ORIGINAL ARTICLE

Palmitate Impairs and Eicosapentaenoate Restores
Insulin Secretion Through Regulation of SREBP-1c in
Pancreatic Islets
Toyonori Kato,1 Hitoshi Shimano,1,2 Takashi Yamamoto,1 Mayumi Ishikawa,1 Shin Kumadaki,1Takashi Matsuzaka,1,2 Yoshimi Nakagawa,1,2 Naoya Yahagi,2 Masanori Nakakuki,1 Alyssa H. Hasty,3
Yoshinori Takeuchi,1 Kazuto Kobayashi,1 Akimitsu Takahashi,1 Shigeru Yatoh,1 Hiroaki Suzuki,1Hirohito Sone,1 and Nobuhiro Yamada1

OBJECTIVE—Chronic exposure to fatty acids causes ␤-cell
failure, often referred to as lipotoxicity. We investigated its
mechanisms, focusing on contribution of SREBP-1c, a key transcription factor for lipogenesis.
RESEARCH DESIGN AND METHODS—We studied in vitro
and in vivo effects of saturated and polyunsaturated acids on
insulin secretion, insulin signaling, and expression of genes
involved in ␤-cell functions. Pancreatic islets isolated from
C57BL/6 control and SREBP-1–null mice and adenoviral genedelivery or knockdown systems of related genes were used.
RESULTS—Incubation of C57BL/6 islets with palmitate caused
inhibition of both glucose- and potassium-stimulated insulin
secretion, but addition of eicosapentaenoate (EPA) restored
both inhibitions. Concomitantly, palmitate activated and EPA
abolished both mRNA and nuclear protein of SREBP-1c, accompanied by reciprocal changes of SREBP-1c target genes such as
insulin receptor substrate-2 (IRS-2) and granuphilin. These
palmitate-EPA effects on insulin secretion were abolished in
SREBP-1–null islets. Suppression of IRS-2/Akt pathway could bea part of the downstream mechanism for the SREBP-1c–mediated insulin secretion defect because adenoviral constitutively
active Akt compensated it. Uncoupling protein-2 (UCP-2) also
plays a crucial role in the palmitate inhibition of insulin secretion, as confirmed by knockdown experiments, but SREBP-1c
contribution to UCP-2 regulation was partial. The palmitate-EPA
regulation of insulin secretion was similarly observed in islets
from C57BL/6 mice pretreated with dietary manipulations. Furthermore, administration of EPA to diabetic KK-Ay mice ameliorated impairment of insulin secretion in their islets.
CONCLUSIONS—SREBP-1c plays a dominant role in palmitatemediated insulin secretion defect, and EPA prevents it through
SREBP-1c inhibition, implicating a therapeutic potential fortreating diabetes related to lipotoxicity. Diabetes 57:2382–
2392, 2008

From the 1Department of Internal Medicine (Endocrinology and Metabolism),
Graduate School of Comprehensive Human Sciences, University ofTsukuba, Tsukuba, Japan; the 2Center for Tsukuba Advanced Research
Alliance, University of Tsukuba, Tsukuba, Japan; and the 3Department of
Molecular Physiology and Biophysics, Vanderbilt University, Nashville,
Tennessee.
Corresponding author: Hitoshi Shimano, shimano-tky@umin.ac.jp.
Received 29 December 2006 and accepted 23 April 2008.
Published ahead of print at http://diabetes.diabetesjournals.org on 5 May 2008.
DOI: 10.2337/db06-1806.
© 2008 by the American Diabetes Association. Readers may use this article aslong as the work is properly cited, the use is educational and not for profit,
and the work is not altered. See http://creativecommons.org/licenses/by-nc-nd/3.0/ for details.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked “advertisement” in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

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M

olecular mechanisms of pancreatic islet
␤-cell failure, a crucial pathological contributor to the development of diabetes, have been
extensively explored (1–3). Impairment of
glucose-stimulated insulin secretion (GSIS) is an earlyfeature of type 2 diabetes, and influx of fatty acids into␤-cells (␤-cell lipotoxicity) has been thought to be involved in its pathogenesis (4,5). The intracellular events
leading to GSIS include glucose metabolism for ATP
production, closure of ATP-dependent K channels, membrane voltage-dependent calcium influx, calcium-dependent vesicle transport, and exocytosis of ␣-granules
containing insulin (6,7). Lipotoxicity has been implicatedin reducing GSIS via many of these steps (8). For example,
uncoupling protein-2 (UCP-2), a mitochondrial membraneprotein involved in energy production, plays an importantrole in fatty acid–induced lipotoxic effects (9 –12). Although ␤-cells have traditionally been thought to simply
produce insulin in response to glucose, more recent studies have highlighted the role of insulin signaling in ␤-cells.
Studies on insulin signaling in ␤-cells, such as targeted
disruption of the insulin receptor (13) and insulin receptor
substrate-2 (IRS-2) (14,15), have shown that this pathway
can influence both ␤-cell mass and insulin secretion.
Sterol regulatory element-binding protein (SREBP)-1c isa membrane-bound transcription factor of the basic helix
loop helix leucine zipper family and has been established
as a regulator of lipogenic enzymes in the liver (16,17).
Expression of SREBP-1c is highly upregulated by dietaryintake of carbohydrates and sugars (18 –21). Conversely,
polyunsaturated fatty acids (PUFAs), such as eicosapentaenoate (EPA), have been shown to inhibit hepatic
SREBP-1c through multiple mechanisms (22,23). Recent
data suggested that hepatic SREBP-1c is also induced bydietary saturated fatty acids (24). The data from SREBP-1ctransgenic and LDL receptor knockout doubly mutant
mice provide evidence that activation of this nutritionallyregulated lipid transcription factor could be involved information of components of metabolic syndrome, such as
hyperlipidemia and atherosclerosis (A. Takahashi, H. Shimano, unpublished data). Furthermore, SREBP-1c directly
represses IRS-2 expression and leads to hepatic insulin
resistance as a part of underlying pathogenesis for metabolic syndrome (25). In pancreatic ␤-cells, activation of
SREBP-1c has been shown to be involved in impaired
insulin secretion and glucose intolerance (26 –28). Features of hepatic SREBP-1c induction by saturated fatty
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T. KATO AND ASSOCIATES

acids, repression by PUFAs, and inhibition of IRS-2 werereproducibly observed in ␤-cells. As the major downstream insulin signaling pathway, the IRS-2/PI3K/Akt pathway has links to cell growth and survival and to glucose
metabolism leading to ATP production (29). Insulin signaling in ␤-cells has been thought to be important for ␤-cellmass based on analyses of ␤-cell–specific transgenic miceof Akt (30 –32) and tissue-specific knockout mice of insulin
receptor (13) and IRS-2 (14,15). More recently, importanceof insulin signaling in ␤-cell function has also been noticed
in the glucose/insulin-signaling/Foxo1 pathway (29,33,34).
Nuclear Foxo1 has a negative effect on ␤-cell mass andinsulin secretion in vivo (34). Insulin signaling phosphorylates nuclear Foxo1 for nuclear exclusion and contributes to ␤-cell protection.
Recently, we reported that granuphilin, a crucial component of the docking machinery of insulin-containing
vesicles to the plasma membrane (35–37), is regulated by
SREBP-1c in ␤-cell (38). Thus, taken together with clinicalimplication of fatty acids as causative factors for ␤-celllipotoxicity, it is conceivable that SREBP-1c is involved in␤-cell lipotoxicity–mediated insulin secretion defects in
GSIS.
In the current studies, we investigated the effects ofpalmitate, a typical saturated fatty acid, on GSIS in isolated islets and found that palmitate impairs GSIS and that
addition of EPA protects against these effects. Analyses ofpalmitate-EPA on gene expression, including SREBP-1c
and their target genes, led to clarification of the molecularmechanisms of palmitate-induced ␤-cell lipotoxicity andprotective effects of EPA.
RESEARCH DESIGN AND METHODS
Palmitate and EPA were purchased from Sigma (St. Louis, MO). Enhanced
chemiluminescence Western blot detection kit, [1-14C]palmitate, and [3H]mannitol were purchased from Amersham Biosciences.
This project was approved by the Animal Care Committee of the Universityof Tsukuba. Male C57BL/6 wild-type and KK-Ay mice at 8 weeks of age werepurchased from Clea Japan (Tokyo). SREBP-1–null mice at 14 –15 weeks ofage were as described previously (39). The mice were housed in colony cages,
maintained on a 12-h light/12-h dark cycle, given free access to water and astandard chow diet (MF; Oriental Yeast, Tokyo), and adapted to their new
environment for 1 week before experiments.
Isolation of mouse pancreatic islets. Isolation of islets from mice wascarried out according to the Ficoll-Conray protocol as described previously(26,38,40). In brief, after clamping the common bile duct at a point close to theduodenum outlet, 2.5 ml Krebs-Ringer bicarbonate buffer (KRBH, pH 7.4)
containing 0.5% BSA and 4 mg/ml collagenase (Sigma) was injected into theduct. The swollen pancreas was removed and incubated at 37°C for 20 min.
The pancreas was then dispersed by pipetting, and after washing twice with
KRBH, the islets were collected manually under stereomicroscope. Isolated
islets were put in culture medium (RPMI 1640 supplemented with 10% FCS,
0.5% BSA, 100 units/ml penicillin, and 100 ␮g/ml streptomycin as antibiotics)
for 16 –18 h at 37°C in a humidified atmosphere containing 5% CO2 before theexperiments.
Analyses of insulin secretion and insulin contents of islets. Insulin
release from islets was measured as described previously (26,38). Batches of
10 islets were incubated for 30 min in 1 ml KRBH, pH 7.4, containing 0.5% BSA
at 2.8 mmol/l glucose for 30 min. Islets medium was replaced with KRBH
containing 20 mmol/l glucose or alternatively 30 mmol/l KCl plus 2.8 mmol/l
glucose to estimate insulin secretion and were incubated for 30 min. At theend of each incubation period, the medium was collected, and islets were
subjected to insulin extraction with acidic ethanol (0.2 mol/l HCl in 75%
ethanol) for insulin measurement with an insulin enzyme-linked immunosorbent assay kit. Hoechst-33258 was used to determine the DNA contents ofsonicated islets.
Determination of ATP-to-ADP ratio and triglyceride contents of islets.
ATP and ADP contents in isolated islets were as described previously(26,38,41). ATP and ADP were extracted from islets with 5% of trichloroacetic
acid. After centrifugation, the supernatants were neutralized with NaOH. ATPcontent was measured using the CellTiter-Glo luminescent cell viability assay
DIABETES, VOL. 57, SEPTEMBER 2008

kit (Promega, Madison, WI). ADP content was estimated after conversion of
ADP to ATP in the reaction buffer (20 mmol/l HEPES and 3 mmol/l MgCl2, pH
7.75) containing 2.3 units/ml pyruvate kinase and 1.5 mmol/l phosphoenolpyruvate at room temperature for 15 min.
Triglycerides (TGs) of islets were measured after extracting lipids with
Folch’s method. After 1–2 min of sonication, islets were mixed with chloroform and methanol (2:1) for lipid extraction, dried up by evaporation, andresuspended in isopropanol. TG concentration was measured using the
GPO-trinder kit (Sigma).
Real-time PCR and immunoblot analysis. Total RNA extraction with the
TRIzol reagent (Invitrogen, Carlsbad, CA) and DNase-I treatment using the
RNeasy Micro kit (Qiagen, Hilden, Germany) were performed according to the
manufacturers’ instructions. cDNA was synthesized with ThermoScript (Invitrogen), and comparative analysis of mRNA levels was performed withfluorescence-based real-time PCR. Real-time PCR analyses were performed
using SYBR-Green Dye (Nippon Gene, Tokyo) in an ABI 7000 PCR instrument(Applied Biosystems, Foster City, CA). The relative abundance for eachtranscript was calculated by a standard curve of cycle thresholds for serial
dilutions of a cDNA sample and normalized to cyclophilin. Primer sequences
are described in supplemental Table 3, which is available in the online
appendix at http://dx.doi.org/10.2337/db06-1806.
Immunoblot analyses were performed as described previously (26,38). Cellextracts from isolated islets were probed with rabbit polyclonal anti–SREBP-1(sc-8984; Santa Cruz Biotechnology, Santa Cruz, CA), anti–IRS-2 (06 –506;
Upstate Technology, Bedford, MA), anti-Akt (no. 9272), anti–phospho Akt(S473; no. 9271), anti–phospho Akt (T308; no. 9275; Cell Signaling, Beverly,MA), anti–UCP-2 (Research Diagnostic, San Antonio, TX), and anti–␣-tubulin(sc-8035; Santa Cruz Biotechnology). Anti-granuphilin a/b antibody was usedas previously described (37,38). Detection was performed using an ECL
advance Western blotting detection kit and ECL Hyperfilm (Amersham
Biosciences).
Treatment of islets with palmitate and EPA. Palmitate and EPA weredissolved to 100 mmol/l in methanol to make stock solutions for later dilution
in RPMI 1640 supplemented with 0.5% BSA to a final concentration of 400
␮mol/l (palmitate) and 50 ␮mol/l (EPA), respectively. Islets were treated for
48 h before indicated experiments.
Cellular uptake of [1-14C]palmitate. [1-14C]palmitate uptake of islets wasmeasured as described previously (42). Briefly, the isolated islets wereincubated for 60 min in culture medium containing 400 ␮mol/l palmitate, 0.3
␮Ci/ml radiolabeled [1-14C]palmitate with or without 50 ␮mol/l EPA, and 0.06
␮Ci/ml [3H]mannitol. The latter was used to calculate correction for nonspecific uptake. Ice-cold 0.5 N NaOH was added to the islets to terminate theuptake reaction and neutralized by 0.5 N HCl. After the removal of thesupernatant by centrifugation at 12,000 ⫻ g for 1 min, the residual radioactivity was determined.
Small interfering RNA for UCP-2. The small interfering RNA (siRNA)
construct for mouse UCP-2 was generated within the coding region of UCP-2:
5⬘-GTCGAAGCCTACAAGACCA-3⬘ (Ad-UCP-2 RNAi). The siRNA for LacZ
(Ad-LacZ RNAi) from Invitrogen (BLOCK-iT U6 RNAi Entry Vector kit;
K4944 – 00) was used as a control according to manufacturer’s instructions.
Oligonucleotide containing this sequence was subcloned into U6/RNAi Entry
vector (Invitrogen). UCP-2 RNAi adenoviruses were generated using
BLOCK-iT Adenoviral RNAi Expression System (Invitrogen).
Adenovirus infections for constitutively active Akt and siRNA of UCP2.
Infection of constitutively active Akt (43) and siRNA of UCP-2 adenovirus
studies were performed as described previously (25,38,44). In brief, generation of recombinant adenoviral plasmid was produced by homologous recombination with the pAdEasy-1 plasmid (Invitrogen). Production of recombinantadenoviruses was performed by CsCl gradient centrifugation as previously
described (25,38,44).
Palmitate-rich diet study and KK-Ay mice study. The in vivo palmitaterich diet study and KK-Ay mice study were described in RESEARCH DESIGN AND
METHODS in the online appendix. Briefly, in the palmitate-rich diet study,
C57BL6 mice were fed with control diet (fish oil–free diet), tripalmitin diet
(20% tripalmitin), and tripalmitin plus EPA-E diet (20% tripalmitin and 5%
EPA-E) for 28 days. In KK-Ay mice study, KK-Ay mice were administeredvehicle (5% gum arabic) or EPA-E at a dose of 1 g 䡠 kg⫺1 䡠 day⫺1 for 28 days.
Statistical analysis. Results are reported as means ⫾ SE. Statistical analyses
were performed using one-way ANOVA followed by Dunnett’s procedure or
two-way ANOVA followed by Tukey’s procedure.

RESULTS

Palmitate impairs and EPA restores insulin secretionin murine islets. To investigate pancreatic lipotoxicity,
we evaluated effects of palmitate (C16:0) on the insulin
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EPA PREVENTS ␤-CELL LIPOTOXICITY THROUGH SREBP-1c

FIG. 1. Lipotoxic effects of palmitate and protective effects of EPA on
insulin secretion in murine-isolated islets. A: Low GSIS (2.8 mmol/l),high GSIS (20 mmol/l), and KSIS from murine-isolated islets incubatedwithout (control, white bars) or with palmitate (black bars), palmitate-EPA (bold hatched bars), or EPA (regular hatched bars). B:
Insulin content of islets incubated without (control) or with palmitate,palmitate-EPA, or EPA. C: Palmitate uptake in islets isolated from
C57BL/6 mice. Three independent experiments were performed using
four sets of islets for each repetition, and results are expressed asmeans ⴞ SE. Statistical analysis was performed using one-way ANOVA
followed by Dunnett’s procedure. **P < 0.01 and *P < 0.05 vs.
palmitate group, respectively.

secretion of isolated mouse pancreatic islets. Althoughpalmitate (400 ␮mol/l) had no effect on basal insulin
secretion (low glucose concentrations), stimulation withhigh glucose concentrations, i.e., GSIS, was inhibited by
the addition of palmitate (Fig. 1A). When 50 ␮mol/l EPA(C20:5, n-3) was combined with palmitate-treated islets(hereafter referred to as palmitate-EPA), the suppressed
insulin secretion was restored to near-normal levels (Fig.
2384

1A). Palmitate inhibition and EPA restoration of insulin
secretion was also observed after addition of KCl, whichbypasses ATP-sensitive channels to stimulate insulin secretion (KCl-stimulated insulin secretion [KSIS]) (Fig. 1A).
These effects of palmitate and EPA on insulin secretion
were dose dependent (supplemental Fig. 1A and B). EPA
by itself did not have any effect on GSIS or KSIS, indicatingthat EPA does not intrinsically increase but cancels palmitate-suppressed insulin secretion. The slight changes of
insulin content compared with GSIS and KSIS by palmitateand EPA indicate that the phenomenon in insulin content
was only a part of the mechanism (Fig. 1B). Considering
the experimental setting, the protective effect of EPA
against palmitate-induced lipotoxicity could be inhibitionof cellular uptake of palmitate. To exclude this possibility,
uptake of labeled palmitate was measured and was notaffected by additional EPA (Fig. 1C). These data indicatethat the EPA does not interfere with palmitate uptake but
rather directly competes with palmitate in intracellular
events.
Palmitate and EPA regulate SREBP-1c and its target
genes. Gene expression in palmitate- and palmitate-EPA–
treated islets was investigated using real-time PCR.
SREBP-1c mRNA was highly induced by palmitate andcompletely suppressed by EPA but not SREBP-1a mRNA
(Fig. 2A). These changes in SREBP-1c mRNA were associated with those in both membrane and nuclear forms of
SREBP-1c protein (Fig. 2D). In accordance, its target
genes, such as fatty acid synthase, stearoy-CoA desaturase
1, and elongation of long-chain fatty acids family number 6
showed similar patterns of regulation by palmitate andpalmitate-EPA (Fig. 2B). TG content, as an indication of
SREBP-1c effect and lipotoxicity, was increased by palmitate and repressed by palmitate-EPA (Fig. 2C).
We recently reported that SREBP directly suppressed
hepatic IRS-2 expression and caused insulin resistance inthe liver (25). In accordance with changes in SREBP-1c inislets, IRS-2 was strongly suppressed by palmitate and waspartially restored by addition of EPA, implicating a role for
SREBP-1c–mediated IRS-2 repression in the palmitateEPA–mediated changes in ␤-cell physiology (Fig. 2D;
supplemental Fig. 2A).
UCP-2 has been shown to play a key role in lipotoxicity
of pancreatic ␤-cells through dissociation of fatty acidoxidation and ATP production (supplemental Fig. 2B)(9 –12). UCP-2 promoter was also reported as an SREBPtarget (11,12). This key regulator of lipotoxicity was modulated by palmitate and palmitate-EPA in a similar mannerat both mRNA and protein levels (supplemental Fig. 2A;
Fig. 2D).
Granuphilin was an effector of Rab27a, and its overexpression was reported to inhibit exocytosis of insulin
granules (35–37). We recently reported that granuphilinpromoter was a direct target of SREBP-1c and that the
SREBP-1c/granuphilin pathway was a potential mechanism for impairment GSIS in diabetes, leading to ␤-celllipotoxicity (38). This key molecule of lipotoxicity was
upregulated by palmitate and suppressed by palmitateEPA at both mRNA and protein levels (supplemental Fig.
2A; Fig. 2D).
SREBP-1c plays a dominant role in palmitate-EPA
effects on insulin secretion. The contribution of
SREBP-1c to palmitate-EPA effects on both GSIS and KSIS
was estimated using islets from SREBP-1–null mice. Basal
insulin secretion was not affected by SREBP-1 deficiency(data not shown); however, GSIS was modestly increased
DIABETES, VOL. 57, SEPTEMBER 2008

500
400
300

500

** **

Control
PA
PA-EPA
EPA

Relative mRNA Levels(Normalized to Cyclophilin)

Relative mRNA Levels(Normalized to Cyclophilin)

T. KATO AND ASSOCIATES

200
100
0

Control
PA
PA-EPA
EPA

400
300

****

****

200
100
0

SREBP-1a

SREBP-1c

FAS
Control**

10
TG Content (ng/islet)

****

**

SCD-1

ELOVL6

PA PA-EPA EPAmSREBP-1

8nSREBP-1

6

UCP-2

4

IRS-2

2

Granuphilin0Control

PA

PA-EPA

EPA-Tubulin

FIG. 2. Gene expression and protein profiles in murine-isolated islets treated with palmitate or palmitate-EPA. A and B: Levels of mRNA ofvarious genes in pancreatic islets isolated from C57BL/6 mice without (control, white bars) or with palmitate (black bars), palmitate-EPA(hatched bars), or EPA (regular hatched bars) as determined by real-time PCR. mRNA quantities were calculated as a ratio to the cyclophilin levelin the each cDNA sample. Data are shown as the relative expression ratio to control samples. C: Cellular TG levels of islets incubated withpalmitate, palmitate-EPA, or EPA. D: Immunoblot analysis of indicated proteins in the islets. Cont., control; mSREBP-1, membrane form of
SREBP-1; nSREBP-1, nuclear form of SREBP-1. ␣-Tubulin protein was used as a loading control. Three independent experiments were performed
using four sets of islets, and results are expressed as means ⴞ SE. Statistical analyses were performed using one-way ANOVA followed byDunnett’s procedure. **P < 0.01 vs. palmitate group.

(Fig. 3A). The absence of SREBP-1 abolished palmitateinduced inhibition of GSIS and KSIS (Fig. 3A). Because ofthis, EPA protection from impairment of GSIS and KSIS, asobserved in wild-type islets, was not detected in SREBP1–null islets (Fig. 3A). These data suggest that palmitateinduced pancreatic lipotoxicity and amelioration of that by
EPA depend on SREBP-1c. Predictably, from the primaryrole of SREBP-1c in lipogenesis, the elevation and repression of TG content in wild-type islets by palmitate and
EPA, respectively, were absent in SREBP-1–null islets(Fig. 3B). Suppression of IRS-2 and stimulation of
granuphilin mRNA expressions caused by palmitate inwild-type islets were both blunted in SREBP-1–null islets(Fig. 4A). Accordingly, the reversal of palmitate effects on
IRS-2 and granuphilin expression by EPA was not observed in SREBP-1–null islets. On the other hand, induction of UCP-2 expression by palmitate was observed evenin SREBP-1–null islets, but EPA treatment reversed thepalmitate effect in both genotypes (Fig. 4A).
IRS-2 suppression by SREBP-1c contributes to palmitate-EPA effects on GSIS. Based on recent cumulative
evidence of the importance of insulin signaling in ␤-cellfunction and our observation of reciprocal changes in
SREBP-1c and IRS-2 by addition of palmitate and/or EPA
in islets (Fig. 2D; supplemental Fig. 2A), we hypothesized
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that palmitate suppression of insulin secretion might bedue to impaired insulin signaling caused by induction of
SREBP-1c, leading to decreased IRS-2 expression. To test
this hypothesis, insulin signaling was estimated in wildtype and SREBP-1–null islets by analysis of Akt phosphorylation status. Consistent with changes at the mRNA level(supplemental Fig. 2A), suppression and restoration of
IRS-2 protein by palmitate and palmitate-EPA, respectively, in wild-type islets was not apparent in SREBP-1–
null islets (Fig. 4B). Consequently, Akt phosphorylation
impaired by palmitate in wild-type islets was completely
abolished by the absence of SREBP-1 (Fig. 4B). These datasuggest that SREBP-1c could be highly involved in palmitate-mediated inhibition of insulin signaling and insulin
secretion.
To explore impacts of insulin signaling on palmitateEPA–regulated insulin secretion, forced activation of insulin signaling downstream of IRS-2 was induced in mouse
isolated islets by adenoviral gene transfer of constitutively
active (dominant-positive) Akt (Akt-CA). Akt-CA overexpression significantly improved both GSIS and KSIS,
which were impaired by palmitate, but did not further
enhance restoration by EPA, indicating that insulin signaling and insulin secretion were linked in palmitate-EPA
effects (Fig. 5A). Akt-CA overexpression only slightly
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EPA PREVENTS ␤-CELL LIPOTOXICITY THROUGH SREBP-1c

Insulin Secretion(pg/ng DNA/hr)

A
1600

**

1400

**

*

SREBP-1(+/+)
SREBP-1(-/-)

1200
1000
800
600
400
200
0
Control PA

PA
-EPA

EPA

Control PA

20 mM glucose

B

12

PA
-EPA

EPA

30 mM KCl**

SREBP-1(+/+)
SREBP-1(-/-)**

TG Content(ng/islet)

10

8

6

4

2

0
Control

PA

PA-EPA

EPA

FIG. 3. Protection from palmitate-induced lipotoxicity in islets isolated from SREBP-1–null mice. Islets were isolated from wild-type littermates(white bars) and SREBP-1–null mice (black bars) and incubated without (control) or with palmitate, palmitate-EPA, or EPA for 48 h. GSIS and
KSIS (A) and cellular TG contents (B) were measured. Three independent experiments were performed using four sets of islets, and results are
expressed as means ⴞ SE. Statistical analyses were performed using two-way ANOVA followed by Tukey’s procedure. **P < 0.01 and *P < 0.05,
respectively.

enhanced phosphorylation of Akt in untreated islets but
completely restored suppressed pAkt in palmitate-treated
islets (Fig. 5B). Insulin signaling downstream of Akt, such
as pAkt, was also consistently suppressed by palmitate.
These signaling molecules were all restored by Akt-CA
overexpression. Islets treated with palmitate-EPA exhibited signals similar to control islets regardless of Akt-CA
overexpression (Fig. 5B). Both SREBP-1 deficiency (Figs.
3A and 4B) and constitutive activation of insulin signaling
by Akt-CA (Fig. 5A and B) cancelled the protective effectsof EPA against palmitate-induced impaired insulin secretion and insulin signaling. Activation of Akt did not change
either SREBP-1c or UCP-2 (Fig. 5C).
Contribution of UCP-2 to palmitate-EPA effects on
GSIS. The contribution of UCP-2 to the effects of palmitate-EPA on GSIS was estimated in knockdown experiments using adenoviral siRNA of UCP-2. A robust
2386

inhibition of mRNA and protein levels of UCP-2 was
obtained (Fig. 6A; supplemental Fig. 3). Gene silencing of
UCP-2 did not effect basal insulin secretion or GSIS. In
contrast, UCP-2 knockdown significantly protected palmitate-mediated impaired GSIS and canceled the EPA protection (Fig. 6B). Palmitate-mediated reduction in ATP-toADP ratio was significantly restored by UCP-2
suppression, and the protective effect of EPA was also
canceled (Fig. 6C). Changes in ATP-to-ADP ratio and GSIS
by modulation of UCP-2 expression were very similar,
confirming that the palmitate-EPA effects on GSIS dependon the UCP-2/ATP system, as was previously suggested byknockout mice studies. Palmitate induction of SREBP-1c
was not affected by UCP-2 knockdown (Fig. 6D). Taken
together with partial regulation of UCP-2 in SREBP-1–null
islets (Fig. 4A), effects of UCP-2 and SREBP-1c on GSIS
are partially connected.
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T. KATO AND ASSOCIATES

Relative mRNA Levels(Normalized to Cyclophilin)

A

UCP-2

SREBP-1c
400

**

400

**

300

300

200

200

100

100
N.D

0

Control

N.D

PA

N.D

N.D

PA
-EPA

EPA

**

Relative mRNA Levels(Normalized to Cyclophilin)**

SREBP-1(+/+)
SREBP-1(-/-)

0
Control

PA
-EPA

PA

EPA

Granuphilin

IRS-2
200

**

300

**

150

**

*

200

100
100

50
0
Control

PA

B
75KDa

PA
-EPA

0
EPA

Control

SREBP-1(+/+)

SREBP-1(-/-)

PA

PA
-EPA

EPA

Cont. PA PE EPA Cont. PA PE EPA
MatureSREBP-1

SREBP-1
50KDa

Aberrant
SREBP-1

IRS-2
pAkt(Ser)pAkt(Thr)
Akt-Tubulin
FIG. 4. Gene expression and protein profiles in islets isolated from SREBP-1–null mice treated with palmitate or palmitate-EPA. Islets wereisolated from SREBP-1–null mice and wild-type littermates and incubated without (control) or with palmitate, palmitate-EPA (PE), or EPA for
48 h. A: mRNA levels of the indicated genes were measured. mRNA levels were determined by real-time PCR, calculated as ratio to cyclophilin
expression levels. B: Immunoblot analysis of SREBP-1 and insulin-signaling proteins. Relative expression ratios to control samples are shown.
Three independent experiments were performed using four sets of islets, and results are expressed as means ⴞ SE. Statistical analyses were
performed using two-way ANOVA followed by Tukey’s procedure. **P < 0.01 and *P < 0.05, respectively.

EPA in vivo exhibits a protective role against palmitate lipotoxicity in islets. To determine whether theeffects of palmitate-EPA on GSIS in isolated islets could be
extended in vivo, mice were fed a fish oil–free diet with orwithout 20% tripalmitin or tripalmitin plus 5% EPA ethyl
ester for 28 days, and GSISs in freshly isolated islets fromthese animals were measured. Palmitate feeding impairedand EPA restored GSIS in conjunction with changes inislet SREBP-1c expression (Fig. 7A and B). These data
demonstrate that dietary palmitate and EPA influenceinsulin secretion in vivo in a similar manner to palmitateEPA effects observed in isolated islets.
DIABETES, VOL. 57, SEPTEMBER 2008

The effect of EPA on GSIS in vivo was further investigated in isolated islets from KK-Ay mice, a model ofobesity and type 2 diabetes (45). In islets from KK-Ay mice,
GSIS was impaired, and SREBP-1c expression was increased. Administration of EPA ethyl ester at a dose of
1 g 䡠 kg⫺1 䡠 day⫺1 for 28 days restored GSIS and suppressed
SREBP-1c expression (Fig. 7C and D), leading to restoration of GSIS and KSIS. In both in vivo experiments, thesedata did not accompany changes in food intake or grossmorphological changes in pancreatic islets (supplemental
Tables 1 and 2; Fig. 4).
2387

EPA PREVENTS ␤-CELL LIPOTOXICITY THROUGH SREBP-1c

Insulin Secretion (pg/ng DNA/hr)

A

**

1000

**
Ad-GFP
Ad-Akt-CA

800
600

*

400
200
0
Control PA

PA EPA-EPA

20 mM glucose

30 mM KCl

ControlB-

Ad-Akt-CA

PA EPA-EPA

Control PA

PA

+

-

PA-EPA

+

-

+

IRS-2
pAkt(Ser)pAkt(Thr)
Akt-Tubulin

Relative mRNA Levels(Normalized to Cyclophilin)C

SREBP-1c
800

**

**

UCP-2
600

*

*

Ad-GFP
Ad-Akt-CA

600
400
400
200

200
0

0
Control

PA

PA-EPA

Control

PA

PA-EPA

FIG. 5. Effects of overexpression of constitutively active Akt in islets treated with palmitate or palmitate-EPA. Islets were isolated from C57BL/6
mice and incubated without (control) or with palmitate or palmitate-EPA for 48 h. Islets were infected (100 multiplicity of infection,
respectively) with adenoviral-GFP (Ad-GFP) or adenoviral– constitutively active Akt (Ad-Akt-CA) for 48 h before incubation with palmitate or
palmitate-EPA. GSIS and KSIS (A) and protein levels of indicated insulin-signaling molecules (B) were measured. Amounts of insulin-signaling
proteins were estimated by immunoblot analysis using indicated antibodies, and ␣-tubulin protein was used as a loading control. Levels of mRNAof SREBP-1c and UCP-2 were determined by real-time PCR (C), calculated as ratio to cyclophilin expression levels. Relative expression ratios tocontrol samples are shown. Three independent experiments were performed using four sets of islets, and results are expressed as means ⴞ SE.
Statistical analyses were performed using two-way ANOVA followed by Tukey’s procedure. **P < 0.01 and *P < 0.05, respectively.

DISCUSSION

It has long been known that chronic exposure of palmitate
to islets or ␤-cell lines causes lipotoxicity leading toblunted GSIS (1,3–5). Our current studies clearly demonstrate that this palmitate-induced impairment of insulin
2388

secretion is restored by supplement of EPA. The results
also indicated that this palmitate-EPA regulation is not due
to cell toxicity or apoptosis (data not shown) but mediatedthrough two major key molecules: SREBP-1c and UCP-2.
Several factors are known to be important for function of
DIABETES, VOL. 57, SEPTEMBER 2008

T. KATO AND ASSOCIATES

A

B

**

**

Ad-LacZ RNAi
Ad-UCP-2 RNAi

200
150
100
50

Insulin Secretion (pg/ng DNA/hr)

UCP-2 mRNA Level(Normalized to Cyclophilin)

250

1000

**

Ad-LacZ RNAi
Ad-UCP-2 RNAi

800
600
400
200
0

PA
EPA
-EPA
2.8 mM glucose

Control PA

0Control

PA

PA-EPA

EPA

C

**

PA
EPA
-EPA
20 mM glucose

Control PA

D

**

**

Ad-LacZ RNAi
Ad-UCP-2 RNAi

15

10

5

350
SREBP-1c mRNA Level(Normalized to Cyclophilin)

ATP/ADP Ratio

20

**

**

300

Ad-LacZ RNAi
Ad-UCP-2 RNAi

250
200
150
100
50
0

0
Control

PA

PA-EPA

EPA

Control

PA

PA-EPA

EPA

FIG. 6. Effects of UCP-2 gene silencing on murine-isolated islets treated with palmitate or palmitate-EPA. Islets were isolated from C57BL/6 miceand infected (500 multiplicity of infection, respectively) with adenoviral siRNA for LacZ (Ad-LacZ RNAi) or UCP-2 (Ad-UCP-2 RNAi) and cultured
without (control) or with palmitate, palmitate-EPA, or EPA for 48 h. A: The effects of UCP-2 siRNA on mRNA levels of UCP-2 in pancreatic islets
isolated from C57BL/6 mice incubated with palmitate, palmitate-EPA, or EPA were determined by real-time PCR. GSIS (B) and ATP-to-ADP ratio(C) from the islets after UCP-2 gene silencing and incubation with palmitate, palmitate-EPA, or EPA were measured. Level of mRNA of SREBP-1c
was determined by real-time PCR (D), calculated as ratio to cyclophilin expression levels. Relative expression ratios to control samples areshown. Three independent experiments were performed using four sets of islets, and results are expressed as means ⴞ SE. Statistical analyses
were performed using two-way ANOVA followed by Tukey’s procedure. **P < 0.01.

␤-cells, such as ATP-to-ADP ratio, IRS-2/Akt insulin signaling, and granuphilin. These factors are all consistently
disturbed by palmitate and improved by additional EPA
through up- and downregulation of SREBP-1c, respectively. Taken together with overexpression and knockout
experiments of SREBP-1, it can be concluded that
SREBP-1c plays a crucial role in ␤-cell lipotoxicity as a
causative upstream factor.
Contribution of UCP-2 to ATP depletion and impaired
insulin secretion has been well established (9 –12,26). Ourcurrent studies also confirm this in palmitate-mediatedsuppression of GSIS. Palmitate led to upregulation of
UCP-2 and reduction of intracellular ATP. Knockdown of
UCP-2 by siRNA restored palmitate-induced impairment of
GSIS. SREBP has been reported to directly bind to andactivate the UCP-2 promoter (11,12). Supportively, weobserved that ␤-cell–specific overexpression of SREBP-1celevated UCP-2 expression contributing to the ␤-cell lipotoxicity in transgenic mice (26). However, based on thecurrent results from SREBP-1–null islets, SREBP-1c only
partially participated in palmitate-induced expression of
UCP-2. Conversely, UCP-2 knockdown did not affect
SREBP-1c expression in islets. Thus, although both key
DIABETES, VOL. 57, SEPTEMBER 2008

molecules play a dominant role in ␤-cell lipotoxicity, there
might not be a definite causative relationship between
SREBP-1c, an indicator of lipogenesis, and UCP-2, an
indicator of energy consumption (Fig. 8).
Our data on Akt-CA overexpression experiments provide further evidence for the importance of insulin signaling in ␤-cell function. Palmitate inhibited and EPA
restored insulin signaling in an opposite manner to
SREBP-1c expression. Based on the potential effect of
SREBP-1c on insulin signaling through regulation of IRS-2(25,26), we explored involvement of insulin signaling inpalmitate-EPA regulation of insulin secretion. Activationof Akt did not change normal insulin secretion but markedly ameliorated palmitate-impaired insulin secretion in
isolated islets. Thus, insulin signaling could be a prerequisite for insulin secretion, and the importance of its role ininsulin secretion becomes overt only with regard to its
impairment. Palmitate suppression of insulin signaling
was cancelled in SREBP-1–null islets. Based on these data,
we conclude that SREBP-1c in ␤-cells plays a crucial rolein the inhibition of insulin signaling via suppression of
IRS-2 and contributes to impaired insulin secretion (26). In
contrast to established effects of insulin signaling on ␤-cell
2389

EPA PREVENTS ␤-CELL LIPOTOXICITY THROUGH SREBP-1c

A

C57BL/6 Control diet
C57BL/6 20% Tripalmitin diet
C57BL/6 20% Tripalmitin + 5% EPA-E dietC

B

C57BL/6 Control diet
C57BL/6 20% Tripalmitin diet
C57BL/6 20% Tripalmitin + 5% EPA-E dietD

FIG. 7. Effect of EPA on insulin secretion in vivo. C57BL/6 mice were fed control diet (white bars), 20% Tripalmitin diet (black bars), andTripalmitin ⴙ 5% EPA-E diet (hatched bars) for 28 days. Islets were isolated from individual animals. GSIS and KSIS (A) and mRNA levels of
SREBP-1c (B) were measured. KK-Ay mice were administered vehicle (black bars) or EPA at a dose of 1 g 䡠 kgⴚ1 䡠 dayⴚ1 (hatched bars) for 28 days.
Islets were isolated from pool pancreas (three to four animals). GSIS (C) and mRNA levels of SREBP-1c (D) were measured. Three independent
experiments were performed using four sets of islets, and results are expressed as means ⴞ SE. Statistical analyses between indicated groups
were performed using one-way ANOVA followed by Dunnett’s procedure. **P < 0.01 and *P < 0.05, respectively.

mass (13–15,29 –34), our data indicated that activation of
Akt could restore insulin secretion impaired by palmitate
in a short term. The precise molecular mechanism for thisis currently unknown, although phosphorylation of Foxo-1
and anti-apoptosis could be involved (29,34).
Dietary PUFAs, such as EPA, have been shown to haveplasma TG-lowering effects and to improve fatty liver andhepatic insulin resistance (22,23,46). We previously reported that PUFAs inhibited hepatic SREBP-1c, which
contributed to beneficial roles of PUFAs against lipotoxicity in the liver (22,23). Current data provide anotherbeneficial role of EPA: protection from lipotoxicity inpancreatic ␤-cells. Our data also suggest that this protective action of EPA is mediated mainly through suppressionof SREBP-1c. EPA reduced mRNA and nuclear protein
levels of SREBP-1c in palmitate-treated islet. In addition, alarge portion of EPA protection against palmitate-induced
impaired GSIS was not reproduced in SREBP-1–null islets.
2390

Amelioration of impaired GSIS by EPA was also confirmed
in vivo with SREBP-1c suppression. EPA also suppressed
overexpression of UCP-2 by palmitate even in SREBP-1–
null islets. This suggests that suppression of UCP-2 also
may contribute to protective effect of EPA against palmitate-mediated suppression of GSIS, which is presumably
independent of SREBP-1c (Fig. 7).
Our data showing that enhancement of insulin signalingin ␤-cells can improve impaired insulin secretion caused
by lipotoxic effects of palmitate have important clinicalrelevance. It has been recognized that hyperglycemia
exacerbates the impairment of insulin secretion, often
referred to as glucotoxicity, and that short-term insulin
treatment often effectively improves insulin secretion.
This has been thought to be due to reducing blood glucose;however, our current findings implicate that stimulation of
insulin signaling in ␤-cells could potentially contribute to
the improvement of insulin secretion, especially in lipoDIABETES, VOL. 57, SEPTEMBER 2008

T. KATO AND ASSOCIATESFree fatty acids

SREBP-1c mRNAEPA
SREBP-1c membrane form

EPA

SREBP-1c nuclear form

Fatty Acidsynthesis

UCP-2

ATP production

IRS-2

Granuphilin

Insulin signal

Insulin secretion
FIG. 8. Mechanism by which palmitate induces and EPA protects
impairment of insulin secretion in pancreatic islets.

toxic states. From a long-term standpoint, our findings
might relate to the onset of type 2 diabetes because intake
of excess saturated fatty acids can cause both insulin
resistance and impaired insulin secretion in ␤-cells. Ourdata also suggest that oral dosing of EPA could contributeto protection from the ␤-cell lipotoxicity. Because ourfindings are based mostly on in vitro studies, further
investigations in vivo are needed to test our conclusions.
ACKNOWLEDGMENTS

This research was partially supported by the Ministry of
Education, Science, Sports, and Culture Grant-in-Aid for
Scientific Research.
REFERENCES
1. Kahn SE: Clinical review 135: the importance of beta-cell failure in thedevelopment and progression of type 2 diabetes. J Clin Endocrinol Metab
86:4047– 4058, 2001
2. Weir GC, Bonner-Weir S: Five stages of evolving ␤-cell dysfunction during
progression to diabetes. Diabetes 53 (Suppl. 3):S16 –S21, 2004
3. Stumvoll M, Goldstein BJ, van Haeften TW: Type 2 diabetes: principles ofpathogenesis and therapy. Lancet 365:1333–1346, 2005
4. Unger RH: Lipotoxicity in the pathogenesis of obesity-dependent NIDDM:
genetic and clinical implications. Diabetes 44:863– 870, 1995
5. Boden G, Shulman GI: Free fatty acids in obesity and type 2 diabetes:
defining their role in the development of insulin resistance and beta-cell
dysfunction. Eur J Clin Invest 32 (Suppl. 3):14 –23, 2002
6. Rorsman P, Renstrom E: Insulin granule dynamics in pancreatic beta cells.
Diabetologia 46:1029 –1045, 2003
7. Prentki M, Tornheim K, Corkey BE: Signal transduction mechanisms innutrient-induced insulin secretion. Diabetologia 40 (Suppl. 2):S32–S41,
1997
8. Girard J: [Contribution of free fatty acids to impairment of insulin
secretion and action. mechanism of beta-cell lipotoxicity]. Med Sci (Paris)
21:19 –25, 2005
9. Chan CB, Saleh MC, Koshkin V, Wheeler MB: Uncoupling protein 2 andislet function. Diabetes 53 (Suppl. 1):S136 –S142, 2004
10. Joseph JW, Koshkin V, Saleh MC, Sivitz WI, Zhang CY, Lowell BB, Chan
CB, Wheeler MB: Free fatty acid-induced beta-cell defects are dependent
on uncoupling protein 2 expression. J Biol Chem 279:51049 –51056, 2004
11. Yamashita T, Eto K, Okazaki Y, Yamashita S, Yamauchi T, Sekine N, Nagai
DIABETES, VOL. 57, SEPTEMBER 2008

R, Noda M, Kadowaki T: Role of uncoupling protein-2 up-regulation andtriglyceride accumulation in impaired glucose-stimulated insulin secretionin a beta-cell lipotoxicity model overexpressing sterol regulatory elementbinding protein-1c. Endocrinology 145:3566 –3577, 2004
12. Medvedev AV, Robidoux J, Bai X, Cao W, Floering LM, Daniel KW, Collins
S: Regulation of the uncoupling protein-2 gene in INS-1 beta-cells by oleic
acid. J Biol Chem 277:42639 – 42644, 2002
13. Kulkarni RN, Bruning JC, Winnay JN, Postic C, Magnuson MA, Kahn CR:
Tissue-specific knockout of the insulin receptor in pancreatic beta cells
creates an insulin secretory defect similar to that in type 2 diabetes. Cell
96:329 –339, 1999
14. Lin X, Taguchi A, Park S, Kushner JA, Li F, Li Y, White MF: Dysregulationof insulin receptor substrate 2 in beta cells and brain causes obesity anddiabetes. J Clin Invest 114:908 –916, 2004
15. Kubota N, Terauchi Y, Tobe K, Yano W, Suzuki R, Ueki K, Takamoto I,
Satoh H, Maki T, Kubota T, Moroi M, Okada-Iwabu M, Ezaki O, Nagai R,
Ueta Y, Kadowaki T, Noda T: Insulin receptor substrate 2 plays a crucialrole in beta cells and the hypothalamus. J Clin Invest 114:917–927, 2004
16. Shimano H, Horton JD, Hammer RE, Shimomura I, Brown MS, Goldstein
JL: Overproduction of cholesterol and fatty acids causes massive liverenlargement in transgenic mice expressing truncated SREBP-1a. J Clin
Invest 98:1575–1584, 1996
17. Shimano H, Horton JD, Shimomura I, Hammer RE, Brown MS, Goldstein
JL: Isoform 1c of sterol regulatory element binding protein is less active
than isoform 1a in livers of transgenic mice and in cultured cells. J Clin
Invest 99:846 – 854, 1997
18. Shimano H, Yahagi N, Amemiya-Kudo M, Hasty AH, Osuga J, Tamura Y,
Shionoiri F, Iizuka Y, Ohashi K, Harada K, Gotoda T, Ishibashi S, Yamada
N: Sterol regulatory element-binding protein-1 as a key transcription factor
for nutritional induction of lipogenic enzyme genes. J Biol Chem 274:
35832–35839, 1999
19. Hasty AH, Shimano H, Yahagi N, Amemiya-Kudo M, Perrey S, Yoshikawa T,
Osuga J, Okazaki H, Tamura Y, Iizuka Y, Shionoiri F, Ohashi K, Harada K,
Gotoda T, Nagai R, Ishibashi S, Yamada N: Sterol regulatory elementbinding protein-1 is regulated by glucose at the transcriptional level. J Biol
Chem 275:31069 –31077, 2000
20. Matsuzaka T, Shimano H, Yahagi N, Amemiya-Kudo M, Okazaki H, Tamura
Y, Iizuka Y, Ohashi K, Tomita S, Sekiya M, Hasty A, Nakagawa Y, Sone H,
Toyoshima H, Ishibashi S, Osuga J, Yamada N: Insulin-independent induction of sterol regulatory element-binding protein-1c expression in the liversof streptozotocin-treated mice. Diabetes 53:560 –569, 2004
21. Horton JD, Bashmakov Y, Shimomura I, Shimano H: Regulation of sterol
regulatory element binding proteins in livers of fasted and refed mice. Proc
Natl Acad Sci U S A 95:5987–5992, 1998
22. Yahagi N, Shimano H, Hasty AH, Amemiya-Kudo M, Okazaki H, Tamura Y,
Iizuka Y, Shionoiri F, Ohashi K, Osuga J, Harada K, Gotoda T, Nagai R,
Ishibashi S, Yamada N: A crucial role of sterol regulatory element-binding
protein-1 in the regulation of lipogenic gene expression by polyunsaturated
fatty acids. J Biol Chem 274:35840 –35844, 1999
23. Sekiya M, Yahagi N, Matsuzaka T, Najima Y, Nakakuki M, Nagai R,
Ishibashi S, Osuga J, Yamada N, Shimano H: Polyunsaturated fatty acids
ameliorate hepatic steatosis in obese mice by SREBP-1 suppression.
Hepatology 38:1529 –1539, 2003
24. Lin J, Yang R, Tarr PT, Wu PH, Handschin C, Li S, Yang W, Pei L, Uldry M,
Tontonoz P, Newgard CB, Spiegelman BM: Hyperlipidemic effects of
dietary saturated fats mediated through PGC-1beta coactivation of SREBP.
Cell 120:261–273, 2005
25. Ide T, Shimano H, Yahagi N, Matsuzaka T, Nakakuki M, Yamamoto T,
Nakagawa Y, Takahashi A, Suzuki H, Sone H, Toyoshima H, Fukamizu A,
Yamada N: SREBPs suppress IRS-2-mediated insulin signalling in the liver.
Nat Cell Biol 6:351–357, 2004
26. Takahashi A, Motomura K, Kato T, Yoshikawa T, Nakagawa Y, Yahagi N,
Sone H, Suzuki H, Toyoshima H, Yamada N, Shimano H: Transgenic miceoverexpressing nuclear SREBP-1c in pancreatic ␤-cells. Diabetes 54:492–
499, 2005
27. Diraison F, Parton L, Ferre P, Foufelle F, Briscoe CP, Leclerc I, Rutter GA:
Over-expressionofsterol-regulatory-element-binding
protein-1c(SREBP1c) in rat pancreatic islets induces lipogenesis and decreases
glucose-stimulated insulin release: modulation by 5-aminoimidazole-4carboxamide ribonucleoside (AICAR). Biochem J 378:769 –778, 2004
28. Kakuma T, Lee Y, Higa M, Wang Z, Pan W, Shimomura I, Unger RH: Leptin,
troglitazone, and the expression of sterol regulatory element binding
proteins in liver and pancreatic islets. Proc Natl Acad Sci U S A 97:8536 –
8541, 2000
29. Martinez SC, Cras-Meneur C, Bernal-Mizrachi E, Permutt MA: Glucose
regulates Foxo1 through insulin receptor signaling in the pancreatic islet
␤-cell. Diabetes 55:1581–1591, 2006
2391

EPA PREVENTS ␤-CELL LIPOTOXICITY THROUGH SREBP-1c

30. Tuttle RL, Gill NS, Pugh W, Lee JP, Koeberlein B, Furth EE, Polonsky KS,
Naji A, Birnbaum MJ: Regulation of pancreatic beta-cell growth andsurvival by the serine/threonine protein kinase Akt1/PKBalpha. Nat Med
7:1133–1137, 2001
31. Bernal-Mizrachi E, Fatrai S, Johnson JD, Ohsugi M, Otani K, Han Z,
Polonsky KS, Permutt MA: Defective insulin secretion and increased
susceptibility to experimental diabetes are induced by reduced Akt activity
in pancreatic islet beta cells. J Clin Invest 114:928 –936, 2004
32. Fatrai S, Elghazi L, Balcazar N, Cras-Meneur C, Krits I, Kiyokawa H,
Bernal-Mizrachi E: Akt induces ␤-cell proliferation by regulating cyclin D1,
cyclin D2, and p21 levels and cyclin-dependent kinase-4 activity. Diabetes
55:318 –325, 2006
33. Kitamura T, Nakae J, Kitamura Y, Kido Y, Biggs WH III, Wright CV, White
MF, Arden KC, Accili D: The forkhead transcription factor Foxo1 links
insulin signaling to Pdx1 regulation of pancreatic beta cell growth. J Clin
Invest 110:1839 –1847, 2002
34. Okamoto H, Hribal ML, Lin HV, Bennett WR, Ward A, Accili D: Role of theforkhead protein FoxO1 in beta cell compensation to insulin resistance.
J Clin Invest 116:775–782, 2006
35. Coppola T, Frantz C, Perret-Menoud V, Gattesco S, Hirling H, Regazzi R:
Pancreatic beta-cell protein granuphilin binds Rab3 and Munc-18 andcontrols exocytosis. Mol Biol Cell 13:1906 –1915, 2002
36. Torii S, Zhao S, Yi Z, Takeuchi T, Izumi T: Granuphilin modulates the
exocytosis of secretory granules through interaction with syntaxin 1a. Mol
Cell Biol 22:5518 –5526, 2002
37. Gomi H, Mizutani S, Kasai K, Itohara S, Izumi T: Granuphilin molecularly
docks insulin granules to the fusion machinery. J Cell Biol 171:99 –109,
2005
38. Kato T, Shimano H, Yamamoto T, Yokoo T, Endo Y, Ishikawa M, Matsuzaka T, Nakagawa Y, Kumadaki S, Yahagi N, Takahashi A, Sone H, Suzuki
H, Toyoshima H, Hasty AH, Takahashi S, Gomi H, Izumi T, Yamada N:

2392

Granuphilin is activated by SREBP-1c and involved in impaired insulin
secretion in diabetic mice. Cell Metab 4:143–154, 2006
39. Shimano H, Shimomura I, Hammer RE, Herz J, Goldstein JL, Brown MS,
Horton JD: Elevated levels of SREBP-2 and cholesterol synthesis in liversof mice homozygous for a targeted disruption of the SREBP-1 gene. J Clin
Invest 100:2115–2124, 1997
40. Scharp DW, Kemp CB, Knight MJ, Ballinger WF, Lacy PE: The use of ficoll
in the preparation of viable islets of Langerhans from the rat pancreas.
Transplantation 16:686 – 689, 1973
41. Schultz V, Sussman I, Bokvist K, Tornheim K: Bioluminometric assay of
ADP and ATP at high ATP/ADP ratios: assay of ADP after enzymatic
removal of ATP. Anal Biochem 215:302–304, 1993
42. Noushmehr H, D’Amico E, Farilla L, Hui H, Wawrowsky KA, Mlynarski W,
Doria A, Abumrad NA, Perfetti R: Fatty acid translocase (FAT/CD36) islocalized on insulin-containing granules in human pancreatic ␤-cells andmediates fatty acid effects on insulin secretion. Diabetes 54:472– 481, 2005
43. Ono H, Shimano H, Katagiri H, Yahagi N, Sakoda H, Onishi Y, Anai M,
Ogihara T, Fujishiro M, Viana AY, Fukushima Y, Abe M, Shojima N, Kikuchi
M, Yamada N, Oka Y, Asano T: Hepatic Akt activation induces marked
hypoglycemia, hepatomegaly, and hypertriglyceridemia with sterol regulatory element binding protein involvement. Diabetes 52:2905–2913, 2003
44. Nakagawa Y, Shimano H, Yoshikawa T, Ide T, Tamura M, Furusawa M,
Yamamoto T, Inoue N, Matsuzaka T, Takahashi A, Hasty AH, Suzuki H,
Sone H, Toyoshima H, Yahagi N, Yamada N: TFE3 transcriptionallyactivates hepatic IRS-2, participates in insulin signaling and ameliorates
diabetes. Nat Med 12:107–113, 2006
45. Iwatsuka H, Shino A, Suzuoki Z: General survey of diabetic features ofyellow KK mice. Endocrinol Jpn 17:23–35, 1970
46. Browning LM: n-3 Polyunsaturated fatty acids, inflammation and obesityrelated disease. Proc Nutr Soc 62:447– 453, 2003

DIABETES, VOL. 57, SEPTEMBER 2008