A R T I C L E

Granuphilin is activated by SREBP-1c and involved in impaired
insulin secretion in diabetic mice
Toyonori Kato,1 Hitoshi Shimano,1,2,* Takashi Yamamoto,1 Tomotaka Yokoo,1 Yuko Endo,1 Mayumi Ishikawa,1Takashi Matsuzaka,1,2 Yoshimi Nakagawa,1,2 Shin Kumadaki,1 Naoya Yahagi,2 Akimitsu Takahashi,1Hirohito Sone,1,2 Hiroaki Suzuki,1 Hideo Toyoshima,1 Alyssa H. Hasty,5 Satoru Takahashi,3 Hiroshi Gomi,4Tetsuro Izumi,4 and Nobuhiro Yamada1
1

Department of Internal Medicine, Endocrinology and Metabolism, Graduate School of Comprehensive Human Sciences
Center for Tsukuba Advanced Research Alliance3
Institute of Basic Medical Sciences and Laboratory Animal Resource Center
University of Tsukuba, 1-1-1 Tennodai, Tsukuba Ibaraki 305-8575, Japan
4
Laboratory of Molecular Endocrinology and Metabolism, Department of Molecular Medicine, Institute for Molecular and Cellular Regulation,
Gunma University, 3-39-15 Showa-cho, Maebashi, Gunma 371-8512, Japan
5
Department of Molecular Physiology and Biophysics, Vanderbilt University Medical Center, Twenty-first Avenue South and Garland Avenue,
Nashville, Tennessee 37232*Correspondence: shimano-tky@umin.ac.jp
2

Summary
Granuphilin is a crucial component of the docking machinery of insulin-containing vesicles to the plasma membrane. Here,
we show that the granuphilin promoter is a target of SREBP-1c, a transcription factor that controls fatty acid synthesis, and
MafA, a b cell differentiation factor. Potassium-stimulated insulin secretion (KSIS) was suppressed in islets with adenoviralmediated overexpression of granuphilin and enhanced in islets with knockdown of granuphilin (in which granuphilin had
been knocked down). SREBP-1c and granuphilin were activated in islets from b cell-specific SREBP-1c transgenic mice,
as well as in several diabetic mouse models and normal islets treated with palmitate, accompanied by a corresponding
reduction in insulin secretion. Knockdown- or knockout-mediated ablation of granuphilin or SREBP-1c restored KSIS in
these islets. Collectively, our data provide evidence that activation of the SREBP-1c/granuphilin pathway is a potential
mechanism for impaired insulin secretion in diabetes, contributing to b cell lipotoxicity.

Insulin secretion by pancreatic b cells involves sequential intracellular events. Initially, glucose oxidation results in ATP production as controlled by intracellular energy-sensing mechanisms(Bratanova-Tochkova et al., 2002). This is followed by closureof ATP-dependent K+ channels leading to a change in membrane voltage (depolarization), Ca2+ influx through voltage-dependent Ca2+ channels, and vesicular transport of insulin-containing granules, culminating in their exocytosis and release of
insulin (Rizzoli and Betz, 2005; Rorsman and Renstrom, 2003).
Theoretically, impaired insulin secretion could be caused by disturbances in any stage of this highly regulated process (Weir andBonner-Weir, 2004). Current literature supports the concept that
disturbances of energy metabolism in b cells leading to impaired
ATP production are the primary pathological cause of impaired
insulin secretion following peripheral insulin resistance in type 2
diabetes mellitus (Kahn, 2003). This pathophysiologic process
can be observed in isolated islets from diabetic animal models
and is characterized by impairment of glucose-stimulated insulin secretion (GSIS) (Diani et al., 2004). Meanwhile, potential perturbations in insulin secretion at steps following ATP production
in islets from the diabetic models as estimated by K+-stimulated
insulin secretion (KSIS) have not been fully investigated.
Studies to unveil the molecular basis for exocytosis of secretory granules have been focused on the interactions between
components of the fusion machinery (Burgoyne and Morgan,

2003; Rizzoli and Betz, 2005; Rorsman and Renstrom, 2003),
including soluble N-ethylmaleimide-sensitive factor attachment
protein receptors (SNAREs: Vamp2/synaptobrevin, Syntaxin1a, and SNAP25) and SNARE-associated proteins (Munc13-1
and Munc18-1) on the plasma membrane, and small Rab
GTPase family members, Rab3 and Rab27a, on the vesicles.
Granuphilin is a Rab27a effecter specific to pancreatic b cells
and the pituitary gland. It has two isoforms, granuphilin-a and-b, which derive from alterative splicing (Coppola et al., 2002;
Wang et al., 1999). Granuphilin directly binds to Rab27a, andalso to syntaxin-1a and Munc18-1, assisting in the docking of insulin granules to the plasma membrane (Coppola et al., 2002;
Torii et al., 2002, 2004; Yi et al., 2002). Contrary to expectations,overexpression of granuphilin in a b cell line inhibited insulin
secretion (Coppola et al., 2002; Torii et al., 2002). In addition,
granuphilin-null islets exhibited inhibition of vesicle docking,yet insulin secretion was increased (Gomi et al., 2005). Thesegain- and loss-of-function experiments implicated granuphilin
as playing a regulatory role in the exocytosis of insulin granules.
To date, the regulation of these vesicle proteins and the potential
role of granuphilin in b cell lipotoxicity and diabetes are largely
unknown.
SREBP-1c is a transcription factor established as a regulator
for biosynthesis of fatty acids and triglycerides (Shimano et al.,1997a, 1997b). In addition to nutritional regulation of lipogenic
enzymes (Horton et al., 1998; Hasty et al., 2000; Matsuzaka
et al., 2004), SREBP-1c is found to play a role in insulin signaling

CELL METABOLISM 4, 143–154, AUGUST 2006 ª2006 ELSEVIER INC.

DOI 10.1016/j.cmet.2006.06.009

Introduction

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by inhibiting IRS-2, the major insulin-signaling mediator in theliver and in b cells (Ide et al., 2004; Takahashi et al., 2005). As
a model for lipotoxicity by endogenous fatty acids in pancreaticb cells, we previously developed transgenic mice overexpressing the active form of SREBP-1c under the insulin promoter.
These mice exhibited impaired insulin secretion in vivo due to
ATP depletion caused by enhanced lipogenesis and direct
UCP2 induction. In addition, these mice demonstrated loss ofb cell mass, presumably due to suppression of IRS-2 and PDX1 in islets from the transgenic mice (Takahashi et al., 2005). In
addition to inhibition of GSIS, SREBP-1c-overexpressing islets
exhibited decreased KSIS, which could indicate dysfunction in
insulin secretion at a phase following ATP production. Our current study demonstrates that granuphilin is an SREBP-1c target
exhibiting a molecular link between b cell lipotoxicity and impaired insulin secretion in the pathogenesis of diabetes development. These data provide a rational to study granuphilin as apotential therapeutic target for treatment of diabetes.
Results
Granuphilin is activated by SREBP-1c in islets
We have previously demonstrated that insulin secretion isblunted in islets isolated from nuclear SREBP-1c transgenic
mice under the control of rat insulin promoter I, whereas
SREBP-1-null islets demonstrated enhanced insulin secretioncompared to wild-type controls (Takahashi et al., 2005). Expression of the transgene was barely detectable in the hypothalamus
and was essentially b cell-specific in this transgenic line (Figure S1A in the Supplemental Data available with this article
online). In the current study, we found that the negative effectof SREBP-1c on insulin secretion encompassed not only GSIS
but also KSIS and induction by arginine (Figure 1A), suggestingthat insulin secretion defects caused by SREBP-1c occurred
post-ATP production. In an attempt to determine the molecularmechanisms responsible, we explored the expression of genes
involved in vesicular transport of insulin granules in islets fromthese mice. Among many different vesicle factors involved inexocytosis of insulin granules, granuphilin was singly upregulated by overexpression of SREBP-1c and suppressed in theabsence of SREBP-1c, as evidenced by real-time PCR (Figure 1B). Immunoblot analysis of islet proteins demonstrated
that wild-type islets mainly express granuphilin-a (Figure 1C),
and the ratio of granuphilin-a/-b mRNA was roughly 8 (data notshown). Consistent with changes at the mRNA level, both granuphilin-a and -b proteins were robustly induced by overexpression of SREBP-1c and reduced in SREBP-1c-null islets (Figure 1C). SREBP-1c slightly reduced Syntaxin-1a and Munc18-1
proteins but did not affect other related vesicle proteins suchas Rab27a, Rab3, Vamp2, and SNAP25 (Figure 1C). Inductionof granuphilin mRNA and proteins by acute overexpression of
adenoviral SREBP-1c was also observed in a murine insulinoma
cell line Min6 (Figure 1D) and islets (Figure 1E), respectively.
Granuphilin promoter is an SREBP target
We next sought to determine whether the granuphilin promoter
is a potential direct target of SREBP. Analysis of the GeneBank
database revealed that the promoter region of the murine granuphilin gene contains two potential binding sites for SREBP(SREs) and a single site for MafA, which was recently established as an important factor for b cell-differentiation and func-

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tion (MARE) (Hagman et al., 2005; Kajihara et al., 2003) (Figure S2). Activation of the granuphilin promoter by transcription
factors such as SREBPs, MafA, and others was tested in Min6
cells using a luciferase reporter gene fused to the 1.2 kb promoter region of mouse granuphilin (Granu-1200-Luc) (Figure 1F).
SREBP-1a and -1c robustly and SREBP-2 moderately stimulated granuphilin promoter activity. MafA was found to be thestrongest activator of the granuphilin promoter. A similar patternof transactivation by these factors was also observed in nonb cells (HEK 293 cells), suggesting that SREBP activation does
not require other b cell-specific factors (data not shown). Human
granuphilin promoter (0.5 kb) luciferase reporter was also activated by SREBPs and MafA (data not shown).
This region of the mouse granuphilin promoter (Figure 2A) was
further analyzed as an SREBP target. Activation of the granuphilin promoter was completely attenuated when SREBPs were mutated (YR mutants) to abolish their binding abilities to authentic
SREs (Figure 2B). Deletion studies suggested that the proximal
SRE is responsible for SREBP activation of granuphilin promoter(Figure 2C). EMSA assays demonstrated direct and specificbinding of SREBPs to this SRE-1 (hereafter referred to as Granuphilin SRE) and not SRE-2 (Figure 2E). Mutation of Granuphilin
SRE (Figure 2A) completely abolished the SREBP activation ofGranuphilin-Luc (Figure 2D). To determine the physiologicalrelevance of SREBP regulation of granuphilin, MIN6 cells weretreated with delipidated serum (DLS) and 25-hydroxycholesterol(25-OH) to activate and suppress endogenous SREBPs, respectively, via modulation of the sterol-regulated SERBP cleavage
system. The cells were also treated with T0901317, an LXR agonist, to induce endogenous SREBP-1c. Granu-1200-Luc activity
was consistently up- and downregulated by these manipulations, respectively, demonstrating that endogenous SREBPs
can also regulate granuphilin promoter activity (Figures 2F and
2G). Chromatin immunoprecipitation assays using MIN6 cells
confirmed direct binding of SREBP-1c to Granuphilin SRE
in vivo. The signal was increased by incubation with DLS and
T0901317 (Figure 2H). Collectively, these data demonstratethat the granuphilin promoter is an SREBP target and that
SREBPs regulate granuphilin expression.
Granuphilin promoter is a MafA target
Next, we focused the MARE downstream of the Granuphilin SRE
in the granuphilin promoter as diagramed in Figure 3A. Activation of the granuphilin promoter by MafA was completely abolished by deletion of the acidic domain of MafA (Figure 3B). Deletion or mutation of the MARE dramatically suppressed thepromoter activity (Figures 3C and 3D). Direct, specific, and invivo binding of MafA to this MARE was confirmed by EMSA
and ChIP assay (Figures 3E and 3F). Considering the two neighboring sites, potential synergism between SREBP-1c and MafA
was estimated. Both factors exhibited dose-dependent activation of granuphilin promoter, although MafA has a curve slightly
steeper and more saturable than SREBP-1c. Cotransfection ofboth SREBP-1c and MafA caused only additive activation of
the granuphilin promoter (Figures 3G and 3H). In immunoprecipitation experiments, no significant protein-protein interactions
between SREBP-1c and MafA were detected (data not shown).
Granuphilin negatively regulates insulin secretion
To determine whether granuphilin could be directly involved
in insulin secretion, its expression was manipulated in islets

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SREBP-1c/granuphilin pathway in insulin secretion

Figure 1. Influence of SREBP-1c on granuphilin
expression
Granuphilin and other fusion machinery proteins andinsulin secretion were estimated in islets from wildtype (WT-Control), b cell-specific nuclear SREBP-1ctransgenic (TgRIP-SREBP-1c), and SREBP-1-nullmice (SREBP-1-KO) mice.
A) Basal (low glucose LG; 2.8 mM), high glucose (HG;
20 mM), KCl- (30 mM), arginine- (3 mM), forskolin(10 mM), or TPA (0.5 mM)-stimulated insulin secretionin isolated islets from age-matched (14- to 17-weekold) male WT-Control (white bars), TgRIP-SREBP-1c(black bars), and SREBP-1-KO (gray bars) mice.
Results were normalized to cellular DNA content.
*p < 0.01 and **p < 0.05 (versus WT-Control).
B) mRNA levels of the fusion machinery for exocytosis of insulin granules in the indicated islets as quantified by real-time PCR. Values were normalized tothe cyclophilin expression level. *p < 0.01 (versus
WT-Control).
C) Immunoblot analysis on total proteins (50 mg/lane)of the fusion machinery for exocytosis of insulin granules in the islets from indicated mice.
D) Northern blot analysis of granuphilin andhSREBP-1 gene expression in mouse insulinoma
cell line, Min6. Cells were infected by adenoviralGFP or -SREBP-1c (500 MOI) for 48 hr. Total RNA(15 mg/lane) was isolated and subjected to blot hybridization with the indicated 32P-labeled probe.
E) Immunoblot analysis on nuclear or total proteins of
SREBP-1 and mouse granuphilin in the islets from
C57BL/6 mice with a-tubulin as a loading control. Islets were infected with adenoviral-GFP or -SREBP1c (500 MOI) for 48 hr.
F) Activation of the granuphilin promoter by SREBPs
and MafA. The mouse granuphilin promoter region(1.2 kb) was fused to a luciferase reporter gene
(Granu-1200-Luc). Min6 cells were cotransfectedwith Granu-1200-Luc as a reporter gene, pSV-40Renilla as a reference, and the indicated expression
plasmids or an empty vector. The luciferase activity
was normalized to Renilla luciferase activity and assays were performed in triplicate. *p < 0.01 (versus
pcDNA3.1 group).
Results are expressed by mean 6 SEM. Studies
were performed in triplicate with sets of islets pooled
from 3–4 mice per replicate (A–C and E).

isolated from C57BL/6 mice using adenoviral overexpression
and RNA interference (Figures S3A and S3B). Adenoviral overexpression of granuphilin-a suppressed GSIS substantiallyand KSIS completely but not basal secretion at low glucose concentration, consistent with previous report in a b cell line (Coppola et al., 2002; Torii et al., 2002) (Figure S3C). Adenoviral
RNAi (RNAi-784) completely inhibited expression of both granuphilin-a and-b. This knockdown of granuphilin resulted in a trend
toward increased GSIS, and significantly increased KSIS, whichis also consistent with a recent report describing granuphilin-nullmice (Gomi et al., 2005) (Figure S3C). Granuphilin-a overexpression and knockdown did not significantly change either insulincontent or ATP/ADP ratio in infected islets, demonstrating thatthe modulation of insulin secretion by granuphilin occurred in
the process post-ATP production (Figures S3D and S3E).

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Granuphilin mediates SREBP-1 inhibition of KSIS
We also evaluated the effect of granuphilin knockdown by RNA
interference in SREBP-1c-overexpressing islets (Figures 4 andS4). Granuphilin mRNA and protein levels were upregulated by
SREBP-1c in these islets in the basal state; however, expression
was completely abolished by granuphilin RNAi (Figures 4A and
4B). This suppression of granuphilin did not affect the protein
level of endogenous membrane SREBP-1c, transgenic nuclear
SREBP-1c, or MafA (Figure 4B). Other vesicle and related molecules were also unchanged at the protein and mRNA levels(Figures 4C and S4A). However, the granuphilin knockdown
substantially ameliorated the decreased GSIS caused by
SREBP-1c overexpression and completely restored the suppressed KSIS without changes in decreased ATP/ADP ratio
and increased triglyceride content (Figures 4D–4F). These data

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Figure 2. Mouse granuphilin promoter analysis as
an SREBP target
A) Schematic representation of the mouse granuphilin promoter with the sequence and location of potential SREBP binding sites (SRE-1 and SRE-2).
The SRE-2 element has an inverted consensus sequence and partially overlaps with an E-box.
B) Transcriptional activities of nuclear SREBP-1a,-1c, and -2 and their respective Tyr-Arg mutants on
Granu-1200-Luc in Min6 cells.
C) Effects of deletion of SREBP-1c activation in the
granuphilin promoter. Sequentially deleted luciferase
constructs of the granuphilin promoter, as indicated,were used for transfection studies in Min6 cells.
D) Effects of a mutation in SRE-1 on SREBP activation of granuphilin promoter. Native (Granu-500Luc) or mutant-SRE-1 (mSRE-Luc) reporter gene
plasmids were cotransfected into Min6 cells with
pSV40-Renilla and expression plasmids for SREBPs.
E) EMSA with radio-labeled probes for the authentic
human LDL receptor SRE and granuphilin SRE-1 andSRE-2. In vitro translated SREBP-1a, -1c, and -2
proteins were incubated with these labeled probes.
Specific binding was confirmed by super-shift using
their respective antibody.
F) Effect of endogenous SREBPs in Min6 cells on
Granu-1200-Luc. Cells were incubated with DMEM
in 7.5% FCS, in 7.5% DLS, an LXR agonist:
T0901317 (1 mM), and 25-hydroxycholesterol (25OH, 0.1 mg/ml).
G) Immunoblot analysis of endogenous SREBP-1
and granuphilin proteins in Min6 cells. Cells were incubated with DMEM in 7.5% FCS, in 7.5% DLS, andT0901317 (1 mM) for 48 hr.
H) In vivo binding of SREBP-1 to Granuphilin-SRE.
Chromatin prepared from the Min6 cells were subjected to the ChIP assay with anti-mouse SREBP-1
antibody and rabbit IgG as a negative control. Immunoprecipitated samples were subjected to PCR using primers to amplify the SRE-element in the granuphilin promoter and for the nonspecific-element (NS).
Mw, molecular weight.
The luciferase activity was normalized to Renilla luciferase activity, and assays were performed in triplicate (B–D and F). Results are expressed as mean 6
SEM.

indicate that SREBP-1c-mediated inhibition of insulin secretioncan be accounted for, in part, by upregulation of granuphilin.
Activation of SREBP-1c/granuphilin inhibits KSIS in islets
from diabetic mice
The inhibitory effect of the SREBP-1c/granuphilin pathway on insulin secretion was tested in islets from murine models of diabetes (Figure 5A). Along with consistent upregulation of SREBP-1c,
both granuphilin mRNA and protein levels were increased in
islets from genetically obese diabetic mice such as ob/ob,
KK-Ay, and db/db, as compared to control C57BL/6 mice. In addition, mice made obese with high-fat or high-fat/high-sucrose
diets also displayed increased SREBP-1c and granuphilin expression. We next sought to determine the effect of granuphilin

146

knockdown on gene expression in islets from KK-Ay mice (Figures 5B–5E). Comparison of LacZi-Ad-infected islets from
C57BL/6 and KK-Ay mice also allowed us to determine basal difference in gene expression under control and diabetic conditions. mRNA levels of SREBP-1c and granuphilin were elevatedin the KK-Ay mice compared to C57BL/6 controls. Granuphilinknockdown resulted in essential abolishment of both granuphilinmRNA and protein levels without impacting SREBP-1c levels(Figure 5B). Islets from KK-Ay mice had higher basal nuclear
SREBP-1 protein with concomitant increases in SREBP-1c target genes FAS and UCP-2 with significant increases in triglyceride content (Figures 5E and S4B). Protein levels of Syntaxin-1aand Munc18-1 were slightly decreased; however, levels of othervesicle proteins remained unchanged by either the increase in

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SREBP-1c/granuphilin pathway in insulin secretion

Figure 3. Mouse granuphilin promoter analysis asa MafA target
A) Schematic representation of the granuphilin promoter with sequence and location of a potential
MafA binding site (MARE).
B) Transcriptional activities of wild-type MafA andmutant MafA (deletion of transactivation domain,amino acid 229–359) on Granu-1200-Luc in Min6
cells.
C) Deletion studies in MafA activation of granuphilinpromoter.
D) Effect of mutation in the MARE on MafA activationof granuphilin. Native (Granu-500-Luc) or mutantMARE (mMARE-Luc) reporter gene plasmids were
cotransfected into Min6 cells with pSV40-Renilla
and MafA.
E) EMSA with radio-labeled probes for rat insulin II
MARE and granuphilin MARE-elements. In vitrotranslated MafA protein was incubated with these labeled probes. Specific binding was confirmed by anexcess of nonlabeled probe.
F) In vivo binding to Granuphilin-MARE. Chromatin
prepared from the Min6 cells were subjected to the
ChIP assay with anti-rabbit MafA antibody and rabbit
IgG as a negative control. Immunoprecipitated samples were subjected to PCR using primers to amplify
the granuphilin promoter containing the MAREelement and of a nonspecific-element (NS). Mw,
molecular weight.
G) Dose-dependent effects of MafA with cotransfection of SREBP-1c on Granu-Luc-1200 in Min6 cells.
H) Dose-dependent effects of SREBP-1c with cotransfection of MafA on Granu-Luc-1200 in Min6
cells.
The luciferase activity was normalized to Renilla luciferase activity and performed in triplicate (B–D, G,
and H). Results are expressed as mean 6 SEM.

SREBP-1c in the diabetic KK-Ay mice or by knockdown of granuphilin (data not shown). Thus, knockdown of granuphilin does
not appear to alter mRNA or protein levels of any other proteins
involved in insulin-containing granule transport in islets. Both
GSIS and KSIS were reduced in KK-Ay islets with a reductionin the ATP/ADP ratio and an increase in the content of triglycerides(Figures 5C–5E). Knockdown of granuphilin completely restored
KSIS but not GSIS without modulating ATP/ADP ratio or triglyceride content. In addition, restoration of decreased KSIS by granuphilin knockdown was also observed in islets from ob/ob anddiet-induced obese (DIO) mice (Figures 5F, 5G, S4C, and S4D).
Knockdown of SREBP-1c by adenoviral RNAi was performed
in islets from KK-Ay and ob/ob mice that exhibit upregulation of

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SREBP-1c and granuphilin (Figure S5). Although acute inhibitionof SREBP-1c was not complete in these islets, reduction of
granuphilin and restoration of decreased GSIS and KSIS werepartially but consistently observed to support the concept of
the contribution of the SREBP-1c/granuphilin pathway to impaired insulin secretion. Combined, these data implicate thespecific involvement of granuphilin in impaired insulin secretionin obesity-related diabetic mice.
Palmitate lipotoxicity involves inhibition of KSIS
through granuphilin
Saturated fatty acids are known to impair insulin secretion, supporting the theory that lipotoxicity plays a role in impaired b–cell147

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Figure 4. Effects of knockdown of granuphilin on
insulin secretion in pancreatic islets from b cellspecific nuclear SREBP-1c transgenic mice
Islets were isolated from male age-matched (13- to
18-week-old) Wild-type-Control or TgRIP-SREBP1c mice and were infected adenoviral-LacZ-RNAior -granuphilin (Granu)-RNAi (500 MOI) for 48 hr.
A) mRNA levels of the human SREBP-1c transgeneand endogenous granuphilin from the indicated islets
as estimated by real-time PCR. hSREBP-1c levels
were calculated as copy numbers.
B) Immunoblot analysis of SREBP-1, MafA, andgranuphilin from the indicated islets.
C) Immunoblot analysis of the fusion machinery for
exocytosis of insulin granules from the indicated islets with a-tubulin as a loading control.
D) Cellular ATP/ADP ratio from the indicated islets.
E) Cellular TG content from the indicated islets.
F) LG-, HG-, and KCl-stimulated insulin secretion in
the indicated islets: WT-LacZ-RNAi (white bars),
WT-Granuphilin-RNAi (black bars), TgRIP-SREBP1c-LacZ-RNAi (light gray bars), and TgRIP-SREBP1c-Granuphilin-RNAi (dark gray bars).
Results were normalized to cellular DNA content (F)and are expressed as mean 6 SEM. *p< 0.01 and**p < 0.05 (versus WT-LacZi). Studies were performed in triplicate with sets of islets pooled from
3–4 mice per replicate.

function (Dubois et al., 2004; Joseph et al., 2004). To test
whether granuphilin expression might contribute to the pathophysiologic consequences of lipotoxicity, we incubated isolated
islets with palmitate (PA) for 48 hr. PA induced both SREBP-1c
and granuphilin in normal islets at both mRNA and protein levels(Figures 6A, 6B, and S4E). These inductions were blunted in
the absence of SREBP-1c, indicating that SREBP-1c plays
a dominant role in PA-induced granuphilin expression. PA
reduced KSIS as well as GSIS in SREBP-1c-normal mice;
however, the PA-suppressed GSIS and KSIS were almost
completely restored in SREBP-1-null mice (Figure 6C). These
results suggested that PA-induced insulin secretion impairment
is mediated through SREBP-1c. Furthermore, we used adenoviral-granuphilin RNAi treatment to completely suppress granuphilin expression in control and PA-treated islets (Figures 6D

148

and S4F). The absence of granuphilin significantly ameliorated
the suppression of both GSIS and KSIS induced by PA(Figure 6E), indicating that granuphilin upregulation is involved
in PA-induced impairment of insulin secretion. PA induction of
SREBP-1c/granuphilin was investigated at the transcriptional
level in MIN6 cells. Both Granu-1200-Luc (Figure 6F) and SRELuc (data not shown) were activated by incubation with PA, indicating that induction of nuclear forms of endogenous SREBPs
and granuphilin was at the transcriptional level. Knockdown of
SREBP-1c led to a strong reduction in PA induction of the granuphilin promoter, but dominant-negative mutant MafA did not.
These data demonstrated that incubation of isolated islets
with PA, a model for lipotoxicity in b cells, caused impaired insulin secretion at least partially through induction of SREBP-1c
and granuphilin.

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SREBP-1c/granuphilin pathway in insulin secretion

Figure 5. Activation of SREBP-1c and granuphilin in
pancreatic islets from different diabetic model miceand effects of granuphilin knockdown on insulin
secretion
Islets were isolated from male C57BL/6, ob/ob, KK,
KK-Ay (10 weeks old), db/misty, and db/db mice
(6 weeks old). C57BL/6, KK-Ay, and ob/ob mice islets were infected adenoviral-LacZ-RNAi or -granuphilin(Granu)-RNAi (500 MOI) for 48 hr.
A) Upper panel: immunoblot analysis of granuphilinfrom the indicated mice with a-tubulin as a loading
control. Lower panel: mRNA levels of SREBP-1c(white bars) and granuphilin (black bars) in the islets
from the indicated mice as estimated by real-time
PCR. HF: high-fat diet, HF/HS: high-fat and high-sucrose diet. *p < 0.01 (versus C57BL/6).
B) Upper panel: immunoblot analysis of granuphilin
and SREBP-1 from the indicated islets. Lower panel:mRNA levels of SREBP-1c (white bars) and granuphilin (black bars), from the indicated islets.
C) LG-, HG-, and KCl-stimulated insulin secretion in
the indicated islets: C57BL/6-LacZ-RNAi (whitebars), KK-Ay-LacZ-RNAi (black bars), and KK-AyGranuphilin-RNAi (gray bars).
D) Cellular ATP/ADP ratio from the indicated islets.
E) Cellular TG content from the indicated islets.
F) Upper panel: immunoblot analysis of granuphilin
and SREBP-1 from the indicated islets. Lower panel:
LG-, HG-, and KCl-stimulated insulin secretion in theindicated islets: C57BL/6-LacZ-RNAi (white bars),
ob/ob-LacZ-RNAi (black bars), and ob/ob-Granuphilin-RNAi (gray bars).
G) Upper panel: immunoblot analysis of granuphilin
and SREBP-1 from indicated islets. Lower panel:
LG-, HG-, and KCl-stimulated insulin secretion in
the indicated islets: LacZ-RNAi (white bars), Granuphilin-RNAi (black bars).
Results were normalized to cellular DNA content (C,F, and G) and are expressed as mean 6 SEM. *p <0.01 and **p < 0.05 (versus C57BL/6-LacZ, B-E).
Studies were performed in triplicate with sets of islets
pooled from 3–4 mice per replicate.

SREBP-1c inhibition of KSIS is abolishedin granuphilin KO islets
Finally, the SREBP-1c effect on insulin secretion was investigated in islets from granuphilin-null mice (Figure 7). Adenoviralnuclear SREBP-1c caused changes in its target genes, ATP/ADP ratio, and triglyceride content in both wild-type and granuphilin KO islets in a similar manner to those in SREBP-1c transgenic islets (Figures 7A–7E). As compared to wild-type islets,
both GSIS and KSIS were increased in the absence of granuphilin. Suppression of KSIS by SREBP-1c overexpression observedin granuphilin-normal islets was cancelled in granuphilin KO
islets. Meanwhile, SREBP-1c-mediated inhibition of GSIS waspartially ameliorated, but remained significantly reduced compared to controls (Figure 7F). Taken together with the data

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from granuphilin knockdown in SREBP-1c transgenic islets,
these data demonstrate that granuphilin contributes to
SREBP-1c-mediated KSIS inhibition.
Discussion
Granuphilin is a target gene of SREBP and MafA
Our current study clearly shows that mouse granuphilin is an
SREBP target. Because SREBP-1c expression is highly regulated by nutrition (Horton et al., 1998; Hasty et al., 2000; Matsuzaka et al., 2004) and is upregulated in pathological states related to over-nutrition and insulin resistance (Biddinger et al.,2005), we propose that SREBP-1-induced granuphilin activation
may contribute to b cell lipotoxicity and diabetes that can

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Figure 6. Involvement of granuphilin in insulin secretion from isolated murine islets treated with palmitate
Islets were isolated from male age-matched (13- to
17-week-old) SREBP-1-null, SREBP-1(2/2), andwild-type littermate, SREBP-1 (+/+), mice. The islets
were incubated with 400 mM palmitate (PA) (A–E) andwere infected with adenoviral-LacZ-RNAi or -granuphilin-RNAi (500 MOI) for 48 hr (D and E).
A) mRNA levels of SREBP-1c (white bars) and granuphilin (black bars) from the indicated islets asestimated by real-time RT-PCR. * p < 0.01 (versus
SREBP-1(+/+)-Control). ND, not detectable.
B) Immunoblot analysis of SREBP-1, MafA, andmouse granuphilin from indicated islets with a-tubulin as a loading control.
C) LG-, HG-, and KCl-stimulated insulin secretion in
SREBP-1(+/+)-control group (white bars), -PA group(black bars), SREBP-1(2/2)-control group (light graybars), and -PA group (dark gray bars). *p < 0.01 and**p < 0.05 (versus SREBP-1(+/+)-Control).
D) Upper panel: immunoblot analysis of mouse granuphilin and SREBP-1. Lower panel: expression of
SREBP-1c (white bars) and granuphilin (black bars)
from the indicated islets as estimated by real-time
RT-PCR. *p < 0.01 (versus Control-LacZ-RNAi).
E) LG-, HG-, and KCl-stimulated insulin secretion in
islets untreated (Control) or treated with palmitate(PA), and infected with Adeno-LacZ-RNAi (LacZiAd) or Adeno-granuphilin RNAi (granuphilin RNAiAd). *p < 0.01 (versus Control-LacZi).
F) Effects of SREBP-1-RNAi and mutant MafA on
Granu-1200-Luc in PA treated Min6 cells. The luciferase activity was normalized to Renilla luciferase
activity and performed in triplicate.
Results were normalized to cellular DNA content (Cand E) and are expressed by mean 6 SEM. Studies
were performed in triplicate with sets of islets pooled
from three to four mice per replicate.

develop in these situations. We also show that the granuphilinpromoter is activated by MafA. MafA has recently been established to be crucial for normal structure and function of b cells
and insulin gene expression (Hagman et al., 2005; Kajihara
et al., 2003) and thus is likely to be responsible for b cell-specificbasal expression of granuphilin. Supportively, basal expressionof granuphilin was severely diminished in islets from MafA
knockout mice (S.T., unpublished data), while it was only 50%
reduced in homozygous SREBP-1-null islets.
Granuphilin activation inhibits insulin secretion
Granuphilin has been thought to be a component of the fusion
machinery for exocytosis of insulin granules. However, activation of granuphilin leads to inhibition of insulin secretion. Al-

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though the exact molecular mechanisms involved are yet unknown, various gain- and loss-of-function experiments fromour current data as well as published reports strongly suggestthat this Rab27a effector is a potent inhibitor of insulin secretion(Coppola et al., 2002; Gomi et al., 2005; Torii et al., 2002).
SREBP-1c and thus granuphilin was activated in islets fromSREBP-1c transgenic, KK-Ay, ob/ob, and DIO mice, as well as
in islets treated with PA. Decreased KSIS in these islets were
all restored by granuphilin knockdown, demonstrating that granuphilin plays a crucial role in the disturbance of insulin secretion
post-ATP production. In combination with granuphilin activation, syntaxin-1a and Munc18-1, components of this docking
machinery, were decreased by SREBP-1c activation and thus,
may also be involved in the secretion impairment. Dysregulation

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SREBP-1c/granuphilin pathway in insulin secretion

Figure 7. Effect of overexpression of nuclear
SREBP-1c on insulin secretion in pancreatic islets
from granuphilin-null mice
Islets were isolated from male age-matched (30- to
32-week-old) granuphilin-null (Granu(2/Y)) andwild-type littermate (Granu(+/Y)) mice. The islets
were infected with adenoviral-GFP or -SREBP-1c(500 MOI) for 48 hr.
A) Immunoblot analysis of SREBP-1, granuphilin,and Rab27a from the indicated islets with a-tubulin
as a loading control.
B) mRNA levels of indicated genes from the indicatedislets as estimated by real-time RT-PCR. Granu(+/Y)GFP (white bars), Granu(+/Y)-SREBP-1c (blackbars), Granu(2/Y)-GFP (light gray bars), andGranu(2/Y)-SREBP-1c (dark gray bars).
C) mRNA levels of human SREBP-1c (hBP-1c) andgranuphilin (Granu) from the indicated islets asestimated by real-time RT-PCR. Lane 1, Granu(+/Y)-GFP; lane 2, Granu(+/Y)-SREBP-1c; lane 3,
Granu(2/Y)-GFP; lane 4, Granu(2/Y)-SREBP-1c.
D) Cellular ATP/ADP ratio from the indicated islets.
E) Cellular TG content from the indicated islets.
F) Upper panel: HG and KCl-stimulated insulin secretion ratio, normalized by LG-stimulated insulin secretion. Lower panel: HG- and KCl-stimulated insulin
secretion in Granu(+/Y)-GFP (white bars), Granu(+/
Y)-SREBP-1c (black bars), Granu(2/Y)-GFP (lightgray bars), and Granu(2/Y)-SREBP-1c (dark graybars).
Results were normalized to cellular DNA content (F)and are expressed by mean 6 SEM. *p <0.01 (versus
Granu(+/Y)-GFP) and #p < 0.01 (versus Granu(2/Y)GFP). Studies were performed in triplicate with setsof islets from 3–4 mice per replicate.

of these three proteins may impair insulin secretion by disturbing
the balance of vesicle transport component concentrations.
Potential involvement of SREBP-1c/granuphilin pathway
in b cell lipotoxicity
We showed that upregulation of granuphilin is observed in b cells
not only from transgenic mice overexpressing SREBP-1c but
also from obesity-associated insulin resistant diabetic micesuch as KK-Ay, ob/ob, and DIO mice, leading to a reduction of
KSIS. It has been believed that impaired GSIS, due to deterioration of glucose metabolism characterized by decreased ATP/ADP ratio and/or insulin production in b-cells, is responsible
for the insulin secretion defect in type 2 diabetes (Weir and Bonner-Weir, 2004). Our current data demonstrate that additional
disturbances can also occur at the exocytosis stage of insulin

CELL METABOLISM : AUGUST 2006

secretion in diabetic models. Impaired KSIS was primarily
mediated through activation of granuphilin, providing anothermechanism for development of diabetes. Overexpression of
granuphilin also inhibited GSIS in normal islets, but this was because the path to GSIS also includes the process of KSIS, anddisturbance causing decreased KSIS should result in impaired
GSIS as well. Supportively, granuphilin knockdown efficiently
restored impaired KSIS in SREBP-1c transgenic and diabetic
islets but only partially restored GSIS, indicating that the
SREBP-1c/granuphilin effect was specific to the process postATP production.
Our data also provide further evidence that SREBP-1c is intimately involved in b cell dysfunction. In islets from the mice withlipotoxicity and diabetes, SREBP-1c is upregulated and contributes to disturbances in ATP production leading to impaired GSIS

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and also to upregulation of granuphilin leading to attenuated
KSIS. Various approaches using gene engineering and adenovirus consistently supported this hypothesis. Thus, the SREBP1c/granuphilin pathway should be added to the list of potential
mechanisms for b cell lipotoxicity in diabetes and could be a potential target for therapy of obesity-related diabetes. In contrast,
it was reported that granuphilin as well as Rab27a and 3 were
suppressed in a glucotoxic state of INS-1 cells through induction of ICER (Abderrahmani et al., 2006). Further studies areneeded to elucidate the precise molecular role of granuphilin
in insulin secretion and to determine the extent to which granuphilin contributes to diabetes in humans.
Experimental procedures
Materials
All chemical compounds were obtained from Sigma chemicals. Enhanced
chemiluminescence Western blot detection kit and redivue [a-32P] dCTP
were purchased from Amersham Pharmacia and restriction enzymes werefrom Takara Bio Inc.
Animals
All animal studies were approved by the Animal Care Committee of Universityof Tsukuba. Male C57BL/6, ob/ob, db/m, and db/db mice were purchasedfrom Charles River Laboratories. KK and KK-Ay were purchased from
Clea. RIP-human nuclear SREBP-1c transgenic mice (Takahashi et al.,2005), SREBP-1-null mice (Shimano et al., 1997b), and Granuphilin-nullmice (Gomi et al., 2005) were generated as previously described. Animals
were adapted to environments for 1 week before isolation of pancreatic
islets. The mice were housed in colony cages and maintained on a 12 hrlight/12 hr dark cycle and given free access to water and a standard chowdiet (MF, Oriental yeast).
Expression plasmids
All expression vectors were produced as previously described (AmemiyaKudo et al., 2000, 2005; Ide et al., 2004; Kajihara et al., 2003; Yamamoto
et al., 2004).
Reporter plasmid
The reporter plasmid Granu-1200-Luc contains a fragment of the granuphilinpromoter from 21200 to +87 bp cloned into the MluI/XhoI sites of the pGL3
basic vector (Promega) containing the coding sequences of firefly luciferase
cDNA. Other constructs were produced by PCR using this construct as a
DNA template, and the PCR products were inserted into pGL3 basic vector.
The primers used for PCR were as follows: 50 primers Granu-1200-Luc
50 -taagcgttgagaaatgaatg-30 , Granu-500-Luc 50 -agggttgcttacagggcgat-30 ,Granu-300-Luc 50 -tcgctgaacaaaaaggcaag-30 , Granu-265-Luc 50 -ttcctacccc
cacccccgac-30 , Granu-174-Luc 50 -tgctaactcaggaggaaattc-30 , Granu-150Luc 50 -aattcctccagtgccttaag-30 , and 30 primer 50 -ggtccgggtgccgaatg-30 . Restriction sites MluI and XhoI were added to each 50 primer and 30 primer,
respectively. The site-directed mutagenesis constructs mSRE- and mMARELuc were produced by PCR with the following primers: mSRE-50 50 -attcccat
tccgtaccccacccccgacccc-30 , -30 50 -ggggtcgggggtggggtacggaatgggaat-30 ,mMARE-50 50 -tcaaactctcagaactcaggaggaaattcc-30 , and -30 50 -ggaatttcctcc
tgagttctgagagtttga-30 . SRE-Luc was produced as previously described(Amemiya-Kudo et al., 2000).
Cell cultures, transfection, and luciferase assays
Min6 cells were cultured at 37 C in atmosphere of 5% CO2 in Dulbecco’s
modified Eagle’s Medium (DMEM) with 25 mM glucose supplemented with
15% fetal bovine serum, 100 U/ml penicillin, 100 mg/ml streptomycin, andb-mercaptoethanol. Each expression plasmid (0–0.5mg), luciferase reporter
plasmid (0.2 mg), and pSV40-Renilla plasmid (0.05 mg) were cotransfectedusing the LipofectAMINE reagent (Invitrogen) according to manufacture’s
instructions. Min6 cells were seeded in 24-well plates at a density of 3 3
105 cells/well. The total amount of DNA was adjusted to 0.5–1 mg/well withempty vector DNA. After 48 hr of transfection, cells were washed with phosphate-buffer saline (PBS) and harvested. Luciferase assays were carried out

152

according to the manufacture’s instructions (Promega), and luciferase activity was quantified by using ALOVA 1420 multilabel counter (Perkin Elmer Life
Sciences). As the internal standard, SV40 Renilla luciferase control vector
was also cotransfected to normalize for transfection efficiency.
Gel mobility shift assay
Gel mobility shift assays (EMSA) were performed as previously described(Amemiya-Kudo et al., 2005; Nakagawa et al., 2006; Yamamoto et al.,2004). In brief, recombinant SREBPs (SREBP-1a, -1c,and -2) and MafA proteins were produced using the TNT T7 quick-coupled transcription/translation system (Promega). The reaction mixture were incubated with in vitro
synthetic protein lysate and then analyzed on 4% polyacrylamide gels.
Super-shift reactions were performed by adding antibody (anti-SREBP-1,
sc-8984 Santa Cruz) to the reaction mixture. The DNA probes for EMSA
are followed: granuphilin-SRE, aactttattcccattcctacccccacccccgaccccaatc
atctaagaga, granuphilin-mSRE, aactttattcccattccgtaccccacccccgaccccaat
catctaagaga, granuphilin-MARE, cctcaaactctgctaactcaggaggaaattcctccagtg
cc, and granuphilin-mMARE, cctcaaactccagtaactcaggaggaaattcctccagtgcc.
Isolation of mouse pancreatic islets and insulin
secretion experiments
Isolation of islets from mice was carried out using the Ficol-Conray protocol
as previously described(Scharp et al., 1973; Takahashi et al., 2005). Briefly,
4 mg/ml collagenase was injected into the pancreatic duct and incubatedat 37 C for 20 min. The islets were subsequently purified by Ficol gradientand hand picking. The remaining islets were cultured for 2 hr at 37 C in a humidified atmosphere containing 5% CO2 and in regular RPMI-1640 medium
supplemented with 10% FCS (pH 7.4) prior to the experiments. Insulin release from islets was measured as previously described (Takahashi et al.,2005). In brief, groups of 10 islets of similar size for each condition were preincubated in Krebs-Ringer bicarbonate buffer (KRBH [pH 7.4]) containing
0.5% BSA at 2.8mM glucose for 30 min. Islets were then incubated with
1 ml of KRBH with 0.5% BSA at 2.8mM glucose (low glucose; LG), 20mM
glucose (high glucose; HG), 30mM KCl with 2.8 mM glucose (KCl), 3mM
Arginine with 2.8 mM glucose, forscolin (10mM) with 2.8 mM glucose, or
TPA (0.5 mM) with 2.8 mM glucose, respectively. Subsequently, medium
were replaced with fresh medium for insulin determination followed by total
islet insulin extraction by 0.5ml of cold acid ethanol mixture (75% ethanol
with 0.2M HCl). Hoechst-33258 staining of sonicated islets was performed
to determine the islet DNA content.
Determination of ATP/ADP ratio and triglyceride contents of islets
ATP and ADP content in isolated islets were determined as previously described (Schultz et al., 1993; Takahashi et al., 2005). In brief, after preincubation at 37 C for 30 min in KRBH buffer containing 2.8 mM glucose, groups of
25 islets were incubated at 37 C for 30 min in KRBH buffer containing 20 mM
glucose. The conversion from ADP to ATP was performed in the reaction
buffer (20mM HEPES and 3mM MgCl2 [pH 7.75]) containing 2.3 U/ml pyruvate kinase and 1.5mM phosphoenolpyruvate at room temperature for
15 min. ATP and ADP were extracted from islets with 100 ml of 5% trichloroacetic acid (TCA). After centrifugation, the supernatants were neutralizedwith NaOH. ATP content was measured using CellTiter-Glo luminescentcell viability assay kit (Promega).
Real-time PCR and immnoblot analysis
Comparative analysis of mRNA levels was performed with fluorescencebased real-time PCR. Total RNA extraction and first-strand cDNA synthesis
were previously as described (Takahashi et al., 2005). Quantitative real-time
PCR was performed using Sybr-Green Dye (Nihon Gene) in an ABI 7000
PCR instrument (Applied Biosystems). Relative abundance for each transcript
was calculated by a standard curve of cycle thresholds for serial dilutions of acDNA sample and normalized to cyclophilin. Primer sequences are available
upon request. For immnoblot blots, total or nuclear cell extracts of isolated
islets (Hagman et al., 2005; Takahashi et al., 2005) and nuclear extracts
from Min6 cells (Amemiya-Kudo et al., 2005) were prepared as described
previously, separated by SDS-PAGE, transferred to immobilon-P membranes(Millipore), and were probed with polyclonal anti-SREBP-1 (sc-8984), antiRab27a (sc-22756), anti-a-tubulin (sc-5282, Santa Cruz), anti-MafA (A-300611, Bethyl Lab. Inc.), anti-syntaxin-1a (S1172, Sigma), anti-Vamp2(627724, Calbiochem), anti-Rab3 (R35520), anti-Munc18-1 (M32320), and

CELL METABOLISM : AUGUST 2006

SREBP-1c/granuphilin pathway in insulin secretionanti-SNAP25 (S35020, BD) antibodies. Detection was performed using an
ECL advance Western blotting ECL detection kit and Hyperfilm (Amersham
Biosciences). Anti-granuphilin-a/b antibody was as previously described(Gomi et al., 2005).
Northern blot analysis
Northern blot analysis was performed as previously described (AmemiyaKudo et al., 2005; Matsuzaka et al., 2004). cDNA probe for granuphilin was
prepared from reverse transcriptase for PCR of Min6 cells total RNA withthe following primers: 50 -cgagatggaaagggatttga-30 for sense and 50 -gtctgc
tgaaggagggactg-30 for antisense of granuphilin.
Preparation of recombinant adenovirus
The construct of granuphilin-a coding cDNA was generated by PCR amplification and into the pShuttle-CMV vector (Ide et al., 2004; Nakagawa et al.,2006). The following primers were used: 50 primer, 50 -atgtcggagatactagacc
tctctt-30 , and 30 primer 50 -catacacccagcttctgcttgaccat-30 . Generation of recombinant adenoviral plasmid was produced by homologous recombination
with the pAdEasy-1 plasmid. The siRNA construct for mouse granuphilin and
SREBP-1 was generated in the coding sequence; Granuphilin-RNAi-784
50 -gagcgagagtctggatagctaca-30 , Granuphilin-RNAi-1955 50 -aggctggaagatct
acaacaaat-30 , and SREBP-1-RNAi 50 -ggcaaaggaggcactacag-30 . Oligonucleotide containing this sequence was subcloned into U6/RNAi empty vector(Invitrogen). Granuphilin RNAi adenoviruses were generated using BLOCKiT Adenoviral RNAi Expression System (Invitrogen). Production of recombinant adenoviruses was performed by CsCl gradient centrifugation aspreviously described (Ide et al., 2004).
Chromatin immunoprecipitation assay
Chromatin immunoprecipitation (ChIP) assays were performed as previously
described (Amemiya-Kudo et al., 2005; Nakagawa et al., 2006). To amplify
the granuphilin promoter region containing SRE and MARE, the following
primer sets were used: for SRE 50 -gtactttactattggacaac-30 for sense, 50 ccttaaggcactggaggaatttc-30 for antisense, and for MARE 50 -gtgagcaaagata
gactaga-30 for sense, 50 -tggagcggggaggagctggactc-30 for antisense. The
PCR conditions were 5 min at 95 C and 30 cycles of 30 s at 94 C, 30 s at
57 C and 1 min 72 C for SRE region, and 10 min at 94 C and 30 cycles of
10 s at 98 C, 30 s at 54 C, and 1 min 72 C for MARE region.
Palmitate treatment
Palmitate was dissolved in ethanol as 100 mM stock solution with 0.5% fatty
acid free BSA (Sigma). For using experiments, palmitate was diluted in
DMEM supplemented with 0.5% fatty acid free BSA to a final concentrationof 400 mM for islets study or 200 mM for Luc-assay.
Statistical analysis
Values are expressed as mean 6 standard error of the mean (SEM). Statistical comparisons were performed using analysis of variance (ANOVA).
Supplemental data
Supplemental data include five figures and can be found with this article
online at http://www.cellmetabolism.org/cgi/content/full/4/2/143/DC1/.
Acknowledgments
This work was supported by grants in aid from the Ministry of Science, Education, Culture, and Technology of Japan.

Received: February 2, 2006
Revised: May 5, 2006
Accepted: June 28, 2006
Published: August 8, 2006
References
Abderrahmani, A., Cheviet, S., Ferdaoussi, M., Coppola, T., Waeber, G., andRegazzi, R. (2006). ICER induced by hyperglycemia represses the expressionof genes essential for insulin exocytosis. EMBO J. 25, 977–986.

CELL METABOLISM : AUGUST 2006

Amemiya-Kudo, M., Oka, J., Ide, T., Matsuzaka, T., Sone, H., Yoshikawa, T.,
Yahagi, N., Ishibashi, S., Osuga, J., Yamada, N., et al. (2005). Sterol regulatory element-binding proteins activate insulin gene promoter directly andindirectly through synergy with BETA2/E47. J. Biol. Chem. 280, 34577–
34589.
Amemiya-Kudo, M., Shimano, H., Yoshikawa, T., Yahagi, N., Hasty, A.H.,
Okazaki, H., Tamura, Y., Shionoiri, F., Iizuka, Y., Ohashi, K., et al. (2000). Promoter analysis of the mouse sterol regulatory element-binding protein-1c
gene. J. Biol. Chem. 275, 31078–31085.
Biddinger, S.B., Almind, K., Miyazaki, M., Kokkotou, E., Ntambi, J.M., andKahn, C.R. (2005). Effects of diet and genetic background on sterol regulatory element-binding protein-1c, stearoyl-CoA desaturase 1, and the development of the metabolic syndrome. Diabetes 54, 1314–1323.
Bratanova-Tochkova, T.K., Cheng, H., Daniel, S., Gunawardana, S., Liu, Y.J.,
Mulvaney-Musa, J., Schermerhorn, T., Straub, S.G., Yajima, H., and Sharp,
G.W. (2002). Triggering and augmentation mechanisms, granule pools, andbiphasic insulin secretion. Diabetes 51(Suppl 1), S83–S90.
Burgoyne, R.D., and Morgan, A. (2003). Secretory granule exocytosis. Physiol. Rev. 83, 581–632.
Coppola, T., Frantz, C., Perret-Menoud, V., Gattesco, S., Hirling, H., andRegazzi, R. (2002). Pancreatic beta-cell protein granuphilin binds Rab3 andMunc-18 and controls exocytosis. Mol. Biol. Cell 13, 1906–1915.
Diani, A.R., Sawada, G., Wyse, B., Murray, F.T., and Khan, M. (2004). Pioglitazone preserves pancreatic islet structure and insulin secretory function inthree murine models of type 2 diabetes. Am. J. Physiol. Endocrinol. Metab.
286, E116–E122.
Dubois, M., Kerr-Conte, J., Gmyr, V., Bouckenooghe, T., Muharram, G.,
D’Herbomez, M., Martin-Ponthieu, A., Vantyghem, M.C., Vandewalle, B.,
and Pattou, F. (2004). Non-esterified fatty acids are deleterious for humanpancreatic islet function at physiological glucose concentration. Diabetologia 47, 463–469.
Gomi, H., Mizutani, S., Kasai, K., Itohara, S., and Izumi, T. (2005). Granuphilin
molecularly docks insulin granules to the fusion machinery. J. Cell Biol. 171,99–109.
Hagman, D.K., Hays, L.B., Parazzoli, S.D., and Poitout, V. (2005). Palmitate
inhibits insulin gene expression by altering PDX-1 nuclear localization and reducing MafA expression in isolated rat islets of Langerhans. J. Biol. Chem.
280, 32413–32418.
Hasty, A.H., Shimano, H., Yahagi, N., Amemiya-Kudo, M., Perrey, S., Yoshikawa, T., Osuga, J., Okazaki, H., Tamura, Y., Iizuka, Y., et al. (2000). Sterol
regulatory element-binding protein-1 is regulated by glucose at the transcriptional level. J. Biol. Chem. 275, 31069–31077.
Horton, J.D., Bashmakov, Y., Shimomura, I., and Shimano, H. (1998). Regulation of sterol regulatory element binding proteins in livers of fasted andrefed mice. Proc. Natl. Acad. Sci. USA 95, 5987–5992.
Ide, T., Shimano, H., Yahagi, N., Matsuzaka, T., Nakakuki, M., Yamamoto, T.,
Nakagawa, Y., Takahashi, A., Suzuki, H., Sone, H., et al. (2004). SREBPs suppress IRS-2-mediated insulin signalling in the liver. Nat. Cell Biol. 6, 351–357.
Joseph, J.W., Koshkin, V., Saleh, M.C., Sivitz, W.I., Zhang, C.Y., Lowell, B.B.,
Chan, C.B., and Wheeler, M.B. (2004). Free fatty acid-induced beta-cell defects are dependent on uncoupling protein 2 expression. J. Biol. Chem. 279,
51049–51056.
Kahn, S.E. (2003). The relative contributions of insulin resistance and betacell dysfunction to the pathophysiology of Type 2 diabetes. Diabetologia
46, 3–19.
Kajihara, M., Sone, H., Amemiya, M., Katoh, Y., Isogai, M., Shimano, H., Yamada, N., and Takahashi, S. (2003). Mouse MafA, homologue of zebrafish
somite Maf 1, contributes to the specific transcriptional activity through theinsulin promoter. Biochem. Biophys. Res. Commun. 312, 831–842.
Matsuzaka, T., Shimano, H., Yahagi, N., Amemiya-Kudo, M., Okazaki, H., Tamura, Y., Iizuka, Y., Ohashi, K., Tomita, S., Sekiya, M., et al. (2004). Insulinindependent induction of sterol regulatory element-binding protein-1c expression in the livers of streptozotocin-treated mice. Diabetes 53, 560–569.

153

A R T I C L E

Nakagawa, Y., Shimano, H., Yoshikawa, T., Ide, T., Tamura, M., Furusawa,
M., Yamamoto, T., Inoue, N., Matsuzaka, T., Takahashi, A., et al. (2006).
TFE3 transcriptionally activates hepatic IRS-2, participates in insulin signaling and ameliorates diabetes. Nat. Med. 12, 107–113.
Rizzoli, S.O., and Betz, W.J. (2005). Synaptic vesicle pools. Nat. Rev. Neurosci. 6, 57–69.
Rorsman, P., and Renstrom, E. (2003). Insulin granule dynamics in pancreatic
beta cells. Diabetologia 46, 1029–1045.
Scharp, D.W., Kemp, C.B., Knight, M.J., Ballinger, W.F., and Lacy, P.E.
(1973). The use of ficoll in the preparation of viable islets of langerhans
from the rat pancreas. Transplantation 16, 686–689.
Schultz, V., Sussman, I., Bokvist, K., and Tornheim, K. (1993). Bioluminometric assay of ADP and ATP at high ATP/ADP ratios: assay of ADP after enzymatic removal of ATP. Anal. Biochem. 215, 302–304.
Shimano, H., Horton, J.D., Shimomura, I., Hammer, R.E., Brown, M.S., andGoldstein, J.L. (1997a). 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.
Shimano, H., Shimomura, I., Hammer, R.E., Herz, J., Goldstein, J.L., Brown,
M.S., and Horton, J.D. (1997b). Elevated levels of SREBP-2 and cholesterolsynthesis in livers of mice homozygous for a targeted disruption of the
SREBP-1 gene. J. Clin. Invest. 100, 2115–2124.
Takahashi, A., Motomura, K., Kato, T., Yoshikawa, T., Nakagawa, Y., Yahagi,N., Sone, H., Suzuki, H., Toyoshima, H., Yamada, N., and Shimano, H. (2005).

154

Transgenic mice overexpressing nuclear SREBP-1c in pancreatic beta-cells.
Diabetes 54, 492–499.
Torii, S., Takeuchi, T., Nagamatsu, S., and Izumi, T. (2004). Rab27 effector
granuphilin promotes the plasma membrane targeting of insulin granules
via interaction with syntaxin 1a. J. Biol. Chem. 279, 22532–22538.
Torii, S., Zhao, S., Yi, Z., Takeuchi, T., and Izumi, T. (2002). Granuphilin modulates the exocytosis of secretory granules through interaction with syntaxin
1a. Mol. Cell. Biol. 22, 5518–5526.
Wang, J., Takeuchi, T., Yokota, H., and Izumi, T. (1999). Novel rabphilin-3-likeprotein associates with insulin-containing granules in pancreatic beta cells.
J. Biol. Chem. 274, 28542–28548.
Weir, G.C., and Bonner-Weir, S. (2004). Five stages of evolving beta-cell
dysfunction during progression to diabetes. Diabetes 53(Suppl 3), S16–
S21.
Yamamoto, T., Shimano, H., Nakagawa, Y., Ide, T., Yahagi, N., Matsuzaka,T., Nakakuki, M., Takahashi, A., Suzuki, H., Sone, H., et al. (2004). SREBP1 interacts with hepatocyte nuclear factor-4 alpha and interferes with PGC1 recruitment to suppress hepatic gluconeogenic genes. J. Biol. Chem.
279, 12027–12035.
Yi, Z., Yokota, H., Torii, S., Aoki, T., Hosaka, M., Zhao, S., Takata, K., Takeuchi, T., and Izumi, T. (2002). The Rab27a/granuphilin complex regulates the
exocytosis of insulin-containing dense-core granules. Mol. Cell. Biol. 22,
1858–1867.

CELL METABOLISM : AUGUST 2006