Distinct Effects of Saturated and Monounsaturated Fatty Acids on ß-Cell Turnover and Function
K. Maedler,
G.A. Spinas,
D. Dyntar,
W. Moritz,
N. Kaiser, and
Marc Y. Donath
From the Division of Endocrinology and Diabetes (K.M., G.A.S., D.D.,
W.M., M.Y.D.), University Hospital, Zurich, Switzerland; and the Department of
Endocrinology and Metabolism (N.K.), Hebrew UniversityHadassah Medical
Center, Jerusalem, Israel.
Address correspondence and reprint requests to Marc Donath, MD, Division of
Endocrinology and Diabetes, Department of Medicine, University Hospital,
CH-8091 Zurich, Switzerland. E-mail:
marc.donath{at}dim.usz.ch
.
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ABSTRACT
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Glucotoxicity and lipotoxicity contribute to the impaired ß-cell
function observed in type 2 diabetes. Here we examine the effect of saturated
and unsaturated fatty acids at different glucose concentrations on ß-cell
proliferation and apoptosis. Adult rat pancreatic islets were cultured onto
plates coated with extracellular matrix derived from bovine corneal
endothelial cells. Exposure of islets to saturated fatty acid (0.5 mmol/l
palmitic acid) in medium containing 5.5, 11.1, or 33.3 mmol/l glucose for 4
days resulted in a five- to ninefold increase of ß-cell DNA
fragmentation. In contrast, monounsaturated palmitoleic acid alone (0.5
mmol/l) or in combination with palmitic acid (0.25 or 0.5 mmol/l each) did not
affect DNA fragmentation. Increasing concentrations of glucose promoted
ß-cell proliferation that was dramatically reduced by palmitic acid.
Palmitoleic acid enhanced the proliferation activity in medium containing 5.5
mmol/l glucose but had no additional effect at higher glucose concentrations
(11.1 and 33.3 mmol/l). The cell-permeable ceramide analog
C2-ceramide mimicked both the palmitic acidinduced
ß-cell apoptosis and decrease in proliferation. Moreover, the ceramide
synthetase inhibitor fumonisin B1 blocked the deleterious effects of palmitic
acid on ß-cell viability. Additionally, palmitic acid but not palmitoleic
acid decreased the expression of the mitochondrial adenine nucleotide
translocator and induced release of cytochrome c from the mitochondria into
the cytosol. Finally, palmitoleic acid improved ß-cellsecretory
function that was reduced by palmitic acid. Taken together, these results
suggest that the lipotoxic effect of the saturated palmitic acid involves an
increased apoptosis rate coupled with reduced proliferation capacity of
ß-cells and impaired insulin secretion. The deleterious effect of
palmitate on ß-cell turnover is mediated via formation of ceramide and
activation of the apoptotic mitochondrial pathway. In contrast, the
monounsaturated palmitoleic acid does not affect ß-cell apoptosis, yet it
promotes ß-cell proliferation at low glucose concentrations,
counteracting the negative effects of palmitic acid as well as improving
ß-cell function.
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INTRODUCTION
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At the initial stages of type 2 diabetes, individuals lose the ability to
produce sufficient quantities of insulin to maintain normoglycemia in the face
of insulin resistance (1). The
capacity to produce insulin is determined by the total ß-cell number and
ß-cell functional activity. The ß-cell mass is capable of long-term
adaptation by increasing the ß-cell number through hyperplasia and
neogenesis
(2,3).
However, ß-cell expansion can be offset by concomitant apoptosis
(4). The failure of these
long-term feedback adaptations in genetically susceptible individuals with
concurrent insulin resistance may result in type 2 diabetes
(3,5).
The etiology and mechanisms leading to apoptosis of ß-cells together with
an inadequately low proliferation rate has not been completely elucidated.
Previously, we analyzed ß-cell turnover in pancreata of Psammomys
obesus, a rodent with a natural tendency to diet-induced type
2like diabetes (6).
Elevated glucose concentrations directly induced ß-cell apoptosis in
cultured islets from diabetes-prone P. obesus, but not in islets from
diabetes-resistant rats
(6,7).
Glucose-induced ß-cell proliferation was observed in both rat and P.
obesus islets; however, P. obesus showed only a limited capacity
of ß-cell proliferation in response to elevated glucose concentrations.
Based on these observations, we suggested the existence of a novel process of
glucotoxicity, in which chronic hyperglycemia is linked to a progressive loss
of ß-cell mass, when genetic susceptibility to diabetes exists.
Apart from hyperglycemia, plasma long-chain free fatty acid levels are
often increased in states of insulin resistance, further impairing
ß-cellsecretory function
(8,9).
In an adipogenic model of diabetes, the Zucker diabetic fatty rat, fatty
acidinduced ß-cell apoptosis was observed
(5). However, it is not known
whether islets of these diabetic models behave differently from those of
normal rats. Furthermore, the effect of fatty acids on ß-cell
proliferation has been investigated only at high fatty acid concentrations (2
mmol/l) in rat islets maintained in suspension for 7 days
(10).
Because ceramide is synthesized from long-chain fatty acids, it has been
postulated that free fatty acidinduced cell death is mediated via
formation of ceramide. Ceramide serves as a second messenger for cellular
functions ranging from proliferation and differentiation to growth arrest and
apoptosis (11). Shimabukuro et
al.
(5,12)
showed that islets from Zucker diabetic fatty rats manifested elevated
ceramide levels. In response to a challenge with fatty acids, these islets
displayed increased incorporation of fatty acids into ceramide, accompanied by
apoptosis. Fumonisin B1, a specific inhibitor of ceramide synthase activity,
blocked both ceramide generation and apoptosis, indicating that ceramide
generation was necessary for the apoptotic response. However, the possible
role of ceramide signaling on ß-cell proliferation has not been addressed
in these studies.
A critical event leading to apoptosis involves changes in mitochondrial
membrane, which culminates in the release of apoptogenic factors, such as
cytochrome c, from the mitochondrial intermembrane space to the cytosol
(13,14).
The loss of barrier function of both mitochondrial membranes is controlled, at
least in part, by the permeability transition pore complex, a polyprotein
complex that includes the most abundant protein of the inner mitochondrial
membranes, the adenine nucleotide translocator (ANT)
(15). ANT catalyses the
exchange of ADP and ATP across the inner mitochondrial membrane. Recent
studies indicate that proapoptotic proteins, such as Bax or Bak, interact with
ANT to facilitate membrane permeabilization
(16,17).
Others have shown that agents that inhibit ANT activity induce mitochondrial
swelling (18). It has also
been postulated that mitochondrial swelling and rupture of the outer membrane
may explain how cytochrome c is released
(19). Palmitoyl-CoA esters are
natural ligands for ANT (20);
however, whether fatty acids affect ANT expression and induce release of
cytochrome c from mitochondria of ß-cells is unknown.
Therefore, we investigated the role of fatty acids at different ambient
glucose concentrations on ß-cell proliferation, apoptosis, and function
in adult rat islets. Pancreatic islets were cultured onto plates coated with
extracellular matrix derived from bovine corneal endothelial cells, allowing
in situ identification of ß-cell apoptosis and proliferation. In
addition, the possible involvement of the ceramide and of the apoptotic
mitochondrial pathway was studied. To elucidate whether possible effects could
be ascribed to the degree of saturation at an identical carbon chain length,
ß-cells were exposed to palmitic (C16:0) and palmitoleic (16:1) acid
alone and in combination.
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RESEARCH DESIGN AND METHODS
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Islet isolation. Male Sprague-Dawley rats (200-220 g) were
anesthetized by ether, and islets isolated from the pancreata using an
adaptation for rat islets of the method of Gotoh et al.
(21). Briefly, after
cannulation of the common bile duct and instillation of 10 ml cold Hank's
balanced salt solution (Gibco, Gaithersburg, MD) containing 0.17 mg/ml
Liberase (Boehringer Mannheim, Mannheim, Germany), 0.1 mg/ml DNAse I
(Boehringer Mannheim), and 25 mmol/l HEPES, each pancreas was removed and
digested in 5 ml of the above described Liberase solution for 35 min at
37°C in a shaking water bath. This was followed by dilution and washing
with Hank's balanced salt solution containing 0.1 mg/ml DNAse I, 25 mmol/l
HEPES, and 10% fetal calf serum (Gibco). Islets from the crude pancreas digest
were purified by centrifugation through a discontinuous Histopaque 1077 (Sigma
Chemical, St. Louis, MO) gradient, washed with RPMI-1640 medium (Gibco), and
dispersed into culture dishes. Islet culture. Details of the procedure
have been described previously
(22,23).
Islets pooled from four rats were suspended in 7 ml RPMI-1640 medium (11.1
mmol/l glucose) containing 100 U/ml penicillin, 100 µg/ml streptomycin, 250
ng/ml amphotericin B, 40 µg/ml gentamicin, and 10% fetal calf serum
(Gibco). Of the islet suspension, 200 µl (30-50 islets) were added to 2 ml
of the above described medium and cultured onto 35-mm plates coated with
extracellular matrix derived from bovine corneal endothelial cells
(23) (Novamed, Jerusalem,
Israel). Two days after plating, when most islets were attached and began to
flatten, the culture medium was changed to RPMI containing 5.5,11.1, or 33.3
mmol/l glucose supplemented with bovine serum albumin (BSA) alone or with
fatty acids (0.5 mmol/l palmitic acid, 0.5 mmol/l palmitoleic acid, or a
mixture of 0.25 mmol/l or 0.5 mmol/l each). Fatty acids (Sigma) were dissolved
at 10 mmol/l in RPMI-1640 medium containing 11% fatty acidfree BSA
(Sigma) under an N2-atmosphere, shaken overnight at 37°C,
sonicated 15 min, and filtrated under sterile conditions (stock solution). For
control incubations, 11% BSA was prepared, as described above. Before use, the
effective free fatty acids concentrations were controlled with a commercially
available kit (Wako, Neuss, Germany). In some experiments, islets were
cultured with 15 µmol/l C2-ceramide (Biomol, Plymouth Meeting,
PA) or 15 µmol/l fumonisin B1 (Sigma); both were first dissolved in
prewarmed 37°C DMSO (Fluka, Buchs, Switzerland) at 5 mmol/l.
ß-cell replication. For ß-cell proliferation studies, a
monoclonal antibody to Ki-67 was used (MIB-5; Dianova, Hamburg, Germany).
Ki-67 is a nuclear antigen expressed by proliferating cells that is used as a
marker for late G1, S, G2, and M phases of the cell cycle
(24). After washing with
phosphate-buffered saline (PBS), cultured islets were fixed in 4%
paraformaldehyde (30 min at room temperature) followed by permeabilization
with 0.5% Triton X-100 (4 min at room temperature). Afterwards, islets were
incubated for 1 h at room temperature with monoclonal mouse antiKi-67
antibody diluted 1:10, followed by detection using a
streptavidin-biotin-peroxidase complex (Histostain-Plus Kit; Zymed, San
Francisco, CA). Subsequently, islets were incubated for 30 min at 37°C
with guinea pig antiinsulin antibody diluted 1:50 (Dako, Carpinteria,
CA), followed by a 10-min incubation with a 1:10 dilution of
fluoresceinconjugated rabbit antiguinea pig antibody (Dako, Glostrup,
Denmark).
Detection of apoptotic ß-cells. The free 3-OH strand breaks
resulting from DNA degradation were detected by the terminal deoxynucleotidyl
transferasemediated dUTP nick-end labeling (TUNEL) technique
(25). After islet cultures
were fixed and permeabilized as described above, the TUNEL assay was performed
according to the manufacturer's instructions (In Situ Cell Death Detection
Kit, AP; Boehringer Mannheim). The preparations were then rinsed with
Tris-buffered saline and incubated (10 min at room temperature) with a
5-bromo-4-chloro-indolyl phosphate/nitro blue tetrazolium liquid substrate
system (Sigma). Thereafter, islets were incubated with a guinea pig
anti-insulin antibody as described above and detection was performed using the
streptavidin-biotin-peroxidase complex (Zymed).
The TUNEL assay detects DNA fragmentation associated with both apoptotic
and necrotic cell death; therefore, islets were also treated with a
fluorescent annexin V probe (Annexin-V-FLUOS staining kit; Boehringer
Mannheim) according to the manufacturer's instructions. Double staining of
cells with propidium iodide and annexin V enables the differentiation of
apoptotic cells from necrotic cells.
After staining for proliferation or apoptosis, islets were embedded in
Kaiser's glycerol gelatin (Merck, Darmstadt, Germany) and analyzed by light
and fluorescent microscopy (microscope Axiolab; Zeiss, Jena, Germany).
Cytochrome c immunofluorescence staining and confocal microscopy.
Islet cultures were fixed and permeabilized as described above, incubated for
2 h at room temperature with mouse anticytochrome c monoclonal antibody
that was diluted 1:50 (PharMingen), and then incubated for 1 h with a 1:50
dilution of fluorescein-conjugated goat anti-mouse antibody (Jackson Immuno
Research Lab, West Grove, PA) and embedded in Dako fluorescent mounting
medium. Images were produced by a confocal laser scanning microscope (Zeiss
Axiophot fluorescence microscope) with a Zeiss Neofluar x40/1.3
objective lens connected to a Bio-Rad MRC-600 confocal scanner (Bio-Rad,
Lasership, Oxfordshire, U.K.) and a Silicon Graphics Personal Iris 4D/25
Work-Station (Silicon Graphics, Mountain View, CA).
Subcellular fractionation. For analysis of rat islet's subcellular
fractions, islets were cultured in suspension in RPMI-1640 medium containing
11.1 mmol/l glucose, as described above. One day after isolation, the medium
was changed, and groups of 200 islets were incubated for 6 or 20 h in medium
containing 11.1 mmol/l glucose with 0.5 mmol/l palmitate, 0.5 mmol/l
palmitoleic acid, or solvent. At the end of the incubations, islets were
washed in PBS, and mitochondrial and cytosolic (S100) fractions were prepared
from islets resuspended in 70 µ1 ice-cold buffer containing 20 mmol/l
HEPES-KOH (pH 7.5), 10 mmol/l KCl, 15 mmol/l MgCl2,1 mmol/l
Na-EDTA, 1 mmol/l dithiothreitol, 0.1 mmol/l phenylmethylsulfonyl fluoride,
and 250 mmol/l sucrose (26).
Mechanical homogenization was achieved by repeated aspiration through a
pipette. Unlysed cells and nuclei were pelleted by a 10-min centrifugation
(750g for 4°C). The supernatant was centrifuged at 10
000g for 15 min at 4°C. This pellet representing the
mitochondrial fraction was then resuspended in 10 µl of the above-described
buffer. Finally, the supernatant was centrifuged at 100,000g for 1 h
at 4°C. The supernatant from this final centrifugation represents the
S-100 fraction (27). Both
fractions were frozen at -80°C until use.
Western blot analysis. Mitochondrial and cytosolic fractions were
diluted 1:3 in sample buffer containing 187.5 mmol/l Tris-HCL, pH 6.8, 6% SDS,
30% glycerol, 150 mmol/l dithiothreitol, and 0.3% Bromphenol blue and were
boiled for 5 min. Equivalent amounts of each treatment group at a ratio of 5:3
cytosolic to mitochondrial fraction were run on 15% SDS polyacrylamide gels.
Proteins were electrically transferred to nitrocellulose filters and incubated
with a mouse anticytochrome c monoclonal antibody (PharMingen, San
Diego, CA) (1µg/ml for 1 h at room temperature), followed by incubation
with horseradish peroxidase linked antimouse IgG (Santa Cruz
Biotechnology, Santa Cruz, CA) (1 h at room temperature). After adding Lumiglo
reagent (Phototope-HRP Western Blot Detection Kit; Biolabs, Beverly, MA), the
emitted light was captured on X-ray film. As a marker, a biotinylated protein
molecular weight standard (Biolabs) was run in parallel according to the
manufacturer's instructions.
For ANT analysis, the nitrocellulose membrane was stripped for 30 min at
50°C in 40 ml of a watery solution containing 280 µl
ß-mercaptoethanol, 5 ml 0.5 mol/l Tris-HCl, pH 6.8, and 10% SDS, then
washed for 1 h in Tris-buffered saline containing 0.1% Tween-20, incubated
with an antiANT antibody (provided by T. Wallimann, Institute of Cell
Biology, Swiss Federal Institute of Technology, Zurich, Switzerland
[28]) and then incubated with
horseradish peroxidaselinked antirabbit IgG (Santa Cruz)
(1:5,000 for 1h at room temperature) and detected as described above.
Insulin release and content. Chronic insulin release was evaluated
in the culture medium collected before the termination of each experiment. To
determine acute insulin release in response to glucose stimulation, islets
were washed in RPMI-1640 medium containing 3.3 mmol/l glucose and were
preincubated for 1 h in the same medium. The medium was then discarded and
replaced with fresh medium containing 3.3 mmol/l glucose for 1 h for basal
secretion, followed by an additional 1 h incubation in medium containing 16.7
mmol/l glucose. Incubates were collected and frozen for insulin assays.
Thereafter, islets were washed with PBS and extracted with 0.18N HCl in 70%
ethanol for 24 h at 4°C; the acid-ethanol extracts were collected and
frozen for determination of insulin content.
Insulin was determined by a human insulin radiommunoassay kit (CIS Bio
International, Gif-Sur-Yvette, France) with rat insulin as the standard.
Crossreactivity of the antihuman insulin antibody used with rat insulin
is 89.5%. Statistical analysis. Data are presented as the means
± SE and were analyzed by Student's t test. P <
0.05 was considered significant. Cultures were evaluated in a randomized
manner by a single investigator (K.M.) who was blinded to the treatment
conditions. Care was taken to score islets of similar size.
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RESULTS
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Modulation of ß-cell proliferation and apoptosis after exposure to
free fatty acids and glucose. Exposure of adult rat islets in long-term
culture to palmitic acid for 4 days resulted in an increased number of
ß-cells with TUNEL-positive nuclei
(Fig. 1A and
B). The increase was 5.2-, 8.9-, and 5.5-fold at 5.5,
11.1, and 33.3 mmol/l glucose, respectively, compared with islets exposed to
identical glucose concentrations and solvent (albumin)
(Fig. 3A). In
contrast, the monounsaturated palmitoleic acid did not induce DNA
fragmentation and, when present with palmitic acid (0.25 mmol/l and 0.5 mmol/l
each), its effect on ß-cell death was inhibited. Baseline ß-cell
death in the absence of fatty acids was minimal at 11.1 mmol/l glucose and
increased 3.6- and 3.0-fold at 5.5 and 33.3 mmol/l glucose, respectively.

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FIG. 1. Characterization of the effect of palmitic acid on ß-cell death by
double staining with the TUNEL assay (alkaline phosphatase) and anti-insulin
antibody (peroxidase) (A and B) and by double fluorescent
staining with Annexin-VFLUOS (green) (C and D) and
propidium iodide (red) (E and F). Islets were plated on
extracellular matrix-coated dishes and exposed for 4 days to media containing
11.1 mmol/l glucose alone (A, C, and E) and with 0.5 mmol/l
palmitic acid (B, D, and F). The arrows indicate nuclei
stained positive for the TUNEL reaction (light and fluorescence microscopy
x 400).
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FIG. 3. Fatty acids and glucose-induced ß-cell DNA fragmentation and
proliferative activity. Islets were cultured for 4 days in the absence or
presence of different fatty acids (0.5 mmol/l) or a mixture of palmitoleic and
palmitic acid (0.25 mmol/l [mixture] or 0.5 mmol/l [mixture 0.5] each) in 5.5,
11.1, and 33.3 mmol/l glucose. Results are means ± SE of the relative
number of TUNEL+ (A) and Ki-67+ ß-cells
(B) per islet, normalized to control incubations at 5.5 mmol/l
glucose alone (100%) (in absolute value: 0.82 TUNEL+ ß-cells
per islet and 4.06 Ki-67+ ß-cells per islet at 5.5 mmol/l
glucose alone). The mean number of islets scored for DNA fragmentation was 40,
68, and 49 and the mean number for proliferative activity by anti-Ki-67
staining was 78, 69, and 63 in media containing 5.5, 11.1, and 33.3 mmol/l
glucose, respectively. Islets were isolated from 40 rats.
*P < 0.01 between control and palmitic or palmitoleic
acid at the same glucose concentration; P < 0.05 between
palmitic acid and fatty acid mixture at the same glucose concentration;
**P < 0.01 relative to islets at 5.5 mmol/l
glucose.
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In parallel with the TUNEL assay, treated islets were incubated with
annexin V and propidium iodide to discriminate apoptotic from necrotic cells
(Fig. 1CF).
Exposure of cultured islets to palmitic acid markedly increased the number of
cells exhibiting phosphatidylserine molecules translocated to the outer
leaflet of the plasma membrane, as revealed by annexin V binding (1.96
± 0.36 Annexin-V-FLUOSpositive cells/islet in control [11.1
mmol/l glucose] vs. 6.78 ± 1.05 in 0.5 mmol/l palmitic
acidtreated islets, P < 0.01). Most of these cells had
intact plasma membranes, impermeable to the DNA-binding dye propidium iodide
(0.8 ± 0.01 propidium iodidepositive cells/islet in control vs.
2.14 ± 0.26 in 0.5 mmol/l palmitic acidtreated islets,
P < 0.01). Therefore, the palmitic acidinduced DNA
fragmentation, as determined by the TUNEL assay, mainly represents apoptotic
cell death.
Exposure to high concentrations of glucose for 4 days resulted in increased
proliferation of ß-cells by 1.9- and 1.8-fold at 11.1 and 33.3 mmol/l
glucose, respectively, compared with islets at 5.5 mmol/l glucose (Figs.
2 and
3B). In contrast,
addition of palmitic acid resulted in a similar decrease in ß-cell
proliferation at all glucose concentrations (45, 33, and 46% decreases at 5.5,
11.1, and 33.3 mmol/l glucose, respectively, compared with islets incubated in
identical glucose concentrations and solvent)
(Fig. 3B). On the
other hand, palmitoleic acid augmented ß-cell proliferation by 65% at 5.5
mmol/l glucose, whereas at higher glucose concentrations, when the maximal
proliferative activity was achieved with glucose alone, no additional effect
was obtained. Treatment of the cells with a mixture of palmitoleic and
palmitic acid had no influence on ß-cell proliferation
(Fig. 3B).

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FIG. 2. Double immunostaining for the Ki-67 nuclear antigen (peroxidase)
(A and B) and insulin (fluorescein) (C and
D). Islets plated on extracellular matrix-coated dishes were exposed
for 4 days to media containing 11.1 mmol/l glucose alone (A and
C) or including 0.5 mmol/l palmitic acid (B and D).
The arrows indicate nuclei stained positive for Ki-67 (light and fluorescence
microscopy x400).
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Ceramide signaling in palmitic acid induced changes in ß-cell
apoptosis and proliferation. In pancreatic islets cultured with 15
µmol/l C2-ceramide in the presence of 11.1 mmol/l glucose,
ß-cell DNA fragmentation was increased 2.6-fold, whereas ß-cell
proliferation was decreased by 71.1%, showing the same trend as that observed
in palmitic acidtreated islets (Fig.
4). Addition of 15 µmol/l of the ceramide synthase inhibitor
fumonisin B1 in cultured medium containing 11.1 mmol/l glucose and 0.5 mmol/l
palmitic acid, reduced the effect of palmitate on ß-cell apoptosis and
had a tendency to reverse its antiproliferative effect
(Fig. 4).

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FIG. 4. Effects of blockade of ceramide synthesis by fumonisin B1 and of
exogenous ceramide on ß-cell DNA fragmentation and proliferative
activity. Islets were cultured for 4 days in 11.1 mmol/l glucose alone
(control) or in the presence of 0.5 mmol/l palmitic acids with or without 15
µmol/l fumonisin B1 (Fumo) or in the presence of 15 µmol/l
C2-ceramide. Results are means ± SE of the relative number
of TUNEL+ (A) and Ki-67+ ß-cells
(B) per islet normalized to the solvent-treated control (100%) (in
absolute value: 0.61 TUNEL+ ß-cells per islet and 7.32
Ki-67+ ß-cells per islet at 11.1 mmol/l glucose alone). The
mean number of islets scored for DNA fragmentation and for proliferative
activity by anti-Ki-67 staining was 85 and 71, respectively. Islets were
isolated from 16 rats. *P < 0.01 relative to
solvent-treated controls.
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Palmitic acidinduced decrease in ANT expression and cytochrome c
release. Exposure of pancreatic islets to palmitic acid, compared with
palmitoleic acid and solvent-treated islets, induced a time-dependent decrease
of mitochondrial ANT expression (Fig.
5). The decrease was already detectable after 6 h of treatment,
and after 20 h, ANT in the mitochondrial fraction of palmitic
acidtreated islets was almost undetectable. At the same time,
cytochrome c of palmitic acidtreated islets was shifted from the
mitochondrial to the cytosolic fraction, whereas most cytochrome c in
palmitoleic acid or control islets was confined to the mitochondria
(Figs.5 and
6). No ANT was detectable in
any cytosolic fraction, thereby confirming the integrity of the mitochondrial
and cytosolic preparations.

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FIG. 5. Subcellular localization of ANT and cytochrome c in fatty acid-treated
islets. Immunoblotting of ANT (molecular weight 30 kDa) and cytochrome c
(molecular weight 15 kDa) was performed on mitochondrial (M) and cytosolic (C)
fractions of islets cultured at 11.1 mmol/l glucose for 6 or 20 h with 0.5
mmol/l palmitic acid or 0.5 mmol/l palmitoleic acid. Control islets were
analyzed after 20 h of incubation. Both antibodies were blotted on the same
membrane after stripping. One representative of three experiments is
shown.
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FIG. 6. Confocal images showing cytochrome c diffusely localized to the
cytoplasm but not significantly to the mitochondria in palmitic acid-treated
islets. Islets were plated on extracellular matrix-coated dishes and were
exposed for 4 days to media containing 11.1 mmol/l glucose alone (A)
and with 0.5 mmol/l palmitic acid (B) and stained with
anti-cytochrome c antibody.
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Insulin release and content. Exposure of rat islets to 0.5 mmol/l
palmitoleic acid for 4 days increased islet insulin content and chronic
insulin secretion as compared to control and palmitic acid treated islets
(Fig. 7A and
B). Incubation with 0.5 mmol/l palmitic acid decreased
islet insulin content; addition of 0.5 mmol/l palmitoleic to 0.5 mmol/l
palmitic acid prevented this effect. An acute glucose challenge of palmitoleic
acidtreated cells induced a higher insulin release rate compared with
control, whereas treatment with palmitic acid abolished the insulin response
to glucose (Fig.
7C).

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FIG. 7. Effect of prolonged exposure of cultured islets to fatty acids on islet
insulin content (A), on chronic insulin release into the culture
medium (B), and on basal and glucose-stimulated insulin secretion
(C). Islets were incubated in 11.1 mmol/l glucose in the absence or
presence of 0.5 mmol/l palmitic or palmitoleic acid or a mixture of both (0.5
mmol/l each) for 4 days. Chronic insulin secretion represents the amount
secreted into the culture medium during the experiment. Basal and stimulated
insulin secretion denotes the amount secreted over 1 h incubation at 3.3 and
16.7 mmol/l glucose, respectively. Each bar represents the mean of 4 separate
experiments ± SE. In each experiment, the data were collected from 4
plates per treatment. *P < 0.05 relative to
solvent-treated controls; P < 0.05 for the difference
between palmitic acid and palmitoleic acid or fatty acid mixture.
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DISCUSSION
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This study shows that the saturated palmitic acid reduces the proliferative
capacity of ß-cells and induces ß-cell death mainly by apoptosis.
Conversely, palmitoleic acid, a monounsaturated fatty acid with identical
carbon chain length, exhibits the opposite effects: it does not affect
apoptosis, but promotes ß-cell proliferation and counteracts the toxic
effects of palmitic acid. The cell-permeable ceramide analog
C2-ceramide mimics the palmitic acidinduced changes in the
cell cycle, which were blocked by the ceramide synthetase inhibitor fumonisin
B1. Thus, formation of ceramide is required to mediate the palmitic acid
effects on ß-cell turnover. Additionally, palmitic acid but not
palmitoleic acid decreases the expression of ANT. This is accompanied by
increased translocation of cytochrome c from the mitochondria to the cytosol.
The overall beneficial effect of palmitoleic acid is also reflected by
improved parameters of ß-cell function: palmitoleic acid increased islet
insulin content as well as chronic and glucose-stimulated insulin secretion.
Furthermore, it prevented the palmitic acidinduced decrease in islet
insulin content and impaired glucose-stimulated insulin secretion.
The distinct effects of the saturated palmitic acid and the monounsaturated
palmitoleic acid on ß-cell turnover and function are striking. The fact
that fatty acids longer than 15 carbons may be harmful to various cell types
has been previously reported
(29,30,31,32,33,34,35).
At the same concentrations, chronic exposure to saturated fatty acids of
different length have similar effects. Likewise, monounsaturated fatty acids
C16:1 and C18:1 induced similar effects
(29,30,31,32,33,34,35).
However, saturated fatty acids were associated with different effects than
unsaturated fatty acids. A putative explanation for this difference is based
on the relative high melting point of saturated fatty acids (63°C for
palmitic acid) compared with unsaturated fatty acids (0.5°C for
palmitoleic acid). Consequently, triacylglycerols synthesized from saturated
fatty chains are insoluble at 37°C. This observation has led to the
hypothesis that, immediately after their formation, saturated triacylglycerol
molecules precipitate at the site of synthesis (i. e., the sarcoplasmatic
reticulum)
(36,37).
These precipitates are thought to hamper sarcoplasmatic reticulum function.
However, it remains to be established whether this process is linked to
apoptosis. A second explanation may be related to changes in membrane
fluidity. Indeed, enrichment of phospholipids by saturated fatty acids lowers
membrane fluidity, which severely hinders membrane function
(38,39).
Moreover, it has been shown in a neuronal cell line that an increase of
saturated fatty acids in the phospholipid pool forms an essential part of the
apoptotic process (40).
Furthermore, alterations in the composition of membrane phospholipids may act
as a trigger for apoptosis, as demonstrated in a promyelocytic cell line, by
inhibition of the remodeling of long-chain unsaturated fatty acids between the
phospholipids of cells (41).
Finally, the specific toxic effects of saturated fatty acids may relate to
ceramide formation. Recent studies indicate that signal transduction through
the ceramide pathway activates apoptosis in various cell types
(11), including islets from
the Zucker diabetic fatty rats
(5). Moreover, an increase in
cellular levels of palmitic or stearic acid but not in levels of palmitoleic
acid is correlated with de novo synthesis of ceramide
(42). In line with these
findings, the present study shows that the ceramide synthetase inhibitor
fumonisin B1 blocked the deleterious effects of palmitic acid.
Palmitic acid has a powerful acute insulinotropic effect
(32). However, exposure to
palmitic acid for 4 days did not increase total insulin secretion. Possibly,
the acute insulinotropic effect gradually disappears in conjunction with
increased ß-cell apoptosis, leading to an overall unchanged insulin
release.
The enhancement of TUNEL+ ß-cells by ceramide is modest
compared with the effect of palmitic acid. The difference is probably not due
to reduced cell permeability of C2-ceramide, because it has a
dramatic effect on ß-cell proliferation, stronger than the palmitic acid
effect. The time course of the C2-ceramide effect possibly differs
from the palmitic acid effect, with an earlier increase in apoptosis that
leads to a faster decrease in proliferation.
Glucose-induced apoptosis was observed in ß-cells of ob/ob
mice and of Wistar rats maintained in medium containing 10% fetal calf serum
(43). In contrast, high
glucose concentrations promoted survival of purified ß-cells from adult
Wistar rats cultured in serum-free conditions
(7). Islets from the
diabetes-prone P. obesus, which were cultured on extracellular
matrix-coated plates in the presence of serum, responded to elevated glucose
levels by increasing the rate of ß-cell apoptosis, whereas no significant
change was observed in similarly treated islets of adult Sprague-Dawley rats
(6). In the present study,
elevated glucose concentrations had only a marginal effect on ß-cells
apoptosis, whereas exposure to palmitic acid resulted in a substantial
increase in the number of ß-cells with TUNEL+ nuclei
independent of the medium glucose levels.
The disruption of mitochondrial ATP/ADP exchange is among the earliest
identified events that may initiate apoptosis
(44). ANT catalyses the
exchange of ADP and ATP across the inner mitochondrial membrane. Here we
demonstrate that palmitic acid decreases the expression of ANT. The loss of
ANT activity induces mitochondrial swelling
(18), as well as rupture of
the outer membrane leading to cytochrome c release and apoptosis
(19), as observed in the
present study. The mechanism leading to decreased ANT expression by palmitic
acid is unknown. Yet, irrespective of the mechanism involved, our results
attest that the mitochondrion is an important target for palmitic acid-induced
apoptosis in ß-cells from adult rat islets.
Although various factors including glucose, fatty acids, and amino acids
have been shown to govern ß-cell proliferation
(2,6,10,45),
to our knowledge, this study is the first to demonstrate the distinct role of
saturated and monounsaturated fatty acids in ß-cell replication. Whereas
palmitoleic acid was shown to stimulate ß-cell proliferation at
normoglycemic glucose concentrations, palmitic acid exhibited an inhibitory
effect independent of a medium glucose level. With the induction of apoptosis,
the deleterious effects of palmitic acid could lead to a reduction in
ß-cell mass, an important determinant of ß-cell functional
activity.
 |
ACKNOWLEDGMENTS
|
---|
This work was supported by grants from the Swiss National Science
Foundation (no. 3200-057595.99 to M.Y.D.) and the Israel Science Foundation
(no. 251/97 to N.K.). M.Y.D. is supported by the Max Cloetta Foundation.
We wish to acknowledge the skillful assistance of Gretha
Siegfried-Kellenberger and Heidi Seiler.
 |
FOOTNOTES
|
---|
ANT, adenine nucleotide translocator; BSA, bovine serum albumin; PBS,
phosphate-buffered saline; TUNEL, terminal deoxynucleotidyl
transferasemediated dUTP nick-end labeling.
Received for publication July 14, 2000
and accepted in revised form October 2, 2000
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