From the a Department of Internal Medicine and Institute of Cellular Biology and Morphology, University Hospital, Lausanne 1011, Switzerland, the d Department of Molecular Genetics, University of Texas, Southwestern Medical Center, Dallas, Texas 75390-9046, the f Department of Molecular Genetics, University of Vienna, Vienna 1030, Austria, g Geriatric Research, Educational Clinical Center, Veterans Affairs Palo Alto Health Care System, Palo Alto, California 94304, and the h Division of Endocrinology and Diabetes, University Hospital, Geneva CH 1211, Switzerland
Received for publication, January 6, 2003, and in revised form, February 4, 2003
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ABSTRACT |
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Diabetes is associated with significant
changes in plasma concentrations of lipoproteins. We tested the
hypothesis that lipoproteins modulate the function and survival of
insulin-secreting cells. We first detected the presence of several
receptors that participate in the binding and processing of plasma
lipoproteins and confirmed the internalization of fluorescent low
density lipoprotein (LDL) and high density lipoprotein (HDL) particles
in insulin-secreting Abnormalities in both glucose and lipid homeostasis are
involved in the pathogenesis of type 2 diabetes (1). The lipid phenotype observed in diabetic patients is characterized by low levels
of HDL1 cholesterol, moderate
elevations in triglyceride-rich remnant particles, borderline to high
LDL cholesterol, and an increase in plasma free fatty acids (2).
Chronically elevated concentrations of free fatty acids have been shown
to induce a state of insulin resistance and to alter Although the contribution of free fatty acids to the development of
diabetes has been studied extensively, the role of lipoproteins in
Lipoprotein Preparation--
Blood was collected from healthy
donors. Plasma VLDL, LDL, and HDL fractions were isolated by sequential
ultracentrifugation (VLDL density, 1.019; LDL density, 1.063; HDL
density, 1.21), dialyzed against PBS, and then filter sterilized using
a 0.22-µm microfilter. Protein and cholesterol concentrations were
measured using the Bio-Rad protein assay and the cholesterol CHOD-PAP
method (Roche Diagnostics). Samples were analyzed by SDS-PAGE to assess the integrity of the apolipoproteins and the purity of the different fractions. The lipoprotein preparations contained less than 0.112 unit
of endotoxin/µmol of cholesterol as determined by the kinetic chromogenic technique (Endotell, Allschwil, Switzerland). Cholesteryl BODIPY-human HDL3 was reconstituted as described elsewhere
(9).
Cell Culture and Pancreatic Islet Isolation--
The
insulin-secreting cell line
Islets were isolated from C57BL/6 wild-type (WT) mice and mice lacking
the LDL receptor (LDLR RNA Extraction, Reverse Transcription PCR, and Quantitative
Reverse Transcription PCR--
Total RNA was extracted from
We performed quantitative real time PCR using the LightCycler
Instrument (Roche Diagnostics) and the QuantiTect SYBR Green PCR kit
(Qiagen). For HMG-CoA reductase quantification, cells were starved for
12 h and then incubated in the presence or absence of 3.0 mM LDL cholesterol for 15 h. For insulin and cyclin B1 quantification, cells were incubated for 48 h with different
lipoprotein subfractions. Total RNA was extracted and reverse
transcribed as described above. The cDNA was subsequently amplified
with the appropriate primers (initial activation at 95 °C for 15 min, denaturing at 95 °C for 15 s, primer annealing at 51 °C
for 20 s, and chain elongation at 72 °C for 18 s) through
40 cycles. Data were analyzed with the LightCycler Software (Roche
Diagnostics). Tubulin and glyceraldehyde-3-phosphate dehydrogenase were
used for normalization.
Western Blotting--
Cells were washed once in cold PBS and
lysed in Promega passive lysis buffer (Promega, Madison, WI). A total
of 40 µg of proteins was subjected to SDS-PAGE and transferred onto a
nitrocellulose membrane by electroblotting. Membranes were blocked by
incubation for 1 h in a buffer containing 0.1% Tween 20 and 5%
milk and incubated overnight at 4 °C with the primary antibodies.
The immunoreactive bands were visualized using a chemiluminescent
substrate (Pierce) after incubation of the nitrocellulose filters for
1 h with secondary anti-rabbit or anti-mouse antibodies conjugated
to horseradish peroxidase. Densitometric scanning of films was
performed using Quantity One software (Bio-Rad).
Antisera directed against SR-BI, LDLR, LRP, VLDLR, and apoER2
have been described previously (10-14). The following antibodies were
obtained commercially: anti-cleaved caspase-3 (Asp-175; Cell Signaling Technology, Beverly, MA), anti-JIP-1 (BD Biosciences, Basel, Switzerland), and anti- Immunohistochemistry--
Murine pancreases were fixed in a
solution containing 1% paraformaldehyde and subsequently 30% sucrose.
12-µm sections were prepared and blocked for 1 h in a solution
containing 1% bovine serum albumin and 0.3% Triton X-100 (Sigma).
Incubation with the primary antisera (dilutions 1:250) was performed at
4 °C for 24 h. After several washes in PBS, sections were
incubated for 30 min with secondary Alexa 488-conjugated
anti-rabbit IgG (Molecular Probes, Eugene, OR) at a dilution of
1:400. Slices were analyzed with a confocal microscope (Leica
Microsystems, Glattbrugg, Switzerland).
Lipoprotein Uptake--
Hoechst 33342 Staining, Immunocytochemistry, and
TUNEL--
For immunocytochemistry, fixed cells were incubated for 1 h in
blocking buffer (1% bovine serum albumin, 0.3% Triton X-100) and
overnight at 4 °C with the primary antisera (dilutions: anti-SR-BI, anti-LDLR, anti-VLDLR, anti-apoER2, 1:250; anti-cleaved caspase-3, 1:40). After four washes in PBS, coverslips were incubated for 30 min
with secondary Alexa 488-conjugated anti-rabbit IgG at a dilution of
1:400. For cleaved caspase-3, cells were counterstained for 3 min with
Hoechst 33342 (dilution 1:1,500), and views were taken under a
fluorescence microscope (100× objective) at 350 and 500 nm. The
resulting images were subsequently assembled by using Photoshop
technology (Adobe Systems). For SR-BI, LDLR, VLDLR, and apoER2, views
were taken with a confocal microscope. For TUNEL, fixed cells were
incubated for 2 min at 4 °C in permeabilization solution (0.1%
Triton X-100, 0.1% sodium citrate). Coverslips were then rinsed, and
50 µl of TUNEL reaction mixture (Roche Diagnostics) was added to each
sample. Slides were incubated in a humidified chamber for 60 min at
37 °C and counterstained for 3 min with Hoechst 33342.
Quantification of Apoptosis in Islets--
Mouse islets were
incubated for 48 h in the presence of lipoproteins, fixed with 1%
paraformaldehyde in PBS, and stained for 7 min with 1 µM
SYTOX Green. For each islet analyzed, 5-15 views spanning the entire
structure, each separated by 2 µm, were taken with a confocal
microscope. The numbers of healthy and apoptotic nuclei were then
evaluated using the Neurolucida software (MicroBrightField, Inc.,
Williston, VT).
In Situ Cell Proliferation--
Akt/PKB Assay--
Akt activity was determined using
a kit from Cell Signaling Technology. Statistical Analysis--
Results are presented as means ± S.E. Unpaired two-tailed Student's t test was used to
compare groups.
Lipoprotein Receptor Expression in Insulin-secreting Cells--
We
first evaluated whether the genes encoding several receptors implicated
in the binding and the internalization of lipoproteins were expressed
in the insulin-secreting cell line Uptake of LDL and HDL Particles in Assessment of Apoptosis in
As shown in Fig. 5A, a 2-day
culture with increasing concentrations of VLDL or LDL in RPMI
containing 11.1 mM glucose induced a
dose-dependent increase in the apoptotic rate in
We then confirmed in freshly isolated islets the effects observed in
Finally, we examined the role of human lipoproteins on
insulin mRNA (Fig. 5D). As measured by quantitative
reverse transcription PCR, insulin mRNA was reduced markedly under
VLDL or LDL exposure. HDL particles had no influence and did not
alleviate the detrimental effects of elevated LDL on insulin transcript
levels. LDL at a lower concentration (3.1 mM) still reduced
insulin mRNA but without inducing cell death.
Caspase-3 Activation and Contribution of the JNK Pathway to
VLDL-induced Apoptosis--
Caspase-3 is one of the key executioners
of programmed cell death. Therefore, we quantified the amount of the
cleaved active form by Western blotting in
Islet-brain 1/JNK-interacting protein-1 (IB1/JIP-1) is a scaffold
protein that interacts with several components of the JNK signaling
pathway (15). We examined whether this pathway is implicated in
apoptosis induced by lipoproteins. Incubations with 3.1 mM
VLDL reduced IB1/JIP-1 levels by 56%, whereas LDL or HDL had no effect
(Fig. 6B). A cell-permeable peptide inhibitor of the JNK
pathway (16) partially inhibited apoptosis induced by VLDL, but not by
LDL particles (Fig. 6C). Taken together, these data suggest
that cell death triggered by VLDL depends on caspase-3, IB1/JIP-1, and
JNK.
Effects of Lipoproteins on Cell Proliferation, Cyclin
B1 Transcription, and Akt/PKB Activation--
Cell
proliferation in This study shows that several members of the LDLR family are
expressed in Exposure of insulin-secreting cells to human VLDL and LDL induces an
increase in apoptosis which depends on both the duration of the
incubation and the concentration of particles. Cell death triggered in
this way appears to occur via two different mechanisms. Caspase-3 is
strongly activated in the presence of elevated VLDL concentrations,
whereas cellular disassembly and packaging into apoptotic bodies, which
take place in the presence of LDL, are independent of caspase-3.
Moreover, VLDL, but not LDL, reduces the levels of IB1/JIP-1. This
protein is expressed at high levels in pancreatic Our study establishes that the LDLR plays an important role in We show that in contrast to VLDL and LDL, HDL promotes We demonstrate that lipoproteins have significant effects on cellular
proliferation. HDL has been identified as potent mitogens in vascular
smooth muscle by stimulating entry into S phase (23). In accordance
with this result, HDL increases insulin-secreting cell proliferation in
our system. VLDL and LDL, in contrast, induce a decrease in
proliferation which is associated with a reduction in cyclin B1 gene
expression. This may explain the reduction in proliferation because
cyclin B1 protein complexes with p34(cdc2) to form the
mitosis-promoting factor.
Finally, we establish that VLDL and LDL, but not HDL, markedly reduce
the level of insulin transcript. These effects are not attributable
solely to apoptosis triggered by these lipoproteins because they occur
even with doses too low to cause apoptosis or when apoptosis is
inhibited with HDL. Deleterious effects of free fatty acids on insulin
transcription are known (24), but our data are the first to demonstrate
a similar role for lipoproteins.
Lipoprotein concentrations used in our study are in the normal or
supraphysiological range observed in human plasma. Because pancreatic
islets are highly vascularized structures with fenestrated capillaries
(25), the settings chosen in our experiments probably reflect the
conditions found in vivo. Recently, Cnop et al.
(26) studied the role of LDL in rat The present study is in line with genetic studies and pharmacological
interventions in humans. It has recently been suggested that a locus on
chromosome 9 is linked to both cholesterol levels and diabetes mellitus
(27). Blood lipoprotein levels have been confirmed as important
predictors for the onset of type 2 diabetes, and preventative
administration of the HMG-CoA reductase inhibitor pravastatin was
associated with a reduction of 30% in the occurrence of type 2 diabetes in the West of Scotland Coronary Prevention Study (WOSCOPS)
(28). In conclusion, we propose that the changes in plasma lipoproteins
observed during type 2 diabetes are not only a consequence of the
disease but may contribute to the pathogenesis of the disease itself.
-cells. Purified human very low density
lipoprotein (VLDL) and LDL particles reduced insulin mRNA levels
and
-cell proliferation and induced a dose-dependent
increase in the rate of apoptosis. In mice lacking the LDL receptor,
islets showed a dramatic decrease in LDL uptake and were partially
resistant to apoptosis caused by LDL. VLDL-induced apoptosis of
-cells involved caspase-3 cleavage and reduction in the levels of
the c-Jun N-terminal kinase-interacting protein-1. In contrast,
the proapoptotic signaling of lipoproteins was antagonized by HDL
particles or by a small peptide inhibitor of c-Jun N-terminal kinase.
The protective effects of HDL were mediated, in part, by inhibition of
caspase-3 cleavage and activation of Akt/protein kinase B. In
conclusion, human lipoproteins are critical regulators of
-cell
survival and may therefore contribute to the
-cell dysfunction
observed during the development of type 2 diabetes.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-cell function.
The susceptibility of these cells to apoptosis is enhanced, and their
insulin content and glucose-stimulated insulin release are
significantly reduced (3, 4). Overt type 2 diabetes is present when the
-cell mass cannot compensate for insulin resistance. In Zucker
diabetic fatty rats, free fatty acids are elevated before the onset of diabetes, leading to triglyceride accumulation in the islets and increased
-cell death (5, 6).
-cell function is only partially documented. Grupping et
al. (7) described the presence of LDL binding sites in rodent and human pancreatic
-cells. More recently, Cnop et al. (8)
demonstrated the uptake of LDL and VLDL in lipid-storing vesicles of
rat and human
-cells. Given the key role of lipid homeostasis in
-cell function and survival, we hypothesized that lipoproteins
modify the susceptibility of
-cells to apoptosis. Here we describe
the presence of functional lipoprotein receptors in mouse pancreatic islets and in a transformed insulin-secreting
-cell line. We demonstrate that human purified VLDL and LDL induce a
dose-dependent increase in the rate of apoptosis and a
decrease in the levels of insulin transcript. In contrast, HDL
efficiently antagonizes cell death by mechanisms that include
activation of protein kinase B (Akt/PKB) and inhibition of caspase-3
cleavage. Finally, we establish that the LDL receptor (LDLR) and the
c-Jun N-terminal kinase (JNK) signaling pathway play an important role
in apoptosis induced by lipoproteins. These results demonstrate how the
modifications in lipoproteins observed in type 2 diabetes could
contribute to the pathogenesis and progression of
-cell failure.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
TC3 (cells over 120 passages) was
cultured in RPMI 1640 supplemented with 10% fetal calf serum, 100 µg/ml streptomycin, 100 units/ml penicillin, and 2 mM
glutamine (Invitrogen). Both 11.1 mM and 5.0 mM
glucose concentrations were used. Interleukin-1
(Alexis,
Carlsbad, CA) was used at a concentration of 0.3 ng/ml, tumor
necrosis factor-
(Alexis) at a concentration of 0.3 ng/ml, and
interferon-
(R&D Systems) at a concentration of 15 ng/ml.
Lipoproteins were used at the following cholesterol concentrations:
VLDL, 1.6 mM, 3.1 mM, 6.2 mM; LDL, 1.6 mM, 3.1 mM, 6.2 mM; HDL, 1.0 mM. Cell-permeable JNK inhibitor 1 (L-stereoisomer; Alexis) was added at a concentration of
1.5 µM 30 min before the addition of lipoproteins and
again after 18 and 36 h.
/
) (ages 2-3 months; Jackson
Laboratory, Bar Harbor, ME) by intraductal digestion of the pancreas
with the Liberase RI Purified Enzyme Blend (Roche Diagnostics)
according to the manufacturer's protocol. Islets were hand-picked to
purity and cultured in
TC3 culture medium supplemented with 1% 1 M HEPES, pH 7.4. and 0.1% 50 mM
-mercaptoethanol (Invitrogen).
TC3
cells using the RNAqueousTM-4PCR kit (Ambion, Huntingdon,
UK) according to the manufacturer's protocol and treated with DNase to
remove residual contaminations with DNA. RNA was reverse transcribed
and cDNA amplified by PCR using Superscript II reverse
transcriptase, Taq polymerase, and oligo(dT) primers
(Invitrogen). The primers used for the VLDL receptor (VLDLR) were
5'-TCA TCA TCT GTG CTT ACA-3' and 5'-ACT TAC AGT GAG ACA AAA G-3'. For
the LDLR, the primer sequences were 5'-CTG TTC CCA CCT CTG TTT AC-3'
and 5'-AGT GAG ATA CGG CGA ATA GA-3'. For LDLR-related protein (LRP),
primer sequences were 5'-CTA CAC CAC ATC CAC CAT-3' and 5'-ACT CAT CCA
GCA CAA AGG-3'. Sequences for apolipoprotein E receptor 2 (apoER2) were
5'-CAG TGG CTG TCC CTC ACT CGG-3' and 5'-CAG GGC AGT CCA TCA TCT TCT
TC-3'. Scavenger receptor class B type I (SR-BI) primer sequences were
5'-GCC TGT TGG TTG GGA TGA-3' and 5'-CTT GCT GAG TCC GTT CCA TT-3'.
3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase)
primer sequences were 5'-TGG GAC CAA CCT TCT ACC TC-3' and 5'-CAT CAA
GGA CAG CTC ACC AG-3'. Tubulin primer sequences were 5'-GGA GGA TGC TGC
CAA TAA CT-3' and 5'-GGT GGT GAG GAT GGA ATT GT-3'.
Glyceraldehyde-3-phosphate dehydrogenase primer sequences were 5'-TCA
AGA AGG TGG TGA AGC AG-3' and 5'-AAG GTG GAA GAG TGG GAG TT-3'. Insulin
primer sequences were 5'-TGG CTT CTT CTA CAC ACC CA-3' and 5'-TCT AGT
TGC AGT AGT TCT CCA-3'. Cyclin B1 primer sequences were 5'-AGC GAA GAG
CTA CAG GCA AG-3' and 5'-CAT GTT CAA GTT CAG GTT CAG G-3'. After
amplification (activation at 95 °C for 5 min, denaturing at 95 °C
for 0.5 min, primer annealing at 51 °C for 0.5 min, and chain
elongation at 72 °C for 0.5 min) through 32 cycles, the whole volume
of the PCR was separated by electrophoresis in a 3% agarose gel.
-tubulin (clone B-5-1-2; Sigma). Antisera were used at dilutions ranging from 1:250 to 1:1,000.
TC3 cells and mouse islets were
seeded onto coverslips, starved for 4 h, and incubated with
cholesteryl BODIPY-human HDL3 FL LDL (10 µg/ml medium;
Molecular Probes) in the presence or absence of excess unlabeled LDL
(100 µg/ml medium). After incubation for 2 h at 37 °C, cells
were fixed and analyzed by light (for
TC3 cells) or confocal (for
islets) microscopy. Microscope settings were kept strictly identical
between different conditions. Similar experiments were performed with
cholesteryl BODIPY-human HDL3 (30 µg/ml medium) in the
presence or absence of excess unlabeled HDL (600 µg/ml medium).
TC3 cells were grown on
laminin/poly-D-lysine-coated coverslips. After 2 days, the
culture medium was changed, and cytokines and/or lipoproteins were
added. Incubations were performed for 48 h. Cells were then fixed
for 15 min with 1% paraformaldehyde in PBS. To evaluate the number of
apoptotic cells, cells were incubated in the presence of Hoechst 33342 (dilution 1:1,500; Molecular Probes) for 3.5 min, and the nuclear
morphology was analyzed under a fluorescence microscope. The number of
cells displaying a pycnotic (highly condensed) nucleus and/or a
fragmented nucleus was evaluated blind by two different investigators.
A minimum of 250 cells in four separate experiments was counted for
each condition. For the analysis of nuclear morphology, fixed cells
were stained for 7 min with 1 µM SYTOX Green
(Molecular Probes). For every cell analyzed, 10 views spanning the
entire nucleus were taken at high magnification with a confocal
microscope, and three-dimensional images were generated using the
Imaris software (Silicon Graphics, Inc., Mountain View, CA).
TC3 cells were seeded onto
coverslips and incubated for 48 h in the absence of serum and/or
in the presence of lipoproteins. BrdUrd (Roche Diagnostics) was added
to the cells for 1 h at a final concentration of 10 µM. Cells were then fixed in 1% paraformaldehyde in PBS
for 15 min and incubated for 1 h with 2 N HCl.
Immunostaining with anti-BrdUrd (clone IIB5; Monosan, Uden, The
Netherlands) diluted 1:100 and counterstaining with Hoechst 33342 were
performed as described above. Counts were made by scoring the number of cells positive for BrdUrd incorporation.
TC3 cells were starved for
15 h and incubated with HDL at a concentration of 1.0 mM for 5 min, 20 min, and 60 min. Akt was
immunoprecipitated from 200 µg of total protein equivalent of cell
lysate with anti-Akt antibody linked to agarose beads overnight at
4 °C. Immunoprecipitates were washed and resuspended in kinase
buffer supplemented with 200 µM ATP and 1 µg of
glycogen synthase kinase-3 fusion protein. After 30 min at 30 °C the
reaction was stopped, and the extent of glycogen synthase kinase-3
phosphorylation was analyzed by Western blotting with a specific
anti-phosphoglycogen synthase kinase-3 antibody.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
TC3. Using reverse transcription
PCR, we detected the transcripts for LDLR and three other members of
the LDLR family: LRP, VLDLR, and apoER2. We also detected the
transcript for SR-BI, which mediates cholesterol uptake from HDL
particles (data not shown). Immunohistochemical analysis of mouse
pancreatic sections, using specific antibodies for these receptors,
revealed a strong labeling in islets (Fig. 1,
A-F). Similar data were
obtained in
TC3 cells (data not shown). Western blot analysis of
protein extracts from
TC3 cells revealed bands of the expected
molecular masses for these receptors (Fig. 1G)
(10-13). Note that the antiserum directed against LRP recognizes two
bands at ~600 and 85 kDa (12).
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Fig. 1.
Lipoprotein receptors are present in
pancreatic islets and TC3 cells. Mice
pancreatic sections were stained with hematoxylin and eosin
(A) and incubated with antisera raised against SR-BI
(B), LDLR (C), LRP (D), VLDLR
(E), and apoER2 (F). Total cell lysates from
TC3 cells were separated on SDS-PAGE, transferred to nitrocellulose
membranes, and incubated with the same antibodies (G).
Arrowheads indicate expected apparent molecular
masses.
TC3 Cells and Murine
Pancreatic Islets and Effects on HMG-CoA Reductase
Transcription--
We next examined the uptake and storage of LDL and
HDL particles in
TC3 cells and in freshly isolated murine pancreatic
islets. After incubation for 2 h with fluorescent HDL or LDL,
TC3 cells accumulated high quantities of labeled lipids (Fig. 2,
A and C). Fluorescence was reduced markedly when cells were incubated with an
excess of nonfluorescent LDL or HDL particles (Fig. 2, B and D), assessing the specificity of the uptake. We performed
similar experiments on murine pancreatic islets isolated from WT and
LDLR-deficient (LDLR
/
) mice. Islets from WT animals
displayed an intense cytoplasmic signal when incubated with fluorescent
LDL (Fig. 2E), whereas islets from LDLR
/
mice
showed only a faint signal (Fig. 2F). The effect of LDL particles on HMG-CoA reductase expression was also investigated. As
shown in Fig. 2G, incubation of
TC3 cells with LDL
reduced HMG-CoA reductase mRNA by 83% (p < 0.01).
Taken together, these data indicate that insulin-secreting cells
express functional LDL receptors.
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Fig. 2.
Assessment of lipoprotein uptake and HMG-CoA
reductase expression. TC3 cells were incubated with fluorescent
LDL (A) and with an excess of unlabeled LDL (B),
or with fluorescent HDL (C) and with an excess of unlabeled
HDL (D). Islets from WT mice (E) and from
LDLR
/
mice (F) were incubated with fluorescent
LDL. HMG-CoA mRNA levels were measured by quantitative PCR in the
presence or absence of 3.0 mM LDL cholesterol
(G). Results are the means ± S.E. of six different
experiments. **, p < 0.01 between treatments.
-Cells Exposed to Lipoproteins and
Effects on Insulin Transcript--
Apoptosis is characterized by
morphological changes that include chromatin condensation, cellular
shrinkage, membrane blebbing, and the formation of apoptotic bodies.
Changes in nuclear morphology are recognized as among the most reliable
criteria to assess apoptosis. Cells were stained either with
Hoechst 33342 or SYTOX Green, and the rate of apoptosis was evaluated
by counting the number of cells displaying a pycnotic (highly
condensed) and/or a fragmented nucleus (Fig.
3A). Both dyes provide the
same information, but SYTOX Green has the advantage of being more
stable than Hoechst and easily detected at 488 nm with a standard
confocal microscope. We studied in detail the morphological changes
that take place in the nuclei of
-cells undergoing apoptosis.
Three-dimensional images were generated showing the differing
morphologies of a normal nucleus (Fig. 3D), an apoptotic
nucleus (Fig. 3E), and a nucleus in early mitosis (Fig.
3F). To assess apoptosis in primary islets accurately, we
developed a novel experimental approach that allowed us to scan the
three-dimensional islet structure and therefore did not require cell
dissociation. Fig. 4 shows the appearance
of an islet incubated in normal medium (Fig. 4, A and
B) and after 2 days treatment with 3.1 mM VLDL
(Fig. 4, C and D), 6.2 mM LDL (Fig.
4, E and F), or 1.0 mM HDL (Fig. 4, G and H). Apoptotic nuclei appear highly
fluorescent at low magnification and display characteristic features at
higher magnification (arrows, Fig. 4). To confirm the data
obtained by morphological studies, we performed a TUNEL assay (Fig.
3C) and immunocytochemistry for cleaved caspase-3 (Fig.
3B). All cells with an altered nuclear morphology assessed
by Hoechst staining were positive for TUNEL. In contrast, cleaved
caspase-3 was detected only in apoptotic cells treated with cytokines
or VLDL, but not with LDL (data not shown).
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Fig. 3.
Assessment of apoptosis in
TC3 cells.
TC3 cells were stained with
Hoechst 33342 alone (A) or in combination with anti-cleaved
caspase-3 antibody (B) or TUNEL reaction (C).
Note that the cells displaying a pycnotic or fragmented nucleus
(arrowheads) are positive for cleaved caspase-3
(arrow) and TUNEL. In parallel,
TC3 cells were stained
with SYTOX Green, analyzed by confocal microscopy, and
three-dimensional images were generated. D, normal nucleus;
E, apoptotic nucleus; F, nucleus in
early mitosis.
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Fig. 4.
Assessment of apoptosis in mouse islets
treated with lipoproteins. Mouse islets were cultured for 48 h in the presence of lipoproteins, stained with SYTOX Green, and
analyzed with a confocal microscope at low (×25) and high (×63)
magnification. A and B, control; C and
D, 3.1 mM VLDL; E and F,
6.2 mM LDL; G and H, 1.0 mM HDL. Arrows indicate apoptotic nuclei.
TC3
cells. The effects increased steadily over a 48-h time course (data not shown). Serum deprivation was used as a positive control and induced a
4.3-fold increased apoptotic rate. In contrast, HDL particles had a
protective effect against
-cell death (Fig. 5B). In the presence of 1.0 mM HDL cholesterol, the basal apoptotic
rate was reduced significantly. In addition, HDL particles antagonized the proapoptotic effects of serum deprivation or of cytokine treatment. Cell death induced by high doses of LDL could be partially abolished by
HDL particles. To determine whether elevated glucose concentrations played a role in the adverse effects of VLDL and LDL, we performed similar experiments with
TC3 cells incubated with medium containing 5.0 mM glucose. No differences were observed in the
apoptotic rate compared with experiments conducted with 11.1 mM glucose concentrations.
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Fig. 5.
Effects of lipoproteins on
-cell apoptosis and insulin transcript.
A and B,
TC3 cells were incubated for 48 h in the absence of serum (starving) or in the presence of cytokines
(interleukin-1
/tumor necrosis factor-
/interferon-
), LDL, and
HDL as indicated. Cells were then stained with Hoechst 33342, and
apoptotic cells were counted. Data are the means ± S.E. of a
minimum of four separate experiments. *, p < 0.05;
***, p < 0.001 compared with CTRL or conditions
without HDL. C, apoptosis was evaluated in islets from WT
and LDLR
/
mice incubated for 48 h with VLDL, LDL, and
HDL. Data represent the means ± S.E. of counts in 6-21
islets. *, p < 0.05 and ***, p < 0.001 compared with WT islets in control conditions. §§§,
p < 0.001 compared with WT islets treated with LDL.
, p < 0.001 compared with WT islets treated
with LDL in absence of HDL. D, insulin mRNA levels were
measured by quantitative PCR in
TC3 cells. Data are the means ± S.E. of a minimum of three independent experiments. **,
p < 0.01 and ***, p < 0.001.
TC3 cells with the exception of HDL, which did not decrease the
basal apoptotic rate in control islets (Figs. 4 and Fig.
5C). To evaluate the role of lipoprotein receptors in
LDL-induced apoptosis, we incubated islets from LDLR
/
and
WT mice with LDL particles. Elevated VLDL or LDL significantly increased the apoptotic rate in WT mice. The increase in apoptosis under LDL treatment was significantly lower in LDLR
/
mice
compared with WT mice. We also confirmed the beneficial effects of HDL
on islet cell survival (Fig. 5C). In WT mice, cell death
induced by LDL particles was decreased by 46% in the presence of HDL.
Taken together, these data demonstrate a deleterious effect of VLDL and
LDL particles which requires, at least in part, the presence of the
LDLR and which can be antagonized by HDL particles.
TC3 cells (Fig.
6A). Serum deprivation, cytokines, and 3.1 mM VLDL were all associated with
increased levels of the activated cleaved protein. Surprisingly, 6.2 mM LDL did not induce any cleavage. Activation of caspase-3
was partially antagonized by HDL particles, confirming the
survival-promoting role of these lipoproteins.
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Fig. 6.
Caspase-3 activation and contribution of the
JNK pathway in VLDL-induced apoptosis. TC3 cells were incubated
for 48 h in the absence of serum (starvation) or in the presence
of cytokines (interleukin-1
/tumor necrosis
factor-
/interferon-
), VLDL, LDL, and HDL. Lysates were subjected
to immunoblot analysis with anti-cleaved caspase-3 (A) and
anti-IB1/JIP-1 antibodies (B). The amount of protein was
quantitated by densitometry. Tubulin was used for normalization. Data
are the means ± S.E. of a minimum of four separate experiments.
**, p < 0.01 and ***, p < 0.001 compared with control conditions.
, p < 0.01 compared with starvation without HDL. §, p < 0.05 compared with cytokines without HDL. C, apoptotic counts
were performed in
TC3 cells incubated for 48 h with
lipoproteins in the presence or absence of a JNK inhibitor
(JNKi). Data are the means ± S.E. of three independent
experiments. *, p < 0.05.
TC3 cells exposed to lipoproteins was evaluated by
quantitating BrdUrd incorporation (Fig.
7A). Elevated concentrations of VLDL (3.1 mM) or LDL (6.2 mM) significantly decreased the number of cells positive
for BrdUrd incorporation, whereas HDL caused a slight increase that was
not statistically significant (p = 0.064). Cyclin B1 is
expressed predominantly in the G2/M phase of cell division
and participates in the control of cell proliferation. Cyclin B1
mRNA was reduced by 39% in cells treated with 3.1 mM
VLDL and by 29% when treated with 6.2 mM LDL (Fig.
7B), suggesting a mechanism by which these lipoproteins reduce cell proliferation. Finally, HDL particles induced activation of
Akt/PKB, known as a key regulator of cellular survival (Fig. 7C).
View larger version (13K):
[in a new window]
Fig. 7.
Effects of lipoproteins on
TC3 cell proliferation, cyclin B1 transcription,
and Akt/PKB activation. A,
TC3 cells were incubated for
48 h in the presence of lipoproteins and subsequently for 1 h
with BrdUrd. The number of nuclei positive for BrdUrd was evaluated in
three independent experiments. Data are the means ± S.E. **,
p < 0.01. B, cyclin B1 mRNA levels were
measured by quantitative PCR. Data are the means ± S.E. of a
minimum of four separate experiments. **, p < 0.01. C,
TC3 cells were incubated with HDL, and Akt/PKB
activity was determined by measuring the in vitro
phosphorylation of glycogen synthase kinase-3 (P-GSK).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-cells. Because islets from LDLR
/
mice
display markedly decreased LDL uptake compared with islets from WT
mice, we propose LDLR to be the main receptor responsible for LDL
internalization in
-cells. ApoER2 can also bind LDL particles with
lower affinity (17), which may explain why low levels of LDL uptake are
still observed in islets from LDLR
/
mice. Down-regulation
of HMG-CoA reductase expression in
-cells treated with LDL confirms
that these cells express functional receptors for LDL particles. SR-BI
is unrelated to LDLR and has been shown to mediate selective uptake of
cholesterol from HDL particles (10). Our data demonstrate for the first
time the presence of SR-BI in
-cells concomitant with the uptake of
HDL.
-cells and has
been shown to play an important role in controlling the activity of the
JNK signaling pathway (18). A mutation in the MAPK8IP1 gene
encoding IB1 has been associated with a familial form of type 2 diabetes (19). Recently, Bonny et al. (16) engineered
cell-permeable peptide inhibitors of JNK corresponding to the minimal
JNK binding domain of IB1/JIP-1 which resulted in a marked inhibition
of
-cell death triggered by interleukin-1
. These peptides could
partially antagonize VLDL-induced apoptosis, but they had no effect on
incubations performed with LDL. These data indicate that VLDL induces
apoptosis concomitant with a reduction in IB1/JIP-1 levels and an
activation of the JNK pathway. A study by Bonny et al. (18)
implicated this reduction in IB1/JIP-1 levels in increased apoptosis.
-cell
death caused by LDL. When exposed to elevated LDL concentrations, islets from LDLR
/
were more resistant to apoptosis than
islets from WT mice. However, apoptotic rates were slightly higher than
in control conditions, which may be explained by the presence of other
lipoprotein receptors that were shown to be present in
LDLR
/
mice.
-cell
survival and protect against apoptosis. HDL efficiently antagonizes the
proapoptotic effects of serum deprivation and incubation with cytokines
or lipoproteins. In the presence of HDL, caspase-3 cleavage is reduced
markedly, and Akt/PKB is activated. Akt inhibits apoptosis by
phosphorylating a variety of substrates, including Bad, FKHR, glycogen
synthase kinase-3, and caspase-9 (20). In the endocrine pancreas, Akt1
has been shown to regulate
-cell growth and survival (21). In
endothelial cells, Akt is a mediator of the antiapoptotic effects of
HDL (22).
-cell death. Surprisingly, LDL toxicity occurred with doses far below physiological concentrations, and the effects on
-cells were described as necrosis. Our study shows that higher concentrations are required to cause death in
TC3 cells and primary mouse islets, with features
characteristic of apoptosis. Differences between the species studied
might explain these discrepancies.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. C. Bonny (CHUV, Switzerland) for the JNK inhibitors and Dr. R. Kraftsik (IBCM, Switzerland) for his help with Imaris and Photoshop. We also thank Dr. A. Zanchi (CHUV), Dr. P. Clarke (IBCM), Dr. G. Knott (IBCM), Dr. C. Widmann (IBCM), and Dr. M. Shaw (IBCM) for help with the manuscript.
![]() |
FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
b Supported by an M.D./Ph.D. program of the Swiss National Science Foundation.
c Supported by Swiss National Science Foundation Grant 32-62623.00 and the Placide Nicod and Octav Botnar Foundation.
e Supported by grants from the National Institutes of Health, the Alzheimer Association, the Perot Family Foundation, and a Wolfgang-Paul award from the Humboldt Foundation.
i Supported by Swiss National Science Foundation Grant 31-068036.02 and the Placide Nicod and Octav Botnar Foundation.
j Supported by Swiss National Science Foundation Grant 32-48916.96, Juvenile Diabetes Research Foundation Grant 1-2001-555/600, and the Placide Nicod and Octav Botnar Foundation. To whom correspondence should be addressed: Dept. of Internal Medicine B, CHUV-University Hospital, BH 10-640, Lausanne 1011, Switzerland. Tel.: 41-21-314-09-60; Fax: 41-21-314-09-28; E-mail: gwaeber@chuv.hospvd.ch.
Published, JBC Papers in Press, February 18, 2003, DOI 10.1074/jbc.M300102200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
HDL, high density
lipoprotein;
Akt/PKB, protein kinase B;
apoER2, apolipoprotein E
receptor 2;
BrdUrd, bromodeoxyuridine;
HMG-CoA reductase, 3-hydroxy-3-methylglutaryl coenzyme A reductase;
IB1/JIP-1, islet-brain
1/c-Jun N-terminal kinase-interacting protein-1;
JNK, c-Jun N-terminal
kinase;
LDL, low density lipoprotein;
LDLR, low density lipoprotein
receptor;
LDLR/
, LDL receptor knock-out;
LRP, LDL
receptor-related protein;
PBS, phosphate-buffered saline;
SR-BI, scavenger receptor class B type I;
TUNEL, terminal nucleotidyl
transferase-mediated UTP nick end labeling;
VLDL, very low density
lipoprotein;
VLDLR, very low density lipoprotein receptor;
WT, wild-type.
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