Lipotoxicity of the Pancreatic ß-Cell Is Associated With Glucose-Dependent Esterification of Fatty Acids Into Neutral Lipids
Isabelle Briaud,
Jamie S. Harmon,
Cynthia L. Kelpe,
Venkatesh Babu G. Segu, and
Vincent Poitout
From the Pacific Northwest Research Institute (I.B., J.S.H., C.L.K.,
V.B.S., V.P.) and the Department of Medicine (V.B.S., V.P.), University of
Washington, Seattle, Washington.
Address correspondence and reprint requests to Vincent Poitout, DVM, PhD,
Pacific Northwest Research Institute, 720 Broadway, Seattle, WA 98122. E-mail:
vpoitout{at}pnri.org
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ABSTRACT
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Prolonged exposure of isolated islets to supraphysiologic concentrations of
palmitate decreases insulin gene expression in the presence of elevated
glucose levels. This study was designed to determine whether or not this
phenomenon is associated with a glucose-dependent increase in esterification
of fatty acids into neutral lipids. Gene expression of
sn-glycerol-3-phosphate acyltransferase (GPAT), diacylglycerol
acyltransferase (DGAT), and hormone-sensitive lipase (HSL), three key enzymes
of lipid metabolism, was detected in isolated rat islets. Their levels of
expression were not affected after a 72-h exposure to elevated glucose and
palmitate. To determine the effects of glucose on palmitate-induced neutral
lipid synthesis, isolated rat islets were cultured for 72 h with trace amounts
of [14C]palmitate with or without 0.5 mmol/l unlabeled palmitate,
at 2.8 or 16.7 mmol/l glucose. Glucose increased incorporation of
[14C]palmitate into complex lipids. Addition of exogenous palmitate
directed lipid metabolism toward neutral lipid synthesis. As a result, neutral
lipid mass was increased upon prolonged incubation with elevated palmitate
only in the presence of high glucose. The ability of palmitate to increase
neutral lipid synthesis in the presence of high glucose was
concentration-dependent in HIT cells and was inversely correlated to insulin
mRNA levels. 2-Bromopalmitate, an inhibitor of fatty acid mitochondrial
ß-oxidation, reproduced the inhibitory effect of palmitate on insulin
mRNA levels. In contrast, palmitate methyl ester, which is not metabolized,
and the medium-chain fatty acid octanoate, which is readily oxidized, did not
affect insulin gene expression, suggesting that fatty-acid inhibition of
insulin gene expression requires activation of the esterification pathway.
These results demonstrate that inhibition of insulin gene expression upon
prolonged exposure of islets to palmitate is associated with a
glucose-dependent increase in esterification of fatty acids into neutral
lipids.
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INTRODUCTION
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According to the lipotoxicity hypothesis, chronic exposure to elevated
lipid levels impairs pancreatic ß-cell function in type 2 diabetic
patients
(1,2).
We (3) and others
(4,5)
have previously shown that prolonged (>1 day) culture of normal islets in
the presence of supraphysiologic concentrations of palmitate decreases insulin
content and impairs insulin gene expression only in the presence of elevated
glucose levels. This occurs, at least in part, via decreased insulin gene
promoter activity in HIT-T15 cells
(3) and decreased binding of
the transcription factor pancreas-duodenum homeobox-1 (PDX-1) to the insulin
gene in islets (4). In Zucker
diabetic fatty (ZDF) rats, it has been postulated that ß-cell dysfunction
is due to increased triacylglycerol (TAG) content in islets
(6,7,8),
which leads to increased production of nitric oxide
(9) and ceramide synthesis
(10). However, the ZDF rat is
an extremely obese genetic model of type 2 diabetes bearing a mutation in the
leptin receptor gene. It remains to be demonstrated that prolonged exposure of
normal ß-cells to fatty acids results in increased esterification into
neutral lipids, eventually leading to intracellular TAG accumulation. In
addition, whether increased neutral lipid synthesis occurs via modulation of
expression of lipogenic and lipolytic enzymes or is simply driven by the
amount of available substrates is unknown. Finally, the differential and
possibly synergistic effects of glucose and palmitate on neutral lipid
metabolism have not been studied in the context of prolonged exposure to both
nutrients.
This study was based on the hypothesis that prolonged exposure to palmitate
results in a glucose-dependent increase in neutral lipid synthesis. It was
designed to 1) determine whether sn-glycerol-3-phosphate
acyltransferase (GPAT), AcylCoA:diacylglycerol acyltransferase (DGAT), and
hormone-sensitive lipase (HSL)three key enzymes of neutral lipid
metabolismare expressed in islets; 2) ascertain whether
prolonged exposure of islets to glucose and palmitate modulates the expression
of these enzymes; and 3) assess the effects of prolonged exposure to
glucose and palmitate on neutral lipid metabolism in islets and
insulin-secreting cells.
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RESEARCH DESIGN AND METHODS
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Reagents. Palmitic acid (sodium salt), palmitate methyl ester,
octanoate, fatty-acidfree bovine serum albumin (BSA), triolein, and the
GPO Trinder reagent (No. 339) were from Sigma (St. Louis, MO).
2-Bromopalmitate was from Aldrich (Milwaukee, WI). Reverse
transcriptasepolymerase chain reaction (RT-PCR) primers and the Gold
RT-PCR kit were from PerkinElmer (Foster City, CA).
[U-14C]Palmitate, [1-14C]cholesteryl oleate, and
[carboxyl-14C]triolein were from NEN (Boston, MA). Thesit was from
Boehringer Mannheim (Mannheim, Germany).
Fatty-acid solutions. Stock solutions were prepared as follows:
palmitic acid was dissolved in ethanol:H2O (1:1, vol:vol) at
50°C at a final concentration of 150 mmol/l; 2-bromopalmitate and
palmitate methyl ester were dissolved in ethanol at a final concentration of
300 mmol/l; and octanoate was dissolved in ethanol:H2O (1:1,
vol:vol) at a final concentration of 500 mmol/l. Aliquots of stock solutions
were complexed with fatty-acidfree BSA (10% solution in H2O)
by stirring for 1 h at 37°C and then diluted in culture media. The final
molar ratio of fatty acid:BSA was 5:1. The final ethanol concentration was
0.33% (vol:vol). All control conditions included a solution of vehicle
(ethanol:H2O) mixed with fatty-acidfree BSA at the same
concentration as the fatty-acid solution, unless indicated otherwise.
Animals. Six-week-old male Wistar rats were purchased from Harlan
Sprague Dawley (Indianapolis, IN). Seven-week-old male Zucker diabetic fatty
(ZDF/Gmi fa/fa) rats and Zucker lean control (ZLC/Gmi +/fa
or +/+) rats were purchased from Genetic Models (Indianapolis, IN). Animals
were housed on a 12-h light/dark cycle with free access to water and standard
laboratory food. All procedures using animals were approved by the
Institutional Animal Care and Use Committee.
HIT-T15 cells and isolated rat islets. HIT-T15 cells (passage 69-78)
were routinely cultured as described
(11). For Northern blot,
labeling, and TAG content experiments, cells were detached and subcultured for
48 h by plating 1 x 106 cells per well in 6-well plates. Rat
islets were isolated by collagenase digestion as described
(3). After an overnight culture
in RPMI 1640 containing 10% fetal bovine serum and 11.1 mmol/l glucose to
ensure optimal recovery (12),
batches of 100-200 islets were incubated in various experimental conditions as
described in RESULTS.
Northern analysis of insulin mRNA. Cultured islets were transferred
to 15-ml conical tubes, rinsed twice with phosphate-buffered saline (PBS) (137
mmol/l NaCl, 2.7 mmol/l KCl, 4.3 mmol/l
NaH2PO4.7H2O, pH 7.3), and resuspended in
denaturing solution (4 mol/l guanidine thiocyanate, 25 mmol/l sodium citrate,
pH 7, 0.5% sarcosyl, 0.1 mol/12-mercaptoethanol). HIT-T15 cells were rinsed
with PBS and scraped with denaturing solution. RNA was extracted according to
Chomczynski and Sacchi (13),
and insulin mRNA was assessed by Northern blot as described
(3). Membranes were stripped
and rehybridized with a ß-actin cDNA to control for variations in the
amount of total RNA loaded on each lane.
Fluorescence-based RT-PCR of GPAT, DGAT, HSL, and insulin mRNAs.
Comparative analysis of GPAT, DGAT, and HSL mRNA levels in isolated islets
cultured with and without palmitate was performed using the Gold RT-PCR kit
and an ABI Prism 7700 sequence detector equipped with a thermocycler (TaqMan
technology), as described
(14). ß-Actin was used as
an internal control, and insulin was used as a positive control for the effect
of palmitate. Primer and probe sequences are given in
Table 1.
Analysis of intracellular lipids by thin-layer chromatography.
Fatty-acid solutions were prepared as described above with the addition of 5
µmol/l (4.25 mCi) [U-14C]palmitate in all conditions. Islets or
HIT-T15 cells were cultured in the various fatty-acid solutions, as described
in RESULTS. At the end of the culture period, islets or cells were transferred
to 12 x 75 mm prechilled glass tubes on ice, centrifuged at 1,200 rpm
for 10 min at 4°C, washed with 2 ml ice-cold PBS, centrifuged as above,
resuspended in 3 ml chloroform:methanol:HCl (200:100:1), and stored at 4°C
overnight under N2. After addition of 750 µl double-distilled
H2O, samples were vortexed and centrifuged for 10 min at 4°C.
The upper (aqueous) phase was removed, and the lower (organic) phase was
washed with 750 µl double-distilled H2O and centrifuged as
above. The upper phase was removed and the lower phase was dried under
N2. The lipid pellet was resuspended in 100 µl
chloroform:methanol:HCl (200:100:1), and 40 µl was spotted in duplicate on
silica gel thin-layer chromatography (TLC) plates (Whatman, Clifton, NJ).
Lipids were resolved in petroleum ether:diethyl ether:glacial acetic acid
(70:30:1). [1-14C]Cholesteryl oleate, [U-14C]palmitate,
and [carboxyl-14C]triolein were used as standards. Results obtained
with this system were initially verified using N-hexane:diethyl
ether:methanol:glacial acetic acid (90:20:2:3). TLC plates were exposed to
X-OMAT AR autoradiography films (Eastman Kodak, Rochester, NY). Individual
bands were scraped, transferred to scintillation vials with 5 ml Ecoscint
scintillation fluid (National Diagnostics, Atlanta, GA), and counted.
Intracellular TAG assay. We have experienced difficulties in trying
to measure TAG content in islet extracts, as previously published
(6,15,16,17,18,19).
Therefore, we have modified this method by 1) extracting the lipids
before the assay, 2) resuspending the extracted lipids in the
detergent Thesit as recommended in other cells
(20), and 3)
calculating the sample TAG concentrations from a triolein standard curve, as
opposed to a single glycerol value. The assay is based on the colorimetric
determination of glycerol produced by hydrolysis of neutral lipids in the
presence of lipoprotein lipase. Islets or cells were transferred to 12 x
75 mm prechilled glass tubes on ice, centrifuged at 1,200 rpm for 10 min at
4°C, washed with 500 µl ice-cold PBS, and centrifuged as above. The
pellet was resuspended in 3 ml of chloroform:methanol (2:1) and stored
overnight at 4°C under N2. After the addition of 1.5 ml
double-distilled H2O, the tubes were vortexed and centrifuged at
1,200 rpm for 10 min at 4°C. The upper phase was discarded, and 750 µl
double-distilled H2O was added to the lower phase. The samples were
vortexed and centrifuged at 1,200 rpm for 10 min at 4°C. The upper phase
was removed, and the lower phase was evaporated under N2. The
samples were resuspended in 50 µl chloroform; 10 µl was quickly
transferred to glass tubes in duplicate and air dried. The dry pellet was
resuspended in 10 µl Thesit. The triolein standard curve (1-50 µg) was
prepared using triolein (Sigma) diluted in chloroform:methanol (2:1). Samples
and standards resuspended in Thesit were air dried; 50 µl H2O
was added; and the tubes were vortexed and incubated in a 37°C shaking
water bath for 10 min as described
(20). One milliliter of GPO
Trinder reagent was added to the tubes, which were then gently mixed and
incubated at 37°C for 5 min. The absorbance was read at 540 nm in a
Beckman (Fullerton, CA) DU-64 spectrophotometer. To test the efficiency of TAG
extraction, samples consisting of 5, 10, or 50 µg total lipid, each
containing 0.9 µg of [carboxyl-14C]triolein, and the remainder
unlabeled triolein underwent the extraction process. Aliquots were taken at
each step during the extraction process and counted. The total loss of
radioactivity during the extraction process was 3.0 ± 1.3% (n
= 4).
Expression of data and statistics. Data are expressed as means
± SE. Intergroup comparisons were performed by paired Student's
t test or analysis of variance with Dunnett's t test for
multiple comparisons, where appropriate. P < 0.05 was considered
significant.
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RESULTS
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Expression of GPAT, DGAT, and HSL in isolated rat islets. We first
sought to determine whether critical enzymes of neutral lipid metabolism are
expressed in islets and whether their level of expression is modulated upon
prolonged exposure to palmitate and glucose. GPAT is a rate-limiting enzyme
for neutral lipid synthesis
(21), and its expression is
increased in islets from ZDF rats
(6). DGAT is the enzyme
responsible for the synthesis of TAG from diacylglycerols (DAGs), but its
expression has never been documented in ß-cells. HSL is responsible for
the hydrolysis of TAG and is expressed and active in ß-cells
(22). Fluorescence-based
RT-PCR demonstrated the expression of GPAT
(Fig. 1A), DGAT
(Fig. 1B), and HSL
(Fig. 1C) in islets
isolated from normal Wistar rats. Semiquantitative analysis of mRNA levels for
each enzyme was then used to determine the effects of a 72-h culture in 2.8 or
16.7 mmol/l glucose with or without 0.5 mmol/l palmitate
(Fig. 2). As expected, insulin
mRNA levels were increased in the presence of 16.7 mmol/l glucose (P
= 0.002, n = 8; Fig.
2A), and this effect was blocked by palmitate (P
= 0.002, n = 8; Fig.
2A). However, neither GPAT
(Fig. 2B), DGAT
(Fig. 2C), nor HSL
(Fig. 2D) mRNA was
significantly affected by prolonged exposure to elevated glucose and/or
palmitate. DGAT mRNA levels tended to be slightly lower in the presence of
palmitate at 2.8 mmol/l glucose, but this difference was not statistically
significant (NS, n = 7; Fig.
2C). The effect of the culture period itself was
investigated by comparing insulin mRNA levels in freshly isolated islets to
those in islets cultured for 72 h in either 2.8 or 16.7 mmol/l glucose.
Insulin mRNA levels were lower in islets cultured in 2.8 mmol/l glucose than
in freshly isolated islets (insulin:ß-actin mRNA ratio 2.5 ± 0.9
vs. 5.7 ± 0.2, n = 3). Islets cultured for 3 days in 16.7
mmol/l glucose had insulin mRNA levels similar to those of freshly isolated
islets (insulin/ß-actin mRNA ratio 4.5 ± 1.5 vs. 5.7 ± 0.2,
n = 3). Therefore, we cannot exclude the possibility that the lack of
effect of palmitate on insulin mRNA levels at 2.8 mmol/l glucose is due to the
fact that insulin mRNA is already downregulated at this glucose concentration
and cannot be decreased further.

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FIG. 1. Detection of GPAT (A), DGAT (B), and HSL (C)
mRNAs by RT-PCR using Taqman technology in isolated rat islets. Rn
designates changes in fluorescence emission. Representative amplification
plots were from experiments performed in triplicate on two separate
occasions.
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FIG. 2. : Effects of prolonged exposure to glucose (Gluc.) and palmitate (Palm.)
on insulin (A), GPAT (B), DGAT (C), and HSL
(D) mRNA levels in isolated rat islets. Isolated islets were
incubated in the absence or presence of 0.5 mmol/l palmitate and 2.8 or 16.7
mmol/l glucose for 72 h. Insulin (n = 8), GPAT (n = 3), DGAT
(n = 7), and HSL (n = 3) mRNA levels were determined by
fluorescence-based RT-PCR as described in RESEARCH DESIGN AND METHODS. Results
(means ± SE) are expressed as the ratio between the signal
corresponding to the gene of interest and the signal for ß-actin mRNA.
*P < 0.01.
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Effects of exogenous palmitate and glucose on incorporation of
[14C]palmitate into neutral lipids. Intracellular neutral lipid
content results from the balance between lipogenic (GPAT and DGAT) and
lipolytic (HSL) enzyme activities. To determine whether neutral lipids
accumulate upon prolonged exposure to elevated palmitate and glucose, isolated
islets were cultured for 72 h in the presence of 5 µmol/l
[U-14C]-labeled palmitate with and without addition of 0.5 mmol/l
unlabeled palmitate, in 2.8 or 16.7 mmol/l glucose. Intracellular lipids were
extracted and analyzed by TLC. In the absence of unlabeled palmitate,
culturing islets with 16.7 mmol/l glucose increased the total number of counts
incorporated from labeled palmitate into complex lipids (phospholipids [PLs] +
DAG + TAG) 3.0 ± 0.9 fold compared with islets cultured in 2.8 mmol/l
glucose (P < 0.05, n = 7;
Fig. 3A). In islets
cultured in 0.5 mmol/l unlabeled palmitate, the total number of counts
incorporated into complex lipids was 2.4 ± 0.8-fold higher in the
presence of 16.7 mmol/l glucose than in the presence of 2.8 mmol/l glucose
(P < 0.05, n = 7; Fig.
3A). The ability of high glucose to increase
incorporation of the counts into complex lipids was therefore similar in the
absence or presence of unlabeled palmitate. The total number of counts
incorporated into complex lipids was artificially lower in the presence of
unlabeled palmitate because of the dilution of the specific activity of the
tracer. The effect of palmitate on partitioning the counts is therefore
expressed as the percentage of the counts incorporated into each lipid
fraction (Fig. 3B). In
the presence of 0.5 mmol/l unlabeled palmitate, the percentage of counts
incorporated into DAG and TAG was increased at the expense of the counts
incorporated into PLs. This effect was similar at 2.8 and 16.7 mmol/l glucose.
These results indicate that glucose and palmitate have different effects on
neutral lipid synthesis in islets upon prolonged exposure: glucose increases
the total number of counts incorporated into complex lipids, whereas palmitate
specifically directs partitioning of the counts toward neutral lipid
synthesis.

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FIG. 3. Effects of palmitate and glucose on incorporation of [14C]
exogenous palmitate in isolated rat islets. Isolated rat islets were incubated
with 5 µmol/l [U-14C]palmitate in the absence or presence of 0.5
mmol/l unlabeled palmitate and 2.8 or 16.7 mmol/l glucose for 72 h. Extracted
lipids were analyzed by TLC. For each lane, the lipid bands were scraped and
counted. Results are means ± SE of seven to nine separate experiments.
A: Effects of glucose on the total number of counts incorporated from
labeled palmitate into complex lipids (PL + DAG + DAG). The effect of
palmitate cannot be ascertained because of the dilution of the specific
activity of the tracer in the presence of unlabeled palmitate. Indeed, the
absolute number of counts is artificially lower in the presence of 0.5 mmol/l
unlabeled palmitate. *P < 0.05. B: Effects of
glucose and palmitate on the distribution of the counts into each complex
lipid fraction. Results are expressed as percent of total counts recovered
from each lane. *P < 0.05; **P <
0.01.
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Effects of palmitate and glucose on TAG content in islets. We next
sought to determine whether prolonged and simultaneous exposure to palmitate
and glucose leads to a net increase in TAG content. We first tested the
modified assay against the previously published method using crude cellular
extracts of islets
(6,15,16,17,18,19)
freshly isolated from normal Wistar, ZDF, and ZLC rats. The results are
presented in Table 2 and indicate
that the highest recovery of intracellular TAG is obtained using our modified
method. TAG content was then measured in Wistar rat islets after a 72-h
culture in the presence of 2.8 or 16.7 mmol/l glucose, with or without 0.5
mmol/l palmitate (Fig. 4).
Palmitate did not affect TAG content in the presence of 2.8 mmol/l glucose
(105 ± 5 ng/islet in the presence of palmitate vs. 76 ± 8
ng/islet in the absence of palmitate, n = 3, NS) but significantly
increased it in the presence of 16.7 mmol/l glucose (303 ± 58 ng/islet
in the presence of palmitate vs. 171 ± 31 ng/islet in the absence of
palmitate, P = 0.03, n = 6). These results indicate that the
presence of high glucose is necessary for prolonged exposure to palmitate to
result in a net increase in TAG content in islets.

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FIG. 4. Effects of palmitate and glucose on TAG content in isolated rat islets.
Isolated rat islets were incubated in the absence or presence of 0.5 mmol/l
palmitate and 2.8 or 16.7 mmol/l glucose for 72 h. TAG content was measured as
described in RESEARCH DESIGN AND METHODS. Results are means ± SE of
three to six separate experiments. *P < 0.05;
**P < 0.01.
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Concentration dependency of the effects of palmitate on neutral lipid
metabolism and insulin mRNA levels in HIT-T15 cells. The concentration
dependency of the effects of palmitate on neutral lipid synthesis, TAG
content, and insulin mRNA levels was assessed in HIT-T15 cells
(Fig. 5). First, HIT-T15 cells
were cultured for 72 h in the presence of 11.1 mmol/l glucose, 5 µmol/l
[U-14C]-labeled palmitate, and increasing concentrations of
unlabeled palmitate at a constant palmitate:BSA molar ratio of 5:1. Analysis
of incorporation of the counts into the various forms of complex lipids showed
that palmitate dose-dependently shifted the partitioning of the counts into
TAG and DAG, at the expense of PL, confirming the observations made in
isolated islets (Fig.
5A). The effect of palmitate was statistically
significant at 0.25 mmol/l and higher. Importantly, culturing HIT-T15 cells in
the presence of increasing concentrations of BSA alone had no effect on the
partitioning of the counts (data not shown). Next, HIT-T15 cells were cultured
for 72 h in the presence of 11.1 mmol/l glucose with increasing concentrations
of palmitate. TAG mass was measured as described above and was found to
gradually augment as the concentration of palmitate was increased, with a
significant and maximal effect observed at 0.5 mmol/l (P < 0.05,
n = 5; Fig.
5B). This was associated with a dose-dependent decrease
in insulin mRNA levels (Fig. 5B
and C) that was statistically significant at and above
0.25 mmol/l. These results indicate that the effect of palmitate promoting
neutral lipid synthesis in the presence of high glucose is concentration
dependent and that there is an inverse relationship between TAG content and
insulin gene expression.

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FIG. 5. Dose-dependent effects of palmitate on lipid partitioning, TAG content,
and insulin mRNA levels in HIT-T15 cells. HIT-T15 cells were incubated for 72
h in RPMI 1640 containing 11.1 mmol/l glucose with increasing concentrations
of palmitate. A: The culture medium contained 5 µmol/l
[U-14C]palmitate. Lipids were extracted and analyzed by TLC. For
each lane, the lipid bands were scraped and counted. Results are means
± SE of three separate experiments and are expressed as percent of
total counts recovered from each lane. All points at and above 0.25 mmol/l are
significantly different from the control (0 palmitate) for PL, TAG, and DAG.
CE, cholesterol ester. B: Inverse correlation between TAG content and
insulin mRNA levels. Results are expressed as means ± SE of five or six
separate experiments. C: Representative Northern blot of insulin
mRNA.
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Effects of palmitate, octanoate, palmitate methyl ester, and
2-bromopalmitate on insulin mRNA levels in HIT-T15 cells. We next
investigated whether increased esterification into neutral lipids was
necessary for the inhibitory effects of fatty acids on insulin mRNA levels
(Fig. 6). After a 24-h exposure
to 0.5 mmol/l palmitate, insulin mRNA was decreased by
40% compared with
control in the presence of BSA alone, confirming our previous findings (3;
Fig. 5). 2-Bromopalmitate,
which irreversibly binds to carnitine-palmitoyl-transferase 1 (CPT-1) and
inhibits longchain fatty acid ß-oxidation
(23), had a similar effect to
palmitate. In contrast, the medium-chain fatty acid octanoate, which does not
require CPT-1 to enter the mitochondria and is readily oxidized, and palmitate
methyl ester, which is not activated into a fatty-acyl CoA in the cytosol, did
not affect insulin mRNA levels. These results suggest that the
fatty-acidinduced decrease in insulin mRNA levels is not related to
ß-oxidation but requires activation of the fatty acid into a fatty-acyl
CoA. Considering that fatty-acyl CoAs undergo either ß-oxidation or
esterification, these results indirectly suggest that activation of the
esterification pathway is necessary for fatty acids to inhibit insulin gene
expression.

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FIG. 6. Effects of fatty acids on insulin mRNA level in HIT-T15 cells. HIT-T15
cells were cultured for 24 h in the presence of 0.5 mmol/l palmitate, 2.5
mmol/l octanoate, 0.5 mmol/l palmitate methyl ester, or 0.5 mmol/l
2-bromopalmitate. The control condition contained an equivalent amount of BSA.
Insulin mRNA was measured by Northern analysis and was normalized to
ß-actin mRNA. Results are means ± SE of three separate experiments
and are expressed as percent control. A representative Northern blot is shown
on the right.
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DISCUSSION
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This study was designed to assess whether the decrease in insulin gene
expression upon prolonged exposure to palmitate in the presence of high
glucose was associated with modulation in the expression of lipogenic and
lipolytic enzymes in ß-cells and with increased esterification of fatty
acids into neutral lipids.
GPAT, DGAT, and HSL mRNA were detected, demonstrating for the first time
that DGAT is expressed in pancreatic islets. In ZDF rat islets, the dramatic
increase in TAG content is associated with a marked elevation in GPAT mRNA
(6). However, we have not found
GPAT mRNA to be increased in normal islets after prolonged exposure to
palmitate. In other cell types, it has been suggested that DGAT, together with
GPAT, is an important enzyme in TAG synthesis because it catalyzes the final
and only committed step in this pathway
(24). DGAT activity is
increased in adipose tissue from obese Zucker rats
(25). Its expression was not
found to be modified in islets cultured in palmitate and glucose for 3 days.
HSL, the rate-limiting enzyme for intracellular TG hydrolysis, is expressed
and active in rat islets and several ß-cell lines
(22). Its expression, however,
does not seem to be modified by prolonged exposure to either glucose or
palmitate.
Pancreatic ß-cells have the capacity to store energy in the form of
TAG from both glucose and palmitate upon short-term incubation
(26). Glucose is mainly
incorporated into the glycerol-3-phosphate backbone of TAG
(27). In the fasting state,
the ß-cell preferentially utilizes fatty acids as metabolic fuels, and
therefore palmitate oxidation is high compared with esterification
(28,29).
At high glucose concentrations, lipid metabolism is switched to preferential
incorporation into TAG and PL
(28), at least in the short
term. Our results show that these observations also apply to long-term
situations and demonstrate that exposing islets to elevated concentrations of
palmitate results in a sustained increase in intracellular TAG stores only if
the concentration of glucose is elevated. The metabolic signals responsible
for the switch from fatty-acid oxidation to esterification in the presence of
high glucose in the ß-cell are thought to be malonyl-CoA and cytosolic
long-chain acyl-CoA (30).
Acceleration of glucose metabolism following a rise in intracellular glucose
concentration leads to increased cytosolic levels of malonyl-CoA
(31,32),
which potently inhibits CPT-1, thereby decreasing fatty-acid oxidation and
resulting in accumulation of fatty acyl-CoA esters in the cytosol
(33). Our results suggest that
this hypothesis, originally proposed to explain the role of fatty acids in
stimulus-secretion coupling in the ß-cell, also applies to situations in
which ß-cells are chronically exposed to elevated levels of glucose and
fatty acids. Furthermore, our results directly demonstrate that esterification
of fatty acids into neutral lipids is increased upon prolonged exposure to
glucose and palmitate, and that both nutrients have synergistic effects on
neutral lipid metabolism. Glucose promotes incorporation of the fatty acid
into complex lipids, whereas palmitate specifically directs partitioning
toward neutral lipid synthesis. As expected, this results in a net increase in
neutral lipid mass only when both fuels are present simultaneously. The fact
that neutral lipid accumulation is not associated with changes in the level of
expression of lipogenic and lipolytic enzymes suggests that activation of the
esterification pathway is driven by the amount of substrates, i.e., fatty
acids and glucose. Intracellular accumulation of neutral lipids results mainly
from the balance between the activities of lipogenic and lipolytic enzymes.
Our results indicate that overall lipogenic activity is augmented upon
prolonged exposure to palmitate in the presence of high glucose, resulting in
increased DAG and TAG content.
Various methods have been used for measuring TAG content in islets,
generating quantitatively variable results. The differences between studies
are in the mode of preparation of the lipid extract utilized for the assay.
Most studies in ZDF animals have used crude cellular extracts and found
intracellular levels of TAG to increase from <50 ng/islet in control rats
to up to 1,000 ng/islet in 12-week-old ZDF animals
(6,15,16,17,18,19).
Two studies using normal islets in culture have extracted the lipids before
the GPO Trinder assay
(34,35).
Our results are quantitatively similar to those of Zhou et al.
(34) and uniquely demonstrate
that the presence of glucose is necessary for TAG content to increase
significantly upon prolonged exposure to exogenous palmitate.
In conclusion, the results presented herein demonstrate for the first time
that neutral lipid synthesis is increased in normal ß-cells exposed to
elevated palmitate and glucose for prolonged periods of time. Glucose is
required for palmitate to increase intracellular TAG mass, and both fuels have
synergistic effects on neutral lipid metabolism. The glucose-dependent
activation of the esterification pathway upon prolonged exposure to palmitate
does not seem to be mediated by modulation of expression of key enzymes
involved in neutral lipid metabolism, and it is therefore likely to be driven
by the amount of available substrates.
 |
ACKNOWLEDGMENTS
|
---|
I.B. was supported in part by the Association de Langue
Française pour l'Etude du
Diabète et des Maladies
Métaboliques (ALFEDIAM) and the Aide aux
Jeunes Diabétiques (AJD).
We thank Drs. Stewart A. Metz, Christopher J. Rhodes, and R. Paul Robertson
for fruitful discussions and careful reading of the manuscript. We acknowledge
the excellent secretarial assistance of Madeline Johnson.
 |
FOOTNOTES
|
---|
BSA, bovine serum albumin; CPT-1, carnitine-palmitoyl-transferase 1; DAG,
diacylglycerol; DGAT, AcylCoA:diacylglycerol acyltransferase; GPAT,
sn-glycerol-3-phosphate acyltransferase; HSL, hormone-sensitive
lipase; PBS, phosphate-buffered saline; PL, phospholipid; RT-PCR, reverse
transcriptase-polymerase chain reaction; TAG, triacylglycerol; TLC, thin-layer
chromatography.
Received for publication May 12, 2000
and accepted in revised form October 11, 2000
 |
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