From the Division of Clinical Biochemistry, Department of Internal Medicine, University Medical Centre, Geneva-4 CH-1211, Switzerland
Received for publication, December 9, 2002, and in revised form, February 21, 2003
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ABSTRACT |
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Accumulation of lipids in non-adipose
tissues is often associated with Type 2 diabetes and its complications.
Elevated expression of the lipogenic transcription factor, sterol
regulatory element binding protein-1c (SREBP-1c), has been demonstrated
in islets and liver of diabetic animals. To elucidate the molecular
mechanisms underlying SREBP-1c-induced The lipogenic transcription factors, sterol regulatory element
binding proteins (SREBPs)1
are transmembrane proteins of the endoplasmic reticulum (ER). In
response to low sterol and other unidentified factors, SREBP cleavage-activating protein escorts SREBPs from the ER to the Golgi, where SREBPs are sequentially cleaved by Site-1 protease and
Site-2 protease. The processed mature SREBPs enter the nucleus and
transactivate target genes (1). Three SREBP isoforms have been
identified: SREBP-1a and -1c (alternatively known as adipocyte determination and differentiation factor-1 (ADD1)) (2), which are derived from the same gene through alternative splicing, and SREBP-2, which is encoded by a distinct gene (1). SREBPs play an
essential role in regulation of lipid homeostasis in animals and have
been shown to directly activate the expression of more than 30 genes
dedicated to the biosynthesis of cholesterol, fatty acids,
triglycerides, and phospholipids (3). SREBP-1 preferentially regulates
genes implicated in fatty acid synthesis, whereas SREBP-2 preferentially activates genes involved in cholesterol synthesis (1).
In particular, SREBP-1c mediates insulin effects on lipogenic gene
expression in both adipocytes and liver (4, 5).
Type 2 diabetes mellitus is a common disorder that affects ~5% of
the population worldwide, especially in industrialized countries (6).
Affected patients are usually obese with accumulation of lipids in
non-adipose tissues such as the pancreatic islets, liver, heart,
skeletal muscle, and blood vessels (7-9). This is associated with
impaired glucose-stimulated insulin secretion, increased hepatic
glucose production, peripheral insulin resistance, and late
complications in various organs (10, 11). The ultimate precipitating
process responsible for the development of Type 2 diabetes is the
failure of the pancreatic Elevated expression of SREBP-1c has been demonstrated in islets or
liver of diabetic animals, such as Zucker diabetic fatty rats,
ob/ob mice, insulin receptor
substrate-2-deficient mice, and a transgenic mouse model of
lipodystrophy (7, 12-16). On the other hand, preventing SREBP-1c
overexpression is a common function of leptin, metformin, and PPAR The present study was designed to evaluate the direct correlation
between SREBP-1c overexpression and Establishment of INS-1 Cells Permitting Inducible Expression of
naSREBP-1c--
Rat insulinoma INS-1 cell-derived clones were cultured
in RPMI 1640 containing 11.2 mM glucose (20), unless
otherwise indicated. The first step stable clone INS-r Immunofluorescence and Western Blotting--
For
immunofluorescence, cells grown on polyornithine-treated glass
coverslips were treated for 24 h with or without 500 ng/ml doxycycline. Cells were then washed, fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 in phosphate-buffered saline containing 1% BSA (PBS-BSA). The preparation was then blocked with
PBS-BSA before incubating with the first antibody, anti-SREBP-1 (Santa
Cruz, Basel, Switzerland) (1:100 dilution), followed by the second
antibody labeling.
For Western blotting cells were cultured with 0, 75, 150, and 500 ng/ml
doxycycline for 24 h. Rat islets were isolated by collagenase
digestion as described (24), and their nuclear proteins were extracted
as previously reported (25). Total cell proteins were prepared by lysis
and sonication of naSREBP-1c#233 and INS-1E cells in buffer
containing 20 mM Tris-HCl, pH 7.4, 2 mM EDTA, 2 mM EGTA, 1% Triton X-100, and 1 mM
phenylmethylsulfonyl fluoride. Nuclear extracts and total cellular
proteins were fractionated by 9% SDS-PAGE. The dilution for SREBP-1
antibody is 1:1,000. Immunoblotting procedures were performed as
described previously (26) using enhanced chemiluminescence
(Pierce) for detection.
Staining of Lipid Accumulation by Oil Red O and Measurements of
Triglyceride Content--
Cells were cultured with 0, 75, 150, and 500 ng/ml doxycycline for 24 h. Cells were fixed and stained as
previously reported (19). Lipid droplets were visualized using
phase-contrast microscopy (Nikon Diaphot). Cellular triglyceride
content was determined using Triglyceride (GPO-TRINDER) kit (Sigma)
according to the manufacturer's protocol.
Measurements of Insulin Secretion and Cellular Insulin
Content--
Insulin secretion in naSREBP-1c#233 cells was
measured in 12-well plates over a period of 30 min, in
Krebs-Ringer-bicarbonate-HEPES buffer (KRBH, 140 mM NaCl,
3.6 mM KCl, 0.5 mM
NaH2PO4, 0.5 mM MgSO4,
1.5 mM CaCl2, 2 mM
NaHCO3, 10 mM HEPES, 0.1% BSA) containing indicated concentrations of glucose. Insulin content was determined after extraction with acid ethanol following the procedures of Wang
et al. (26). Insulin was detected by radioimmunoassay using rat insulin as a standard (26).
Total RNA Isolation and Northern Blotting--
Cells were
cultured with or without 500 ng/ml doxycycline in 2.5 mM
glucose medium for 16 h and then incubated for a further 8 h
at 2.5, 6, 12, and 24 mM glucose concentrations.
Alternatively, naSREBP-1c#233 cells were cultured with or
without 75 ng/ml doxycycline in standard medium (11.2 mM
glucose) for 0, 24, 48, and 96 h. Total RNA was extracted and
blotted to nylon membranes as described previously (22). The membrane
was prehybridized and then hybridized to 32P-labeled random
primer cDNA probes according to Wang and Iynedijian (22). To ensure
equal RNA loading and even transfer, all membranes were stripped and
re-hybridized with a "housekeeping gene" probe cyclophilin.
cDNA fragments used as probes for SREBP-1c, hepatocyte nuclear
factor (HNF)-1 Mitochondrial Membrane Potential
( Cell Proliferation/Viability and
Apoptosis--
Quantification of cell proliferation/viability was
measured by a Quick Cell Proliferation Assay Kit (LabForce/MBL,
Nunningen, Switzerland) according to the manufacturer's protocol. This
assay is based on the reduction of a tetrazolium salt WST-1 to formazan by cellular mitochondrial dehydrogenases. Expansion in the number of
viable cells results in an increase in the overall activity of the
mitochondrial dehydrogenases and subsequently an augmentation in the
amount of formazan dye formed. The formazan dye produced by viable
cells was quantified with a multiwell spectrophotometer by measuring
the absorbance at 440 nm.
Experiments for DNA fragmentation were performed using a Quick
Apoptosis DNA Ladder Detection Kit (LabForce/MBL) following the
manufacturer's protocol. Detection of mitochondrial cytochrome c release into cytosol was performed as described previously
(29). Briefly, cells in 15-cm dishes were harvested and suspended in Buffer A (20 mM HEPES-KOH, pH 7.5, 10 mM KCl,
1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 250 mM sucrose, 1 mM phenylmethylsulfonyl fluoride)
and homogenized by a Dounce homogenizer. Cell debris and nuclei were
removed by centrifugation at 1000 × g for 10 min at
4 °C. The supernatant was further centrifuged at 10,000 × g for 20 min. The cytosolic proteins (supernatant fractions)
were separated by 15% SDS-PAGE and analyzed by Western blotting with a
specific antibody against cytochrome c (LabForce/MBL).
Establishment of a Stable INS-1 Cell Line Permitting Inducible
Expression of naSREBP-1c--
A rat insulinoma INS-1-derived stable
cell line, INSr All Cells Uniformly Express the Nuclear Localized naSREBP1c Protein
in a Doxycycline Dose-dependent
Manner--
Immunofluorescence (Fig.
1A) with an antibody against
the N terminus of SREBP1c illustrated that nuclear localized naSREBP1c protein was induced homogeneously in naSREBP-1c#233 cells
cultured with 500 ng/ml doxycycline for 24 h. Western blotting
(Fig. 1B) with the same antibody demonstrated that
naSREBP-1c#233 cells expressed the transgene-encoded
protein in a doxycycline dose-dependent manner. As
predicted, naSREBP-1c#233 cells did not show detectable
expression of naSREBP-1c protein under non-induced conditions (Fig. 1).
The protein levels of naSREBP-1c in cells cultured with 75, 150, and
500 ng/ml were ~10%, 25%, and 150%, respectively, of endogenous
level of SREBP-1c precursor protein (Fig. 1B).
Induction of naSREBP-1c Causes Rapid Accumulation of Lipid Droplets
by Increasing Lipogenic Gene Expression--
Oil Red O staining showed
that induction of naSREBP-1c with 75, 150, and 500 ng/ml doxycycline
for 24 h resulted in accumulation of lipid droplets in INS-1 cells
cultured in standard medium (10% fetal calf serum) (Fig.
2A). Cellular triglyceride
content was increased by 39% ( Induction of naSREBP-1c Impairs Nutrient-stimulated Insulin
Secretion--
Induction of naSREBP1c with 500 ng/ml doxycycline for
24 h (Fig. 3A) or 75 ng/ml doxycycline for 96 h (Fig. 3B) caused blunted glucose- and leucine-stimulated but not
K+-depolarization-induced insulin secretion. Glucose
generates ATP and other metabolic-coupling factors important for
insulin exocytosis through glycolysis and mitochondrial oxidation (30).
Leucine stimulates insulin release through direct uptake and metabolism by the mitochondria, thereby providing substrates for the tricarboxylic acid cycle (30). K+ causes insulin secretion by
depolarization of the Induction of naSREBP-1c Alters the Expression of Genes Important
for Glucose Metabolism and
Induction of naSREBP1c also suppressed the expression of Pdx-1,
HNF-4 Induction of naSREBP-1c Disrupts Mitochondrial Membrane
Potential--
Mitochondrial membrane potential ( SREBP-1c Targets Multiple Genes Implicated in Induction of naSREBP-1c Results in Cell Growth Arrest and Apoptosis
in INS-1 Cells--
To assess the impact of naSREBP-1c on INS-1 cell
growth and viability, we performed the WST-1 assay. This assay is based
on the reduction of a tetrazolium salt WST-1 to formazan by cellular mitochondrial dehydrogenases. Expansion in the number of viable cells
results in an increase in the overall activity of the mitochondrial dehydrogenases and subsequently an augmentation in the amount of
formazan dye formed. Induction of naSREBP-1c with 500 ng/ml doxycycline
for 24 h or with 75 ng/ml doxycycline for 4 days in naSREBP-1c#233 cells significantly inhibited INS-1 cell
growth/viability. The measurements of optical density at 440 nM were reduced by, respectively, 31.6 ± 4.2% and
36.3 ± 5.5% relative to non-induced cells (n = 4, p < 0.001). Mitochondrial cytochrome c
release and DNA fragmentation are characteristic hallmarks of cells
undergoing apoptosis (37, 42). Extensive DNA fragmentation was observed in naSREBP-1c#233 cells cultured with 500 ng/ml doxycycline
for 48 h or with 75 ng/ml doxycycline for a week (Fig.
7A). Consistently, similar induction of naSREBP-1c also induced mitochondrial cytochrome c release (Fig. 7B). These results suggest that
naSREBP-1c induces INS-1 cell apoptosis in a dose- and
time-dependent manner.
SREBP-1c Processing in
Low sterol-mediated cleavage of SREBP precursor proteins is not the
only mechanism for the processing of SREBPs. It has been suggested that
insulin and cytokines stimulate phosphorylation and transcriptional
activity of SREBPs via the mitogen-activated protein kinase cascade
(44-48). Caspase-3/CPP32, a cysteine protease and mediator of
apoptosis, also induces cleavage of SREBPs and release of their
transactive N-terminal fragments (42). In addition, TNF-
Hyperglycemia, hyperlipidemia, elevated TNF- Intracellular lipid accumulation, termed lipotoxicity (metabolic
syndrome X), in skeletal muscle, myocardium, blood vessels, kidney,
liver, and pancreatic islets has been speculated to cause, respectively, insulin resistance, cardiac dysfunction, vascular complications, nephropathy, fatty liver (hepatic steatosis), and Conditional expression of naSREBP-1c, a nuclear localized N-terminal
form of SREBP-1c with intact transcriptional activity, results in
massive accumulation of lipid-droplets in INS-1 cells, a clonal
In addition, the present study also suggests that, in contrast to
hepatocytes and adipocytes, islet We hypothesize that SREBPs have evolved to allow animals adapting to
starvation by mediating insulin action on energy storage and promoting
cell survival by maintaining constant lipid composition of membranes.
Overnutrition and sedentary life-style in industrialized modern society
may lead to lipotoxicity in non-adipose tissue. The important
contribution of our study is that the transcription factor SREBP-1c is
instrumental in the pathogenesis of -cell dysfunction, we
employed the Tet-On inducible system to achieve tightly controlled and
conditional expression of the nuclear active form of SREBP-1c
(naSREBP-1c) in INS-1 cells. Controlled expression of naSREBP-1c
induced massive accumulation of lipid droplets and blunted
nutrient-stimulated insulin secretion in INS-1 cells.
K+-evoked insulin exocytosis was unaltered.
Quantification of the gene expression profile in this INS-1 stable
clone revealed that naSREBP-1c induced
-cell dysfunction by
targeting multiple genes dedicated to carbohydrate metabolism, lipid
biosynthesis, cell growth, and apoptosis. naSREBP-1c elicits cell
growth-arrest and eventually apoptosis. We also found that the SREBP-1c
processing in
-cells was irresponsive to acute stimulation of
glucose and insulin, which was distinct from that in lipogenic tissues.
However, 2-day exposure to these agents promoted SREBP-1c processing.
Therefore, the SREBP-1c maturation could be implicated in the
pathogenesis of
-cell glucolipotoxicity.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-cells to compensate for insulin
resistance (7, 10, 11). However, the mechanism by which the
-cells
become unable to meet increased insulin demands remains to be established.
agonists, which is well correlated with their antidiabetic effects (7,
12-15). An important function of leptin in the regulation of fatty
acid homeostasis is to restrict the lipid storage in adipocytes and to
limit lipid accumulation in non-adipocytes, thereby protecting them
from lipotoxicity (7, 9). Infusion of recombinant leptin reverses
insulin resistance and hyperglycemia in the transgenic model of
congenital generalized lipodystrophy and in ob/ob
mice (13). Adenovirus-mediated hyperleptinemia also decreases the
expression of SREBP-1c and lipogenic genes in liver and islets of
wild-type fa/fa rats (12). Leptin also prevents
the SREBP-1c overexpression in insulin receptor substrate (IRS)-2-null
mice (15). It is noteworthy that metformin, an oral antihyperglycemic
agent, also corrects fatty liver disease in ob/ob
mice by inhibition of obesity-related induction of SREBP-1c (14). The
inhibitory effects of metformin on hepatic SREBP-1c expression involves
activation of AMP-activated protein kinase (17). Similarly,
Troglitazone, an antidiabetic agent and a high-affinity ligand for the
PPAR
, prevents SREBP-1c overexpression in Zucker diabetic
fatty rats (12). In addition, it has recently been reported that
adenovirus-mediated overexpression of SREBP-1c in MIN6 cells results in
impaired glucose-stimulated insulin secretion (18). Therefore, these
studies suggest that overexpression of SREBP-1c may be the cause of
both liver steatosis and islet
-cell dysfunction.
-cell dysfunction and to
elucidate the underlying molecular mechanism. The Tet-On system was
employed in INS-1 cells to achieve tightly controlled and inducible
expression of a nuclear active form of SREBP-1c (naSREBP-1c; N-terminal
1-403 amino acids) (19). Quantification of the gene expression profile
in this INS-1 stable cell line revealed that naSREBP-1c induced
-cell dysfunction by targeting multiple genes implicated in
carbohydrate metabolism, lipid biosynthesis, cell growth, and
eventually apoptosis.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
cell line,
which carries the reverse
tetracycline/doxycycline-dependent transactivator (21) was
described previously (22, 23). The plasmid used in the secondary stable
transfection were constructed by subcloning the cDNA encoding the
nuclear active form of SREBP-1c (naSREBP-1c/ADD1-(1-403) (19),
kindly supplied by Dr. B. M. Spiegelman) into the expression vector PUHD10-3 (21) (a generous gift from Dr. H. Bujard). The procedures for stable transfection, clone selection and screening were
described previously (22).
, HNF-4
, glucokinase, Glut-2, insulin, Sur1,
Kir6.2, and pancreas duodenum homeobox (Pdx-1) mRNA
detection were digested from the corresponding plasmids. cDNA
probes for rat islet amyloid polypeptide, Nkx6.1, adenine nucleotide
translocator 1 and 2, mitochondrial uncoupling protein 2 (UCP2),
mitochondrial glutamate dehydrogenase, citrate synthase,
glyceraldehydes-3 phosphate dehydrogenase, fatty acid synthase,
acetyl-CoA carboxylase, glycerol-phosphate acyltransferase,
carnitine palmitoyltransferase-1, acyl-CoA oxidase, 3-hydroxy-3-methylglutaryl-CoA reductase, P21WAF1/CIP1,
P27KIP1, Bax, Bad, APO-1, Cdk-4, IRS-2, and glucagon-like
peptide-1 receptor were prepared by reverse transcription-PCR
and confirmed by sequencing.
m)--
Cells seeded in 24-well plates were
cultured with or without 500 ng/ml doxycycline in 11.2 mM
glucose medium for 24, 48, and 96 h (day 1, day 2, and day 4, respectively). Cells were then maintained for 2 h in 2.5 mM glucose medium at 37 °C before loading with 10 µg/ml rhodamine-123 for 20 min at 37 °C in KRBH (28). The
m was monitored in a plate-reader fluorimeter
(Fluostar Optima, BMG Labtechnologies, Offenburg, Germany) with
excitation and emission filters set at 485 and 520 nm, respectively, at
37 °C with automated injectors for glucose (addition of 13 mM on top of basal 2.5 mM) and carbonyl cyanide
4-trifluoromethoxyphenylhydrazone (FCCP).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(23), which expresses the reverse
tetracycline-dependent activator (21), was used for the
secondary stable transfection with an expression vector, PUHD10-3
(21), carrying the naSREBP-1c and a plasmid pTKhygro containing the
hygromycin-resistant marker. Five-round stable transfection experiments
were performed, and a total of 400 hygromycin-resistant clones were
screened by Northern blotting for clones positively expressing
naSREBP-1c after doxycycline induction. Our persistent effort was
rewarded. Only one clone, termed naSREBP-1c#233, which
actually represented undetectable background expression under
non-induced conditions, showed remarkable expression of naSREBP-1c
mRNA after doxycycline induction. From experience, we would have
anticipated that 10-20% of hygromycin-resistant clones should
positively express the transgene. The unexpected results indicate that
expression of naSREBP-1c even at leakage level was sufficient to cause
"
-cell toxicity." The INS-1 stable cell line
naSREBP-1c#233 provided a unique chance to study the impact
of naSREBP-1c expression on
-cell function.
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Fig. 1.
Induction of naSREBP-1c protein in a
dose-dependent and an all-or-none manner.
A, immunofluorescence staining with an antibody against the
N terminus of SREBP-1c. The phase contrast image is shown in the
upper panel. The naSREBP-1c#233 cells were
cultured with (+Dox) or without ( Dox) 500 ng/ml doxycycline for
24 h. B, Western blotting of total cell extracts (100 µg) with the same antibody. Cells were cultured with 0, 75, 150, and
500 ng/ml doxycycline for 24 h.
Dox: 0.536 ± 0.08; +Dox:
0.745 ± 0.10 mg/mg protein, p < 0.001, n = 14) after 24 h of induction with 500 ng/ml doxycycline. Similar results were obtained in cells cultured in serum-free medium (data not shown), suggesting that the
naSREBP-1c-induced lipid accumulation was not due to uptake of fatty
acids by the cells. This contention was supported by results of
quantitative Northern blotting. Induction of naSREBP-1c significantly
increased the expression of genes dedicated to biosynthesis of fatty
acids and cholesterol but did not alter the expression of genes
involved in
-oxidation of fatty acids (Fig. 2, B and
C). The increased mRNA levels of fatty acid synthase,
acetyl-CoA carboxylase, glycerol-phosphate acyltransferase, and
3-hydroxy-3-methylglutaryl-CoA reductase would explain the
naSREBP-1c-induced lipid accumulation. In addition, the increased
lipogenesis did not raise the transcript levels of carnitine
palmitoyltransferase-1 and acyl-CoA oxidase, which are important for
fatty acid metabolism.
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Fig. 2.
Accumulation of lipid droplets and
increased lipogenic gene expression are shown in INS-1 cells expressing
naSREBP-1c. A, Oil Red O staining
naSREBP-1c#233 cells cultured with 0, 75, 150, and 500 ng/ml doxycycline for 24 h. B, Northern blotting was
performed using total RNA isolated from cells cultured with 500 ng/ml
doxycycline in 2.5 mM glucose medium for 16 h and
continued in 2.5, 6, 12, and 24 mM glucose medium for a
further 8 h. The experiments were repeated two times with similar
results. C, Northern blotting was conducted using total RNA
isolated from cells cultured in standard glucose medium (11.2 mM glucose) with 75 ng/ml doxycycline for 0, 24, 48, and
96 h. Two independent experiments are shown side by side to
demonstrate the consistency of the results.
-cell membrane, resulting in increased
cytosolic Ca2+ (30). To explore the molecular targets of
naSREBP-1c responsible for the impaired metabolism-secretion coupling,
we examined the gene expression profile in naSREBP-1c#233
cells.
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Fig. 3.
Induction of naSREBP-1c results in impaired
nutrient-stimulated insulin secretion. A, cells were
cultured with (+Dox) or without ( Dox) 500 ng/ml doxycycline in
standard medium (11.2 mM glucose) for 19 h and then
equilibrated in 2.5 mM glucose medium for a further 5 h. B, cells were cultured with (+Dox) or without (
Dox) 75 ng/ml doxycycline in standard glucose medium (11.2 mM) for
91 h followed by an additional 5 h of equilibration in 2.5 mM glucose medium. Cellular insulin content in A
and B was reduced, respectively, by 24.6 ± 4.8 (n = 6) and 21 ± 5.3 (n = 6)
after naSREBP-1c induction. Insulin release from
naSREBP-1c#233 cells stimulated by 21.5 mM
glucose, 20 mM leucine, and 20 mM KCl in KRBH
buffer (140 mM NaCl, 3.6 mM KCl, 0.5 mM NaH2PO4, 0.5 mM
MgSO4, 1.5 mM CaCl2, 2 mM NaHCO3, 10 mM HEPES, 0.1% BSA)
containing 2.5 mM glucose (Basal) was quantified
by radio-immunoassay and normalized by cellular insulin content. Data
represent mean ± S.E. of six independent experiments. **,
p < 0.0001.
-Cell Function--
Quantitative
Northern blotting revealed that similar induction of naSREBP1c also
caused marked down-regulation of glucokinase and Glut2 but
up-regulation of mitochondrial UCP-2 (Fig. 4,
A and B). In
contrast, naSREBP-1c did not alter the mRNA levels of
glyceraldehydes-3-phosphate dehydrogenase, mitochondrial adenine nucleotide translocator-1 and -2, glutamate dehydrogenase, citrate synthase, KATP channel subunits SUR1 and KIR6.2 (Fig. 4,
A and B). Glucokinase is the rate-limiting enzyme
for glycolysis and thereby determines
-cell glucose sensing (22,
31). The effect of naSEBP-1c on
-glucokinase expression is opposite
to its action on liver glucokinase, which is transcribed from a
distinct liver-specific promoter (5, 32). UCP-2 may act as a
protonophore and dissipate the mitochondrial membrane potential,
thereby uncoupling respiration from ATP synthesis (33-36).
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Fig. 4.
Induction of naSREBP-1c causes defective
expression of genes implicated in glucose metabolism and
-cell function. A and C,
cells were cultured with or without 500 ng/ml doxycycline in 2.5 mM glucose medium for 16 h and then incubated for a
further 8 h at indicated glucose concentrations. The experiments
were repeated two times with similar results. B and
D, cells were cultured with or without 75 ng/ml doxycycline
in standard medium (11.2 mM glucose) for 0, 24, 48, and
96 h. Two independent experiments are shown side by side to
demonstrate the consistency of the results. The gene expression
patterns in naSREBP-1c#233 cells were quantified by
Northern blotting. Total RNA samples (20 µg/lane) were analyzed by
hybridizing with indicated cDNA probes.
, and CCAAT/enhancer-binding protein-
in a dose- and time-dependent manner (Fig. 4, C and
D). The HNF-1
expression was unaffected, whereas the
mRNA level of a
-cell transcription factor Nkx6.1 was increased
by naSREBP-1c (Fig. 4, C and D). High-level induction of naSREBP-1c also decreased the transcript levels of insulin
and islet amyloid polypeptide (Fig. 4C).
m),
reflecting electron transport chain activity, was measured in attached
cells by monitoring rhodamine-123 fluorescence. In non-induced control
cells, the addition of 13 mM glucose (15.5 mM
final) hyperpolarized
m and 1 µM the
protonophore FCCP deploarized
m. After induction of naSREBP1c, glucose-induced mitochondrial hyperpolarization was markedly
inhibited as early as day 1 (24 h of induction) and completely abolished at days 2 and 4 (Fig. 5). Basal
activity of the electron transport chain was progressively lost, as
indicated by the dissipation of
m by FCCP, and
completely abrogated at day 4 (Fig. 5). The mitochondrion is not only
the powerhouse for cell growth and survival but also the arsenal for
cell apoptosis (37). Disruption of mitochondrial membrane potential is
an essential event that commits a cell to undergo apoptosis (38, 39).
Consistently, we also found that expression of naSREBP-1c even at
leakage level was sufficient to cause "cell toxicity" during the
screening procedure for naSREBP-1c-positive clones. To elucidate the
molecular mechanism underlying naSREBP-1c-induced cell toxicity,
we investigated the effect of naSREBP-1c on the expression of genes
important for cell proliferation and apoptosis.
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Fig. 5.
Induction of naSREBP-1c dissipates
mitochondrial membrane potential
( m) in
naSREBP-1c#233 cells. Cells were cultured
without (
Dox) or with (+Dox) 500 ng/ml doxycycline for 24, 48, and
96 h (day 1, day 2, and day 4, respectively). The
m was measured on attached cells in KRBH containing
basal 2.5 mM glucose using rhodamine-123 fluorescence.
Electron transport chain activation observed as hyperpolarization of
m was induced by the addition of 13 mM
glucose (15.5 mM final), followed by the complete
depolarization of
m using 1 µM of the
uncoupler FCCP. Values are means plus standard deviations
(n = 4) of one of a total of four to six independent
experiments.
-Cell Growth and
Survival--
As shown in Fig. 6,
induction of naSREBP-1c decreased the expression of
cyclin-dependent kinase 4 and IRS-2 in a dose- and time-dependent manner. In contrast, the mRNA level of
glucagon-like polypeptide-1 receptor was barely changed (Fig. 6). Both
cyclin-dependent kinase 4 and IRS-2 have been reported to
promote
-cell development, proliferation, or survival (40, 41). In
addition, the mRNA level of P21WAF1/CIP1 was
dramatically increased by naSREBP-1c induction, whereas the P27KIP1 remained constant (Fig. 6). The increased
expression of the P21WAF1/CIP1, a
cyclin-dependent kinase inhibitor, could also lead to INS-1 cell growth arrest. Furthermore, as demonstrated in Fig. 6, naSREBP-1c up-regulated the expression of proapoptotic genes, APO-1/Fas/CD95 and
Bax (37, 38), but did not alter the mRNA levels of Bad and tumor
necrosis factor (TNF)-1
. The up-regulation of APO-1 and Bax could
contribute to the naSREBP-1c-induced INS-1 cell apoptosis (see
below).
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Fig. 6.
Induction of naSREBP-1c alters the expression
of genes important for -cell growth and
survival. A, cells were cultured with or without 500 ng/ml doxycycline in 2.5 mM glucose medium for 16 h
and then incubated for a further 8 h at indicated glucose
concentrations. The experiments were repeated two times with similar
results. B, cells were cultured with or without 75 ng/ml
doxycycline in standard medium (11.2 mM glucose) for 0, 24, 48, and 96 h. Two separate experiments are demonstrated in
parallel. The gene expression profile in naSREBP-1c#233
cells was quantified by Northern blotting. Total RNA samples (20 µg/lane) were analyzed by hybridizing with indicated cDNA
probes.
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Fig. 7.
Apoptosis in INS-1 cells induced by
naSREBP-1c and maturation of SREBP-1 protein in INS-1E cells regulated
by factors causing -cell dysfunction.
A, DNA fragmentation in naSREBP-1c#233 cells
cultured either with 500 ng/ml doxycycline for 1, 2, and 4 days
(left panel) or with 75 ng/ml doxycycline for 1, 4, and 7 days (right panel). B, cytochrome c
released in cytosolic fractions isolated from
naSREBP-1c#233 cells cultured either with 500 ng/ml
doxycycline for 1, 2, and 4 days (left panel) or with 75 ng/ml doxycycline for 1, 4, and 7 days (right panel).
Cytosolic proteins (100 mg/lane) were separated with 15% SDS-PAGE and
analyzed by immunoblotting with an anti-cytochrome c
antibody. C, immunoblotting with antibody against the N
terminus of SREBP-1. Total cell extracts (100 µg of protein/lane)
from INS-1E cells (lanes 1-8) or nuclear proteins (50 µg
of protein) from freshly isolated rat islets (lane 9) were
separated by 9% SDS-PAGE. INS-1E cells were treated for either 6 h (lanes 1 and 2) or 48 h (lanes 3 and
4) with 30 mM glucose or 100 nM
exogenous insulin as indicated. Lanes 5-7 represent,
respectively, total cell extracts from INS-1E cultured for 48 h in
standard medium (11.2 mM glucose) containing 1.5 mM long-chain free fatty acids (2:1 oleate/palmitate), 10 ng/ml TNF-
, and 10 µM H2O2.
Total cell lysates from INS-1E cells under standard culture conditions
are shown in lane 8 as control.
-Cells Is Distinct from Lipogenic
Tissues--
Unlike liver and adipose tissue, pancreatic
-cells
have evolved to secrete insulin rather than store energy in response to rising blood glucose and nutrient levels. The released insulin subsequently promotes the lipogenic gene expression in liver and adipocytes through up-regulation of SREBP-1 expression and increase of
its processing (4, 5, 43). The molecular mechanism by which
-cells
restrict lipogenesis in response to physiological glucose and insulin
stimulation remains undefined. We performed Western blotting using an
antibody specific for the N terminus of SREBP-1, which should recognize
both precursor and mature SREBP-1. As shown in Figure 7C,
the mature nuclear form of SREBP-1 was not detectable in nuclear
extracts from freshly isolated rat islets. Consistently, only the
precursor but not the mature nuclear form of SREBP-1 protein was
detected in total cell extracts from native INS-1E cells (Fig.
7C). Similar results were obtained in INS-1E cells treated
for 6 h with 30 mM glucose and 100 nM
exogenous insulin (Fig. 7C).
stimulates
the maturation of SREBP-1c in human hepatocytes (49). Furthermore,
hyperglycemia in animal models of diabetes causes increased SREBP-1
maturation and renal lipid accumulation leading to diabetic nephropathy
(50). Moreover, ethanol, ER stress, shear stress, and cytokines have
been reported to up-regulate the lipogenic gene expression in
hepatocytes and/or vascular endothelial cells by activation of SREBPs
(14, 49, 51-54).
, and increased
oxidative stress have been linked to
-cell dysfunction in Type 2 diabetes (9, 55-63). We therefore studied the SREBP-1 maturation in
native INS-1E cells, the most differentiated INS-1 cell clone (64). The
cells were exposed for 48 h to 30 mM glucose, 100 nM insulin, 1.5 mM long-chain free fatty acids
(2:1 oleate/palmitate), 10 ng/ml TNF-
, and 10 µM
H2O2 (Fig. 7C). The mature nuclear
form of SREBP-1 was detected in INS-1E cells treated for 48 h with glucose, insulin, TNF-
, or H2O2 (Fig.
7C). In contrast, free fatty acids were without effects
(Fig. 7C). The level of the mature form of SREBP-1 in INS-1E
cells treated with high glucose for 48 h is ~10% of that of the
endogenous precursor protein (Fig. 7C). This corresponds to
the induction observed with 75 ng/ml doxycycline in
naSREBP-1c#233 cells (Fig. 1B).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-cell dysfunction in Type 2 diabetes (9, 50). However the molecular
mechanism underlying the pathogenesis of lipotoxicity in pancreatic
-cells and other non-adipose tissues remains undefined. In fact,
lipotoxicity is a confusing concept. Some investigators refer to
lipotoxicity as the consequence of increased circulating free fatty
acids and cellular lipid accumulation (7-9), whereas others suggest
that glucotoxicity is the prerequisite for lipotoxicity, at least in
-cells (58, 59, 65-68). The present study and other
observations2 support the
latter (see bellow). In creased expression of SREBP-1c has been
demonstrated in islets and/or liver in several animal models of
diabetes (7, 12-16). Overexpression of SREBP-1 has been suggested to
be the cause of hepatic steatosis (14, 16, 51, 53, 69, 70) and diabetic
nephropathy (50). The present study provides direct and strong evidence
proving that the transcriptional activity of SREBP-1c also mediates
-cell dysfunction.
-cell line. This could be explained by marked increases in lipogenic
gene expression and unaltered mRNA levels of genes involved in
fatty acid
-oxidation. The lipogenic transcription factor naSREBP-1c
also induces impairment of nutrient-induced insulin secretion,
suggesting defective glucose metabolism. This could be caused by the
down-regulation of glucokinase, up-regulation of UCP-2, and disrupted
mitochondrial membrane potential observed in these INS-1 cells
expressing naSREBP-1c. In addition, we also show that naSREBP-1c
suppresses the expression of insulin, Pdx-1, and HNF-4
. Induction of
naSREBP-1c also leads to INS-1 cell growth arrest and apoptosis
possibly by both suppressing the expression of
cyclin-dependent kinase 4 and IRS-2 and promoting the
expression of P21WAF1/CIP1, APO-1/Fas/CD95, and Bax. Since
the SREBP-1c precursor is a known substrate for caspase 3, we postulate
that SREBP-1c may function as a proapoptotic gene in
-cells.
Similarly, lipid accumulation, impaired glucose-stimulated insulin
secretion, defective
-cell gene expression (insulin, Pdx-1,
glucokinase, and Glut-2), disorganized mitochondrial ultrastructure,
and "lipoapoptosis" have been reported in islet
-cells of
diabetic animals (7, 71-73). We have, therefore, not only established
an in vitro cellular model for
-cell glucolipotoxicity but also elucidated the underlying mechanism.
-cells have evolved to limit the
lipogenic gene expression in response to acute stimulation of glucose
and insulin by restricting the SREBP-1c processing. Our data are in
disagreement with a previous study, claiming that SREBP-1c maturation
in MIN-6 cells is acutely regulated by glucose (18). Whereas 6 h
of stimulation with glucose had no effect in the present study, we
could indeed detect the mature form of SREBP-1c in INS-1E cells after
48 h of exposure to high concentrations of glucose or insulin. In
contrast, 48 h of treatment with free fatty acids did not increase
the levels of the active form of SREBP-1c. Glucotoxicity, a phenomenon
occurring after prolonged exposure to high concentrations of glucose,
has been reported to induce lipid accumulation in INS-1 cells by
raising lipogenic gene expression (58). Our study may have established
a link between glucotoxicity and lipotoxicity and provided an
explanation for the predominant role of high glucose in
-cell
dysfunction (59, 65). A similar effect may account for the increased
apoptosis and subsequent decreased
-cell mass in Type 2 diabetic
patients compared with obese subjects (27). We also found that a 48-h culture of INS-1E cells with 30 mM glucose caused cell
apoptosis and defective gene expression similar to naSREBP-1c
induction, whereas 72 h of incubation with 1.5 mM free
fatty acids (2:1 oleate/palmitate) did not have such deleterious
effects.2 Therefore, naSREBP-1c may induce
-cell
dysfunction independent of lipid accumulation. Hyperglycemia and
hyperinsulinemia are characteristics of Type 2 diabetes. We hypothesize
that as a result of the persistent hyperglycemia and hyperinsulinemia
present in diabetes, SREBP-1c protein is processed and modified to an
active form. Activated SREBP-1c alters the expression of various target genes that contribute to
-cell dysfunction and therefore exacerbates the progression of Type 2 diabetes. Furthermore, our results suggest that the SREBP-1c maturation regulated by cytokines and oxidative stress may also play a role in
-cell dysfunction in Type 2 diabetes. It should be noted that the present work was performed in a rat insulinoma cell line and therefore may not entirely reflect the situation in native
-cells, necessitating caution in the interpretation.
-cell lipotoxicity by targeting
multiple genes implicated in carbohydrate metabolism, lipid
biosynthesis, cell growth, and apoptosis. Similar mechanisms may also
apply to the lipotoxicity in other non-adipose tissues. Development of
chemical compounds acting like leptin, PPAR
agonists, and metformin
through suppression of SREBP-1c function should have therapeutic
potential in the treatment of Type 2 diabetes and its complications.
![]() |
ACKNOWLEDGEMENTS |
---|
We are grateful to D. Cornut-Harry, D. Nappey, Y. Dupré, S. Polti, and V. Calvo for expert technical assistance. We are indebted to Drs. B. M. Spiegelman (SREBP-1c/ADD1-(1-403) cDNA), H. Bujard (PUHD10-3 vector), and N. Quintrell (pTKhygro plasmid).
![]() |
FOOTNOTES |
---|
* This work was supported by Swiss National Science Foundation Grant 32-49755.96 (to C. B. W.) and the Leenaards Foundation (to P. M.).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.
To whom correspondence should be addressed. Tel.:
41-22-702-5548; Fax: 41-22-702-5543; E-mail:
Haiyan.Wang@medicine.unige.ch.
§ Fellow of the Dr. Max Cloetta Foundation.
¶ Present address: Cellular Biochemistry and Biophysics, Memorial Sloan-Kettering Cancer Center, 1275 York Ave., Box 251, New York, NY 10021.
Present address: Dept. of Biochemistry, School of
Pharmacy, University of Barcelona, E-08028 Barcelona, Spain.
** Present address: Swiss Institute for Experimental Cancer Research (ISREC), CH-1066 Epalinges, Switzerland.
Present address: Rolf Luft Center of Diabetes
Research, Endocrine and Diabetes Unit, Dept. of Molecular Medicine,
Karolinska Institute, Karolinska Hospital, Stockholm, Sweden.
Published, JBC Papers in Press, February 24, 2003, DOI 10.1074/jbc.M212488200
2 H. Wang and C. B. Wollheim, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: SREBP, sterol regulatory element-binding protein; ER, endoplasmic reticulum; ADD1, adipocyte determination and differentiation factor-1; PPAR, peroxisome proliferator-activated receptor; naSREBP, nuclear active form of SREBP-1c; PBS, phosphate-buffered saline; BSA, bovine serum albumin; KRBH, Krebs-Ringer-bicarbonate-HEPES buffer; UCP, uncoupling protein; FCCP, carbonyl cyanide 4-trifluoromethoxyphenylhydrazone; Pdx, pancreas duodenum homeobox; HNF, hepatocyte nuclear factor; IRS, insulin receptor substrate; TNF, tumor necrosis factor.
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