-cell signal
transduction and the incretin effect of GLP-1.
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RESEARCH DESIGN AND METHODS
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Growth and incubation of cells. Clonal pancreatic ß-cells
(HIT-T15) were cultured in a RPMI-1640 medium supplemented with 50 U/ml
penicillin, 50 µg/ml streptomycin, and 10% fetal calf serum (used between
passages 64 and 80), harvested with phosphate-buffered saline containing 0.02%
EDTA or with 0.25% trypsin and 0.06% EDTA diluted in phosphate-buffered
saline, and washed in a buffer (pH 7.4)
(18).
The effect of GLP-1 on FFA oxidation was measured as the
14CO2 released from cells that were labeled overnight in
RPMI-1640 with [U-14C]oleic acid (1 µmol/l), washed free of
remaining labeled FFAs, and incubated in the absence of added labeled or
unlabeled FFAs (19).
For insulin secretion measurements, HIT-T15 cells were grown under standard
conditions in 24-well plates and then used 2-4 days after passaging for
secretion assays. Cells were washed twice in Krebs-Ringer bicarbonate
(containing 2 mmol/l CaCl2 and 0.25% bovine serum albumin) and
buffered with 10 mmol/l HEPES at pH 7.4. The cells were preincubated with the
above assay buffer for 30 min at 37°C. The buffer was replaced and the
cells were then incubated for 30 min. Secretion was stopped by cooling the
plates on ice. The sample solution was removed and centrifuged to remove any
loose cells, then the insulin level was measured by radioimmunoassay using the
assay protocol for rat insulin distributed by Linco Research.
Fluorescence measurements of pHi, redox state, intracellular
Ca2+, and extracellular FFA. Cells were loaded with 1
µmol/12',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein
(BCECF), acetoxymethyl ester (AM) for 30 min at 37°C with continuous
shaking (20). For fluorescence
recordings, cells (0.2 mg protein/ml) were maintained in suspension by
continuous stirring in a modified Krebs buffer at 30°C in a
computer-controlled Hitachi F-2000 spectrofluorometer. BCECF fluorescence was
monitored at excitation wavelengths of 440 and 495 nm and an emission
wavelength of 535 nm. The average basal pHi (mean ± SD) was
7.0 ± 0.1 (n = 14). The pHi was calibrated by
measuring the fluorescence of the dye at different pH values between 6 and 8
as determined with a pH electrode after the cells were permeabilized to
equilibrate pHi with extracellular pH
(16). Traces shown are those
obtained at the excitation wavelength of 495 nm. NH4Cl (10 mmol/l
final concentration) was added at the end of each experiment to confirm cell
viability (16); the increase
in pHi followed by the restoration of the basal pHi
indicated viable cells and an intact plasma membrane. The engineered
intestinal fatty acidbinding protein ADIFAB (0.2 µmol/l)
(dissociation constant [KD] for oleic acid = 390 nmol/l
[21]), was monitored at an
excitation wavelength of 390 nm and emission wavelengths of 432 and 505 nm
(21,22)
to measure the release of FFAs into the media. The redox state of the cells
was determined by measuring reduced pyridine and oxidized flavin nucleotide
fluorescence at the excitation and emission wavelengths of 340 and 460 nm and
460 and 540 nm, respectively, as described previously
(20). Intracellular
Ca2+ changes were measured in cells loaded with the Ca2+
indicator fura 2, as described previously
(20). When pHi and
extracellular FFAs were measured simultaneously, only single emission and
excitation wavelengths were used. Baseline drift was subtracted from traces
when single wavelength measurements were used.
HSL. The total RNA from pancreatic islets was isolated using the
guanidinium isothiocyanate-phenol method and was reverse transcribed using
random hexamers. Polymerase chain reaction (PCR) was performed using the rat
HSL sense and antisense primers 5'-gaacactacaaacgcaacgag-3' and
5'-caagggggtgagatggtaac-3'. The PCR cycles included a hot start at
97°C, followed by 30 cycles of 94°C for 30 s, 62°C for 20 s, and
72°C for 1 min. Experimental samples were also processed in the absence of
reverse transcriptase to rule out amplification from genomic DNA. The PCR
products were displayed on a 1.2% agarose gel, stained with ethidium bromide,
and photographed with UV light. Negative control reactions lacking cDNA were
amplified simultaneously with the experimental samples.
Materials. BCECF-AM, fura 2-AM, and ADIFAB were purchased from
Molecular Probes (Eugene, OR). Radioactive tracers were from New England
Nuclear (Boston, MA), and triacsin C was from Biomol Research Laboratories
(Plymouth Meeting, PA). Other reagents were from Sigma (St. Louis, MO).
Orlistat (Xenical, Roche) capsule material was extracted with ethanol to make
a nominally 500 mmol/l stock solution. Forskolin (Research Biochemicals
International, Natick, MA) was dissolved in DMSO. Cells used for secretion
studies contained 0.025% ethanol and 0.1% DMSO in Krebs-Ringer bicarbonate
solution containing 0.25% FFA-free bovine serum albumin.
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RESULTS
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GLP-1 decreases pHi. An acidification in clonal
ß-cells was observed when GLP-1 was added
(Fig. 1A). The
decrease in pHi began immediately after the addition and continued
over a period of minutes. A similar but larger decrease in pHi was
caused by the addition of forskolin (Fig.
1B), which indicated that this change was probably caused
by an increase in cAMP. The extent of the decrease in pHi was
similar to that observed in adipocytes and attributed to the generation of
FFAs from triglycerides after the stimulation of lipolysis
(17). The data in
Fig. 1A and B
were obtained in the absence of glucose. Glucose has been shown to alkalinize
pHi (23), and this
effect appears to dominate pHi in the presence of glucose, although
a transient acidification occurred when GLP-1 was added
(Fig. 1C).

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FIG. 1. The effect of GLP-1 and forskolin on pHi in clonal pancreatic
ß-cells. HIT cells (0.2 mg/ml) loaded with the pH indicator BCECF were
suspended in 1.3 ml Krebs buffer containing no albumin (pH 7.4) at 30°C.
Traces A and B show the acidification caused by the addition of GLP-1 or
forskolin. Trace C shows the alkalinization caused by glucose addition
followed by a small and transient acidification caused by GLP-1. The
illustrations shown are representative of experiments repeated at least three
times.
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Elevation of Ca2+ does not influence pHi.
Agents that elevate cAMP may also elevate Ca2+
(24). Thus, if acidification
results from the release of intracellular FFAs, the FFAs could originate from
either triglyceride stores (via lipolysis) or from phospholipids (via
Ca2+-activated phospholipase activity).
Figure 2 shows that the
elevation of cytosolic free Ca2+ by bombesin is not accompanied by
a change in pHi (top trace). As noted above, previous studies from
our laboratories have shown that glucose stimulation of ß-cells, which
also elevates cytosolic free Ca2+, causes alkalinization
(23). Other agonists (e.g.,
carbachol and bradykinin) that increase Ca2+ do not also decrease
pHi (data not shown). These findings make it very unlikely that
Ca2+-stimulation of a phospholipase is responsible for the
cAMP-induced decrease in pHi.

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FIG. 2. The effect of bombesin on Ca2+ and pHi.
Experiments were performed and cells were incubated as described in
Fig. 1. In addition, half of
the cells were loaded with the Ca2+ indicator fura 2 and monitored
separately to assess changes in cytosolic free Ca2+. The
illustrations shown are representative of experiments repeated at least three
times.
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GLP-1 stimulates lipolysis. If GLP-1 stimulates lipolysis, there
should be evidence for an increase in FFA production. Evidence supporting an
action of GLP-1 to stimulate lipolysis is presented in
Fig. 3. Cells were incubated
overnight with trace amounts of 14C-oleic acid to label the
internal triglyceride pool, then the production of 14CO2
was measured in cells that had been thoroughly washed to remove free oleate.
We expected the stimulation of triglyceride breakdown to lead to increased
oxidation of FFAs released from the labeled triglyceride pool. Indeed, cells
acutely treated with GLP-1 exhibited more than a twofold increase in FFA
oxidation, whereas the addition of glucose inhibited FFA oxidation in both
control and GLP-1treated cells. Inhibition of FFA oxidation by glucose
(in the absence of GLP-1) was previously shown
(19).

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FIG. 3. The effect of GLP-1 and glucose on FFA oxidation. Clonal ß-cells
(0.2 mg/ml) preloaded for 24 h with [U-14C]oleate were washed three
times and preincubated in 1.3 ml Krebs buffer containing 5 mmol/l glutamine
without glucose or FFAs (pH 7.4) at 30°C. After changing to a test medium
containing no glucose, 10 mmol/l glucose, 10 nmol/l GLP-1, or GLP-1 plus 10
mmol/l glucose, 14CO2 evolution was determined during a
period of 60 min. The data are means ± SE from six or eight flasks from
two separate experiments. *P < 0.01 vs. basal or GLP-1
alone.
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To determine whether FFAs were released from the ß-cell into the
medium, we used ADIFAB, a fluorescent fatty acidbinding protein that
reports the binding of FFAs to the protein
(21).
Figure 4 directly demonstrates
the basal FFA efflux from clonal ß-cells by showing the increase in the
ADIFAB fluorescence ratio. Glucose prevented FFA efflux
(Fig. 4A), presumably
by stimulating re-esterification. Lipolysis yields FFAs, which are converted
to their active metabolite LC-CoA by acyl CoA synthetase (ACS). Triacsin C, an
inhibitor of some isoforms of ACS
(25), increased the release of
FFAs when added under basal conditions
(Fig. 4B) or after
glucose addition (Fig.
4A). Control experiments showed that triacsin C itself
neither altered the redox state nor qualitatively prevented the redox changes
induced by glucose (Fig. 5).
Also, it did not alter the Ca2+ changes induced by a variety of
agonists (data not shown).

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FIG. 4. Illustration of the effect of glucose and triacsin C on FFA efflux from
clonal ß-cells. Experiments were performed and HIT cells were incubated
as described in Fig. 1 but
without BCECF. The relative FFA concentration in the medium was monitored with
0.2 µmol/l ADIFAB; an increase in the fluorescence ratio indicates an
increase in medium FFAs. The illustrations shown are representative of
experiments repeated at least three times.
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FIG. 5. Triacsin C does not prevent glucose-mediated redox changes. HIT cells
(0.2 mg/ml) were suspended in 1.3 ml Krebs buffer containing no albumin (pH
7.4) at 30°C. The top trace shows a decrease in oxidized flavin and the
bottom trace shows an increase in reduced pyridine nucleotide fluorescence
caused by the addition of glucose. Reduced pyridine (NADH) and oxidized flavin
nucleotide (FAD) fluorescence were measured at excitation and emission
wavelengths of 340 and 460 nm and 460 and 540 nm, respectively. The
illustration shown is representative of experiments repeated at least three
times.
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To determine whether the pHi effects of GLP-1 (which we attributed
to the stimulation of lipolysis) corresponded to FFA exit from the cells, we
monitored the release of FFAs to the external buffer using ADIFAB while
simultaneously measuring the pHi.
Figure 6 directly demonstrates
that GLP-1 increased FFA release into the medium; as in
Fig. 1, GLP-1 caused
acidification of the cytosol (Fig.
6A, bottom trace) and a small release of FFAs into the
medium (Fig. 6A, top
trace). FFA release in the presence of glucose plus GLP-1 was quite low,
presumably because of glucose-promoted re-esterification similar to that seen
in Fig. 4; however, the FFA
production was unmasked by the addition of triacsin C, which sharply increased
FFA release and intracellular acidification
(Fig. 6B). The
increases in ADIFAB fluorescence (top traces) showed a time dependence for the
release of FFAs into the medium that corresponded to the time course of the
decreases in pHi (bottom traces). This implies that the FFAs can
leave cells almost as quickly as they are generated.

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FIG. 6. The effect of GLP-1 and triacsin C on pHi and FFA release.
Experiments were performed and HIT cells were incubated as described in
Fig. 1. In addition, the
relative FFA concentration in the medium was monitored with 0.2 µmol/l
ADIFAB; an increase in fluorescence indicates an increase in medium FFA. The
illustration shown is representative of experiments repeated at least three
times.
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HSL in the ß-cell. To explain the cAMP-mediated stimulation of
lipolysis, the presence of an HSL-like activity must be postulated. To confirm
that the RNA for adipocyte HSL was expressed in islets, rat islet cDNA was
used in a PCR with HSL-specific sense and antisense primers. The PCR products
were separated by agarose gel electrophoresis and the gel was stained with
ethidium bromide. An amplification product of the expected size (280 bp) was
present in the rat islet cell lane (Fig.
7). DNA sequencing of the isolated band confirmed its identity to
HSL. There was no DNA amplification in the negative control lane, which was
lacking a cDNA template. There was also no amplification product in RNA
samples processed without reverse transcriptase enzyme (data not shown),
ruling out amplification from genomic DNA.

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FIG. 7. Reverse transcriptionPCR of rat pancreatic islet RNA (lane
I) using HSL-specific PCR primers. The products were separated on a 1.2%
agarose gel stained with ethidium bromide and photographed with UV
transillumination. The figure also shows the negative control lane (-) and the
molecular weight marker (MW). The band of the expected size was excised from
the gel, then the DNA was extracted and sequenced using Taq cycle sequencing
(with the original PCR primers) on an ABI 373 DNA sequencer.
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Inhibition of the incretin effect by a lipase inhibitor. Forskolin
increased glucose-stimulated insulin secretion, and this incretin effect was
progressively and largely diminished by 30 min preincubation of the cells with
increasing concentrations of the lipase inhibitor orlistat
(tetrahydrolipstatin)
(26,27,28)
(Fig. 8). Similar results were
seen in a second experiment with a different passage of cells.
Glucose-stimulated secretion itself was less potently affected by orlistat in
the experiment illustrated by Fig.
8, and it was not affected at all in the second experiment. There
was no effect of orlistat on basal secretion (data not shown). It should be
noted that though orlistat acts in the intestinal lumen in vivo and is not
taken up systemically (29),
orlistat can nevertheless cross biological model membranes
(30). Furthermore, orlistat
inhibits HSL (28) in addition
to pancreatic lipase, but it does not inhibit phospholipase A2
(31).
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DISCUSSION
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The data presented here are consistent with a model
(Fig. 9) in which GLP-1
activates adenyl cyclase, generating cAMP, which consequently stimulates HSL
via PKA, thereby causing the breakdown of triglycerides and the generation of
FFAs. The FFAs are converted to LC-CoA, either for oxidation in the
mitochondria or for the synthesis of complex lipids (e.g., DAG or PA).
Triacsin C interferes with this process by partially blocking the generation
of LC-CoA and favoring the accumulation and efflux of FFAs. Support for this
model is indicated by the actions of GLP-1 to 1) decrease
pHi, 2) increase the release of FFAs from the cell, and
3) stimulate FFA oxidation after overnight labeling of the
triglyceride pool. Further support comes from the recent finding that high-fat
feeding, which increases islet triglyceride content, leads to an exaggerated
response to GLP-1 (32).

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FIG. 9. Model of the lipolytic pathway in ß-cells. The model shows that
GLP-1 activates HSL as a consequence of PKA stimulation via cAMP. HSL causes
triglyceride breakdown to FFAs. This is followed by activation of FFAs via ACS
in a triacsin Cinhibitable step with production of the effector signal
LC-CoA. Malonyl CoA prevents the loss of this effector signal (or its products
DAG and PA), by blocking its entry and subsequent oxidation in the
mitochondria. CPT-1, carnitine palmitoyl transferase-1; TG,
triglycerides.
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The suggestion that lipolysis (with the resulting production of FFAs and
LC-CoA) may be involved in the incretin effect of GLP-1 is amenable with our
hypothesis that both FFAs and cytosolic LC-CoA and other complex lipids are
major effector signals in insulin secretion
(12,13).
The data also suggest that an important source of these lipid molecules is
internal triglyceride stores, the regulation of which has not been previously
investigated. The current studies indicate that cAMP may regulate lipolysis,
and ultimately FFA and LC-CoA production, via HSL. This may explain why the
pancreatic islet contains large triglyceride stores
(9,10)
and relies on basal FFA oxidation to provide most of its fuel needs
(9). These triglyceride stores
and their modulation may also play an important role in glucose homeostasis in
vivo
(14,15,33,34).
During the course of this work, similar and more extensive evidence of HSL in
islets was published by Mulder et al.
(11).
It should be noted that PKA phosphorylation of either purified HSL or HSL
in fat cell homogenates causes only a 2- to 3-fold increase in activity, far
less than the 20- to 50-fold change in lipolysis in intact cells
(35,36).
This is attributed to the much greater importance of translocation of HSL to
the substrate and changes in the accessibility of the substrate, probably also
involving PKA-mediated phosphorylation of perilipin
(37). Interestingly, PKA
phosphorylation of purified lipase increases its activity only toward certain
substrates (e.g., triglyceride and cholesteryl oleate), but not toward the
diglyceride substrates that are commonly used in lipase assays and yield the
highest rates of hydrolysis by HSL
(35,38).
Diglyceride substrates were used in the studies of HSL-like activity in
ß-cells by Mulder et al.
(11). Although we have
confirmed lipase activity in HIT cell homogenates using a diglyceride-based
commercial kit, for the above reasons we have not pursued studies of assayed
activity in homogenates. Monitoring the FFA release from intact cells, as we
have done here, appears to be the more physiologically relevant means of
measurement. Admittedly, although the HSL-like enzyme shown to be present in
ß-cells is the most likely candidate for mediating GLP-1induced
FFA release, it is possible that another lipase (e.g., a
Ca2+-independent phospholipase) might be involved. Regardless of
the identity of the lipase, the important finding for the proposed
lipidmediated pathway of signal transduction in the ß-cell is that GLP-1
causes FFA release.
GLP-1 acidified the ß-cell under the conditions of our experiments;
however, the acidification is unlikely to be important for its incretin action
because secretion was not stimulated by GLP-1 in the absence of glucose where
the decrease in pHi was greatest, whereas in the presence of
glucose the ability of GLP-1 to acidify was minimal. In contrast,
glucose-stimulated secretion is accompanied by alkalinization of the
ß-cell
(20,23).
This alkalinization results from an early stimulation of pyruvate transport
into the mitochondria (cotransport with a proton), as suggested by
observations that blockage of the pyruvate transport with
3-hydroxycyanocinnamate prevented the pH change but not the subsequent
glucose-induced rise in Ca2+
(20,23).
Thus, the changes in pHi may serve as useful indicators of
metabolic events without necessarily playing an important role in
stimulus-secretion coupling. In addition, our data indicate that the
pHi change did not act indirectly by causing changes in
Ca2+ and that agonists that elevated cytosolic free Ca2+
did not change the pHi.
The rise in FFA levels caused by GLP-1, as indicated by acidification or
FFA efflux from the cell, is insufficient by itself to cause insulin secretion
in the absence of glucose. Furthermore, glucose reduced the rate of release of
FFAs, and hence probably the cellular level of FFAs, as well as the rate of
FFA oxidation; these effects are presumably due to the ability of glucose to
promote complex lipid formation and re-esterification. This postulation may
suggest that a product formed from FFA but requiring glucose is involved in
stimulus-secretion coupling.
Triacsin C was used here to help unmask FFA production by inhibiting both
the activation of FFAs to LC-CoA and their further removal by either
mitochondrial oxidation or complex lipid formation in the presence of glucose.
In theory, triacsin C could be used to distinguish between effects mediated
directly by FFAs or by LC-CoA or complex lipids. It has been reported that
acutely administered triacsin C largely inhibited oxidation of exogenous FFAs
yet had no effect on glucose-stimulated secretion
(39); we have confirmed this
and, likewise, we have found no consistent effect on the enhancement of
secretion by GLP-1 or FFAs (data not shown). On the surface, this might seem
to indicate that the FFAs, but not LC-CoA, were involved in the stimulation of
secretion. However, the situation is complicated by the existence of pools of
LC-CoA and various isoforms of ACS with differing sensitivity to triacsin C
(25,40).
Indeed, although FFA oxidation is strongly inhibited, the incorporation of
FFAs into phospholipids is much less affected by triacsin C
(25,39,40).
Because glucose itself reduces FFA oxidation, the more relevant path of FFA
metabolism for signal transduction is likely to be the formation of complex
lipids. We propose that GLP-1 causes the release of FFAs and that this effect
can provide more substrate for both FFA oxidation and complex lipid formation,
as well as the observed enhanced FFA efflux from the cell; as noted above,
high glucose levels tend to direct FFA carbon into the complex lipids, at the
expense of the other two possible outcomes.
The potent inhibition of the incretin effect by the lipase inhibitor
orlistat (Fig. 8) provides
strong evidence for the involvement of PKA-stimulated lipolysis, as indicated
in Fig. 9. Orlistat had less
effect on glucose-stimulated secretion alone. This does not imply that glucose
signaling does not involve a lipid-mediated component, merely that glucose
modulation of lipid signals may not arise primarily from direct effects on
lipolysis and thus FFA provision for LC-CoA synthesis. Instead, as discussed
previously (12), glucose
increases the malonyl CoA level, which in turn inhibits carnitine palmitoyl
transferase-1 and reduces mitochondrial uptake and oxidation of LC-CoA. This
leads to a rise in cytosolic LC-CoA and, together with the increased
production of
-glycerophosphate from glucose, to increases in complex
lipid formation.
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ACKNOWLEDGMENTS
|
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These experiments were supported by National Institutes of Health Grants
DK50662 (G.C.Y.), DK35914 (B.E.C.), DK46200 (B.E.C.), HL26335 (J.A.H.), and
DK53064 (K.T.).
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FOOTNOTES
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A.E.B. is deceased.
ACS, acyl CoA synthetase; AM, acetoxymethyl ester; BCECF,
2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein; DAG,
diacylglycerol; FFA, free fatty acid; GLP-1, glucagon-like peptide 1; HSL,
hormone-sensitive lipase; LC-CoA, long-chain acyl CoA; PA, phosphatidic acid;
PCR, polymerase chain reaction; pHi, intracellular pH; PKA, protein
kinase A.
Received for publication February 17, 2000
and accepted in revised form September 11, 2000
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