Cholesterol accumulation and diabetes in pancreaticb-cell-specific SREBP-2 transgenic mice: a new model
for lipotoxicity
Mayumi Ishikawa,2,* Yuko Iwasaki,2,* Shigeru Yatoh,* Toyonori Kato,* Shin Kumadaki,*
Noriyuki Inoue,* Takashi Yamamoto,* Takashi Matsuzaka,*,† Yoshimi Nakagawa,*,†Naoya Yahagi,† Kazuto Kobayashi,* Akimitsu Takahashi,* Nobuhiro Yamada,*and Hitoshi Shimano1,*,†

Abstract To determine the role of cholesterol synthesis inpancreatic b-cells, a transgenic model of in vivo activation ofsterol-regulatory element binding protein 2 (SREBP-2) specifically in b-cells (TgRIP-SREBP-2) was developed and analyzed. Expression of nuclear human SREBP-2 in b-cells
resulted in severe diabetes as evidenced by greater than
5-fold elevations in glycohemoglobin compared with
C57BL/6 controls. Diabetes in TgRIP-SREBP-2 mice was primarily due to defects in glucose- and potassium-stimulated
insulin secretion as determined by glucose tolerance test.
Isolated islets of TgSREBP-2 mice were fewer in number,smaller, deformed, and had decreased insulin content.
SREBP-2-expressing islets also contained increased esterified cholesterol and unchanged triglycerides with reduced
ATP levels. Consistently, these islets exhibited elevated expression of HMG-CoA synthase and reductase and LDL receptor, with suppression of endogenous SREBPs. Genes
involved in b-cell differentiation, such as PDX1 and BETA2,
were suppressed, explaining loss of b-cell mass, whereas
IRS2 expression was not affected. These phenotypes weredependent on the transgene expression. Taken together,
these results indicate that activation of SREBP-2 in b-cells
caused severe diabetes by loss of b-cell mass with accumulation of cholesterol, providing a new lipotoxic model and apotential link of disturbed cholesterol metabolism to impairment of b-cell function.—Ishikawa, M., Y. Iwasaki, S. Yatoh, T.
Kato, S. Kumadaki, N. Inoue, T. Yamamoto, T. Matsuzaka, Y.
Nakagawa, N. Yahagi, K. Kobayashi, A. Takahashi, N. Yamada,and H. Shimano. Cholesterol accumulation and diabetes inpancreatic b-cell-specific SREBP-2 transgenic mice: a newmodel for lipotoxicity. J. Lipid Res. 2008. 49: 2524–2534.
Supplementary key words transcription factors • sterol-regulatory element binding protein • triglycerides

This work was supported by grants-in-aid from the Ministry of Science, Education, Culture, and Technology of Japan.
Manuscript received 8 May 2008 and in revised form 15 July 2008 and in
re-revised form 1 August 2008.
Published, JLR Papers in Press, August 8, 2008.
DOI 10.1194/jlr.M800238-JLR200

Molecular mechanisms of pancreatic islet b-cell failure,a crucial pathological contributor to the development ofdiabetes mellitus, have been extensively explored (1). Impairment of glucose-stimulated insulin secretion (GSIS) isan early feature of type 2 diabetes. Chronic influx of FAs
into b-cells, i.e., b-cell lipotoxicity, has been thought to be
involved in its pathogenesis (2).
Sterol-regulatory element binding protein 1c (SREBP-1c)
is a membrane-bound transcription factor of the basic HLH(bHLH) leucine zipper family and has been established
as a nutritional regulator of lipogenic enzymes in the liver(3, 4). Expression of SREBP-1c is highly upregulated bydietary intake of carbohydrates, sugars, and saturated FAs,
whereas PUFAs, such as eicosapentaenoic acid, have beenshown to inhibit hepatic SREBP-1c through multiple mechanisms (5, 6). These nutritional regulations of SREBP-1c
are also observed in a cultured b-cell line and in isolated
islets of mice (7, 8). SREBP-1c also plays a role in insulin signaling by inhibiting insulin receptor substrate 2 (IRS-2),
the major insulin-signaling mediator in the liver and in
b-cells (9, 10).
As a model for lipotoxicity by endogenous FAs in pancreatic b-cells, we previously developed transgenic miceoverexpressing the active form of SREBP-1c under the insulin promoter expression (10). These mice exhibited impaired glucose tolerance in vivo due to both decreased
b-cell mass and impaired insulin secretion estimated in isolated islets, which was enhanced by feeding the mice ahigh-fat, high-sucrose diet. The SREBP-1c-overexpressing
islets had ATP depletion caused by enhanced lipogenesis
and increased uncoupling protein 2 (UCP-2). Explaining
the loss of b-cell mass, these islets had decreased expression of IRS-2 and PDX1. In addition to inhibition of GSIS,
1
2

To whom correspondence should be addressed.
e-mail: shimano-tky@umin.ac.jp
M. Ishikawa and Y. Iwasaki contributed equally to this work.

Copyright © 2008 by the American Society for Biochemistry and Molecular Biology, Inc.

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Department of Internal Medicine (Endocrinology and Metabolism),* Graduate School of Comprehensive
Human Sciences; and Center for Advanced Research Alliance,† University of Tsukuba, 1-1-1 Tennodai,
Tsukuba-City, Ibaraki, Japan, 305-8575

level was measured using an HbA1c immunoassay kit (DCA2000
system; Bayer Medical Co., Ltd., Tokyo, Japan). The insulin level
was measured with a mouse insulin ELISA kit (Shibayagi Co., Ltd.,
Gunma, Japan). The total cholesterol, triglycerides, and FFA levels
were measured with commercial kits (Wako). Leptin levels were
measured with a mouse leptin ELISA kit (Morinaga Institute of
Biological Science, Inc., Kanagawa, Japan).

Glucose tolerance test
Intraperitoneal glucose tolerance test and intravenous glucose
tolerance test were performed as previously described (10).

Isolation of pancreatic islet
Mice between 16 to 24 weeks of age were used for isolation of
pancreatic islets to avoid aging effects. The isolation of islets was
carried out according to the Ficoll-Conray protocol (10, 18).

Analysis of insulin secretion, insulin content, and DNA
content of islets
Isolated islets were incubated for 2 h in RPMI-1640 medium
supplemented with 10% FBS, 100 U/ml penicillin, and 100 mg/ml
streptomycin. Ten islets were then preincubated for 30 min in
2.8 mM glucose in 0.5% BSA Krebs-Ringer Bicarbonate Hepes(KRBH) buffer. Finally, they were incubated in 2.8 mM glucose, 20 mM glucose, or 2.8 mM glucose with 30 mM KCl in
0.5% BSA KRBH buffer for 30 min, and insulin secretion was
analyzed using a mouse insulin ELISA kit (Shibayagi Co., Ltd.).
Insulin and DNA content of islets were measured as described
previously (10).

Measurements of ATP and ADP of islets

METHODS
Generation and maintenance of transgenic mice
The expression plasmid pRIP-SREBP-2 (1–468) containing therat insulin I promoter (RIP; bp 2715 to 31) was fused to a cDNA
encoding amino acids 1–468 of human SREBP-2 (16) followed bya 3′ polyadenylation signal from the human growth hormone(hGH) cDNA. Techniques used for generating transgenic mice
have been described previously (17). Briefly, the NotI-XhoI fragment (Fig. 1A) of pRIP-SREBP-2 (1–468) was microinjected into
C57BL6/J 3 SJL hybrid eggs to generate transgenic mice. Foundermice were subjected to isolation of pancreatic islets, and presence of the nuclear, truncated form of human SREBP-2 was determined by RT-PCR as described below. Two independent linesof SREBP-2 transgenic mice under the control of rat insulin promoter (TgRIP-SREBP-2) were established and designated lines A
and B. To obtain higher expression, hemizygotes of line A weremated to produce mice homozygous for the transgene. Mice were
genotyped by PCR and Southern blot analysis. Mice were housedin colony cages and maintained on a 12 h light/12 h dark cycle.
Transgenic mice and nontransgenic littermates were fed a regularchow diet (MF; Oriental Yeast, Tokyo, Japan). All experiments
were performed according to the Guide for the Care and Use ofLaboratory Animals at the University of Tsukuba, and the project
was approved by the Institutional Review Board.

Measurement of glucose, HbA1c, insulin, total cholesterol,
triglycerides, FFA, and leptin levels
Blood was taken from the tail vein of age-matched mice fed ad
libitum. The glucose level was measured with a glucose oxidase kit(Wako Pure Chemical Industries, Ltd., Osaka, Japan). The HbA1c

ATP and ADP levels per 10 isolated islets were measured as described previously (19), using the CellTiter-Glo™Luminescent
Cell Viability Assay (Promega, Madison, WI).

Measurements of triglycerides and cholesterol content
of islets
One hundred isolated islets were used for lipid extraction.
Triglycerides were measured by extracting lipids using themethod of Folch, Lees, and Sloane Stanley (20), followed bytriglyceride determination with the GPO-trinder kit (Sigma, St.
Louis, MO). For cholesterol deterimination, lipids were extractedwith hexane-isopropanol (3:2; v/v). The enzymatic and fluorometric method was used for the picomole determination of free
and total cholesterol (21).

Histological and immunohistochemical analysis of Islets
Mice were anesthetized, and pancreata were rapidly dissected
and fixed in 4% paraformaldehyde solution, embedded in paraffin, sectioned, and stained with hematoxylin and eosin for histological analysis. For immunohistochemistry, 2 mm sections ofparaffin-embedded pancreas were dewaxed using xylene, rehydrated through serial dilutions of ethyl alcohol, and subjected
to antigen retrieval using 0.1% Triton X-100 with PBS. The sections were washed and stained with the respective antibodies
in staining buffer with 150 mM NaCl, 0.05% Tween-20, and10 mM Tris-HCL (pH 7.4). Primary antibodies included guinea
pig anti-insulin (DAKO Japan Co., Ltd., Kyoto, Japan) and rabbit
anti-glucagon (DAKO). The secondary antibodies used included
FITC-conjugated sheep anti-guinea pig IgG (P.A.R.I.S.) and Cy3conjugated goat anti-rabbit IgG (Rockland Immunochemicals,Inc., Gilbertsville, PA).

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SREBP-1c transgenic islets exhibited decreased potassiumstimulated insulin secretion (KSIS), which could indicate
dysfunction in a process of insulin secretion following
ATP production. We have recently found that granuphilin,
an effecter of Rab27 involving exocytosis of insulin granules, is an SREBP target and contributes at least partiallyto the impaired insulin secretion of islets with SREBP-1coverexpression and to diabetes (11). Conversely, islets isolated from SREBP-1-null mice exhibited increased basal insulin secretion and immunity to the impairment in insulin
secretion caused by saturated FAs, establishing the contribution of SREBP-1c to lipotoxicity (7).
SREBP-2 is another member of the membrane-boundtranscription factor SREBP family, and plays a crucial rolein the regulation of cholesterol metabolism (12). Cellular
sterol levels are controlled by feedback regulation of cholesterol biosynthetic and LDL receptor pathways. Genes inthese pathways are transcriptionally regulated by SREBP-2
activation, which requires cleavage of its N-terminal bHLH
portion for nuclear translocation to activate promoters ofgenes such as HMG-CoA reductase and synthase as well as
the LDL receptor gene (13–15). FA metabolism has beenwell-studied with regard to its contribution to b-cell lipotoxicity; however, cholesterol metabolism and the role of
SREBP-2 in b-cells have not been fully investigated. Ourcurrent study investigated whether perturbation in sterol
regulation by SREBP-2 could be involved in impaired insulin secretion and b-cell function.

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Fig. 1. Phenotype of sterol-regulatory element binding protein (SREBP)-2 transgenic mice expressing
SREBP-2 under the control of rat insulin promoter (RIP). A: DNA construct for microinjection to generate
Tg RIP-SREBP-2. RIP I was fused to a hybrid-adenovirus-immunoglobulin intron (Intron), and a cDNA encoding amino acids 1–468 of human SREBP-2 (nuclear-hSREBP-2) followed by a 3′ polyadenylation signal
from the human growth hormone cDNA (hGH poly A tail) was added. B: mRNA levels of the human
SREBP-2 transgene in islets (left lower panel) and hypothalamus (left upper panel) from high-expression
line homo (Line A homo), high-expression line hemi (Line A hemi), low-expression line hemi (Line Bhemi), and wild-type (WT) littermate as estimated by real-time PCR. Results were normalized to mRNA levelsof cyclophilin. Immunoblot analysis of SREBP-2 from line A hemi, line B hemi, and WT islets (right panel).
a-Tubulin is a loading control. C: Time course of body weight in male line A homo (Tg homo, n 5 5), line A
hemi (Tg hemi, n 5 8), and WT (n 5 5). D: Appearance (top) and abdominal view (bottom) of male line A
homo (Tg homo) and WT (24 weeks). E: Diurnal food intake was measured in 14 week-old male mice of line
A homo (Tg homo, n 5 6) and WT (n 5 6). *P , 0.05, compared with WT. F: Twenty-four hour water intake
and urine volume in 12 week-old male mice of line A homo (Tg homo, n 5 5) and WT (n 5 5) were measured in individually housed mice. *P , 0.05, compared with WT. G: Intraperitoneal glucose tolerance tests
in 8 week-old male mice. Plasma glucose and plasma insulin levels of line A hemi, line A WT, line B hemi, andline B WT (n 5 4 for each group) were measured 15 min after intraperitoneal injection of glucose (1 g/kg
body weight) following an overnight fast. *P , 0.05, compared with WT of each group.

Preparation of islet RNA and estimaion ofgene expression
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 manufacturersʼ instructions. Northern blot analysis was
performed with the indicated cDNA probes using Rapid-hyb
buffer (GE Healthcare UK, Ltd.). cDNA was synthesized with
the ThermoScript™RT-PCR system (Invitrogen) and real-time
fluorescent detection PCR analysis was performed using SybrGreen Dye (ABgene, UK) with an ABI 7000 PCR instrument (Applied Biosystems Japan, Ltd., Tokyo, Japan) as previously described(11). mRNA level was normalized to cyclophilin expression.

Immunoblot analysis

Body weight (g)Liver (% BW)
Kidney (% BW)
White adipose tissue (% BW)

WT

Tg Hemi

Tg Homo35.9 6 3.5
5.1 6 0.8
1.0 6 0.1
3.6 6 0.9

29.8 6 0.7
6.0 6 0.5
1.5 6 0.1
1.6 6 0.4a

28.6 6 0.7
6.2 6 0.2
1.9 6 0.1a
0.4 6 0.4a

WT, wild type; BW, body weight; Tg, transgenic; TgRIP-SREBP-2
mice of high-expression line (line A, n 5 4 for each group) were sacrificed in a fed condition. Data represent means 6 SEM.
a
P , 0.05, compared with WT.

transgene caused a robust increase in SREBP-2 protein in
islets as estimated by immunoblot analysis (Fig. 1B, right).
A high expressor, line A, and a low expressor, line B, were
established based upon consistent increases in both mRNAand protein levels of human SREBP-2 in islets. Leaky gene
expression from the RIP promoter is often noted, however.
SREBP-2 in the hypothalamus was essentially negative inboth lines (Fig. 1B, inset). In addition, there was no change
in hypothalamic expression of HMG-CoA synthase, an
SREBP-2 target gene (data not shown). Line A transgenic

Expression plasmid and reporter plasmids
For generation of a human nuclear SREBP-2 expression plasmid,
the same cDNA fragment used for producing TgRIP-SREBP-2 constructs was cloned into cDNA3.1(1) (Invitrogen). RIP-luciferase
reporter plasmid and sterol-regulatory element (SRE)-luciferase
reporter plasmid were produced as previously described (22, 23).

Cell culture, transfection, and luciferase assays
MIN6 cells were maintained in DMEM containing 25 mmol/l glucose supplemented with 15% FBS, b-mercaptoethanol (5 ml/l),
penicillin (100 U/ml), and streptomycin (100 mg/ml) at 37°C
in an atmosphere of 95% air/5% CO2. The cells were seeded onto
24-well plates at a density of 3 3 105 cells/ml and cultured overnight to 80% confluency. The cells were transfected with an RIPluciferase reporter plasmid or SRE-luciferase reporter plasmid,
and an expression vector for nuclear SREBP-2 using Lipofectamine
2000 reagent (Invitrogen) according to the manufacturerʼs instructions. Total amount of plasmid DNA was always 300 ng/ml. After a
48 h incubation, the amount of firefly luciferase activity in transfectants was measured.

Statistical analysis
Data are expressed as the mean 6 standard error of the mean
(SEM) except for the data of the estimation of gene expression
and the luciferase assays that were expressed as the mean 6 standard deviation (SD).

RESULTS
Phenotypic features of TgRIP-SREBP-2 miceTransgenic mice overexpressing the nuclear form of human SREBP-2 under control of the RIP I (TgRIP-SREBP-2)
were created (Fig. 1A). Specificity of transgene expression
was verified by RT-PCR to demonstrate that human SREBP2 was expressed in pancreatic islets (Fig. 1B, left). The

Fig. 2. Plasma glucose, insulin, and HbA1c levels of TgRIP-SREBP-2
mice. A: Plasma glucose levels of line A homo (TgBP2 homo), line
A hemi (TgBP2 hemi), and WT (n 5 4–6). *P , 0.05 and **P ,0.01, compared with WT of each group. B: Plasma insulin levels ofline A homo (TgBP2 homo), line A hemi (TgBP2 hemi), and WT(n 5 4–6). C: Time course of HbA1c levels in male line A homo
(TgBP2 homo), line A hemi (TgBP2 hemi), and WT (n 5 6–10).

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Islets were harvested and dissolved in lysis buffer containing
20 mM Hepes (pH 7.6), 150 mM NaCl, 1% NP-40, 0.5% sodium
deoxycholate, 0.1% SDS, 10% glycerol, 25 mg/ml calpain inhibitor,
10 mg/ml aprotinin, and 10 mg/ml leupeptin. Immunoblot analysis
was as previously described (10). Cell extracts from isolated islets
were probed with polyclonal antibodies against SREBP-2 (K0180-3;
Medical and Biological Laboratories Co., Ltd, Nagoya, Japan),
PDX1 (#07-696; Upstate), a-tubulin (sc-5286; Santa Cruz Biotechnology, Santa Cruz, CA), and Lamin A/C (#2032; Cell Signaling).
Detection was performed using an ECL advance Western blotting
detection kit and Hyperfilm ECL (GE Healthcare).

TABLE 1. Body weights and organ weights in 24 week-old miceFig. 3. Intravenous glucose tolerance tests in TgRIPSREBP-2 mice. Plasma glucose and plasma insulinlevels of 8 week-old mice were measured after intravenous injection of glucose (1 g/kg body weight)
following an overnight fast. A: Plasma glucose levelsof male line A hemi (TgBP2 hemi, n 5 5) and WT(n 5 5) mice. B: Plasma insulin levels of male line
A hemi (TgBP2 hemi, n 5 5) and WT (n 5 5) mice.
C: Plasma glucose levels of female line A hemi
(TgBP2 hemi, n 5 5) and WT (n 5 5) mice. D: Plasma
insulin levels of female line A hemi (TgBP2 hemi, n 5 5)
and WT (n 5 5) mice. *P , 0.05 and **P , 0.01, compared with WT of each group.

Insulin-deficient diabetes in TgRIP-SREBP-2
Consistent with these typical signs of diabetes (polyuria,
polydipsia, and hyperphagia), TgRIP-SREBP-2 mice (line A)
were diabetic, with homo more severe. Hemizygotes of line
B (low expressors) were not diabetic, but still exhibited
impaired plasma insulin and higher glucose levels basedon intraperitoneal glucose tolerance tests (Figs. 1G, 2).
In both TgRIP-SREBP-2 hemi and TgRIP-SREBP-2 homo
mice from line A, plasma glucose was markedly increased,

whereas plasma insulin was diminished (Fig. 2A, B). Glycohemoglobin, as a chronic marker for hyperglycemia, wassteadily increased up to 16 weeks, and sustained at the highlevels of 7% and 11% in TgRIP-SREBP-2 hemi and homo
mice, respectively (Fig. 2C). Thus, these diabetic signs andsymptoms depended upon the expression of the SREBP-2
transgene. Intravenous glucose tolerance tests on TgRIPSREBP-2 hemi mice showed extended hyperglycemia andmarkedly impaired insulin levels after glucose loading as
compared with wild-type mice (Fig. 3). The diabetes inthe transgenic mice was milder in females than in males.
Plasma lipids in TgRIP-SREBP2 homo were changed
consistent with the pattern seen in insulin-depleted diabetic patients, often referred to as diabetic lipemia, i.e., increased plasma triglycerides and FFAs and no marked
change in cholesterol (Table 2). Plasma leptin levels weredramatically decreased, reflecting emaciation in TgRIPSREBP-2 homo mice (control: 14.5 6 4.0 ng/ml vs. TgBP2
homo 1.5 6 0.5 ng/ml, n 5 5).
Characterization of pancreatic islets from
TgRIP-SREBP-2 micePancreatic islets from TgRIP-SREBP-2 hemi and TgRIPSREBP-2 homo mice were isolated and morphologicallycompared with islets from wild-type control animals (Fig. 4).

TABLE 2. Plasma parameters in 12 week-old miceMale

TC (mg/dl)
TG (mg/dl)
FFA (mEq/l)

FemaleWTTg Hemi

Tg Homo

WT

Tg Hemi

Tg Homo87.9 6 6.8
73.5 6 13.1
0.53 6 0.04

87.8 6 6.8
82.6 6 8.2
0.54 6 0.11

87.5 6 19.6
129.3 6 54.7
0.97 6 0.28

67.2 6 2.5
39.9 6 1.8
0.49 6 0.06

72.8 6 2.2
38.0 6 2.7
0.60 6 0.05

61.6 6 6.5
85.0 6 15.4
0.96 6 0.18a

TG, triglyceride; TC, total cholesterol; The blood of TgRIP-SREBP-2 mice of high-expression line (line A, n 5 5 for each group) was drawn infed condition. Data represent means 6 SEM.
a
P , 0.05, compared with WT.

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hemizygous mice (hereafter referred to as TgRIP-SREBP-2
hemi) were used to produce mice homozygous for thetransgene (hereafter referred to as TgRIP-SREBP-2 homo)for further studies. Growth curve was slightly retarded in
TgRIP-SREBP-2 homo mice as compared with wild-type
control littermates (Fig. 1C). Gross appearance of TgRIPSREBP-2 homo mice was characterized by a thinner bodywith a distended flank (Fig. 1D, upper panel), which wascaused by enlarged urine-filled bladder as was apparent
upon anatomical inspection (Fig. 1D, lower panel). Adipose tissue mass was markedly diminished, explaining the
body weight loss, whereas kidneys were enlarged (Table 1).
Body length was not changed. TgRIP-SREBP-2 homo mice
exhibited increased food intake, water intake, and urine
volume compared with controls (Fig. 1E, F).

The number and size of islets from the transgenic mice
were both decreased (Fig. 4A, B). Islets from TgRIP-SREBP-2
mice did not display the typical rounded shape of a normal
islet, and the color was changed from dark to pale brown,
presumably due to reduced cellularity in TgRIP-SREBP-2
islets, which was more prominent in homozygote comparedwith hemizygote mice (Fig. 4B). Histological section demonstrated that smaller and deformed islets of TgRIPSREBP-2 mice were located close to connective tissues
containing pancreatic ducts (Fig. 4C). Immunologicalstaining showed that insulin immunoreactivity was diminished and scattered, whereas peripheral staining of glucagon observed in a normal islet was altered to a scatteredpattern (Fig. 4D). These changes in the pattern of a- andb-cells were more severe in homozygotes than in hemizygotes. These data indicate that islets of TgRIP-SREBP-2
mice were structurally abnormal and had less b-cells andinsulin content.
Impaired insulin secretion from TgRIP-SREBP-2 islets
Isolated islets were tested for insulin secretion (Fig. 5).
Basal (low glucose), glucose-stimulated, and KSIS were severely impaired in both TgRIP-SREBP-2 homo and TgRIPSREBP-2 hemi mice when determined on the basis of islet

number (Fig. 5A). Other insulin secretagogues, such as
GLP-1 and glibenclamide, also failed to enhance insulin
secretion in TgRIP-SREBP-2 hemi islets (Fig. 5B). DNA
and insulin content per islet were slightly and severely
suppressed, respectively. Thus, when normalized to insulin content, impaired insulin secretion was obscured in
TgRIP-SREBP-2 islets (Fig. 5C, D). Both ATP and ADP contents were also decreased (Fig. 5E).
To estimate the consequences of nuclear SREBP-2activated lipid synthesis, lipid content in islets was measured (Fig. 6). TgRIP-SREBP-2 hemi islets from line A
exhibited significant increases in cholesteryl ester (Fig. 6A),
whereas triglyceride content was not changed (Fig. 6B). It
was in good contrast to TgRIP-SREBP-1c islets that exhibited
increases in triglycerides but not in cholesterol (Fig. 6B)(10). Hemizygous mice from the low expressor, line B,
showed no significant increases in islet cholesterol or triglyceride content. Gene expression of the islets was examined.
Cholesterol biosynthetic genes, HMG-CoA synthase and reductase, and LDL receptor are major targets of SREBP-2
and were consistently elevated in TgRIP-SREBP-2 homo
islets (Fig. 7A). TgRIP-SREBP-2 hemi islets also exhibitedan increasing trend in expression of SREBP-2 target genes,
with the exception of HMG-CoA synthase. As shown in
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Fig. 4. Isolated islet number and morphology of pancreas sections from TgRIP-SREBP-2 mice. A: Pancreatic islets were isolated from male mice of line A
homo (homo), line A hemi (hemi), and WT (n 5
8–10) age 16 to 24 weeks and counted manually.
**P , 0.01, compared with WT. B: Representative isolated islets were photographed through stereomicroscopy. C: Representative pancreas sections of line A
homo, line A hemi, and WT were stained with hematoxylin and eosin for histological analysis. Original
magnification, 340 (top panels); 3200 (bottom panels). D: Representative islets from pancreas sectionsof line A homo (homo), line A hemi (hemi), andWT stained with immunofluorescent antibodies forinsulin (green) and glucagon (red) as described in
the Methods section. Original magnification, 3200.

Fig. 7B, SREBP-2 overexpression also increased FA synthasegene expression. Endogenous SREBP-1 and -2 genes were
downregulated. Liver X receptor (LXR)-a, but not -b wasincreased; however, ABCA-1 and ABCG-1, targets of LXRs,
were decreased in TgRIP-SREBP-2 homo islets. These results suggest that unregulated activation of cholesterogenic
genes caused by SREBP-2 overexpression in islets of TgRIPSREBP-2 was still sustained despite over-accumulation ofcholesterol. b-cell-specific transcription factors PDX1 andBETA2/NeuroD were suppressed, whereas neurogenin 3(ngn3) was upregulated (Fig. 8A). PDX1 protein was also
decreased in SREBP-2 overexpression (Fig. 8B). Unlike
TgRIP-SREBP-1c islets and SREBP-1 overexpressing livers(9, 10), IRS-2 was not decreased in TgRIP-SREBP-2 islets.
Granuphilin, a recently identified SREBP-1 target gene involved in insulin secretion impairment (11), was not significantly changed in TgRIP-SREBP-2 islets. Suppression of
PDX1 and BETA2 could explain deformed shape and altered components of insulin and glucagon-staining cells
in pancreatic islets of aged TgRIP-SREBP-2 mice. These abnormal structures of islets were already observed in TgRIPSREBP-2 mice by the age of 2 weeks (Fig. 8B), indicatingthat impairment of b-cell differentiation was a primary
event, and not secondary to the emergence of diabetes in
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TgRIP-SREBP-2 mice. Insulin mRNA was also decreased in
TgRIP-SREBP-2 hemi islets from line A (Fig. 8C). Downregulation of insulin expression could be due at least partially to inhibition of transcription by overexpression of
nuclear SREBP-2, because the insulin promoter was downregulated by overexpression of SREBP-2 in a luciferase reporter assay in MIN6 cells (Fig. 8E).

DISCUSSION
Our current studies clearly demonstrated that chronic
activation of SREBP-2 in b-cells caused severe diabetes withinsulin depletion. The insulin depletion in vivo was due toboth reduction of b-cell mass and impaired insulin secretion per islet as observed in high expressor (line A) andlow expressor (line B), respectively. Comparing the severityof diabetes among homo and hemi line A and line B mice,decreased b-cell mass is probably the major cause of diabetes
in line A. Overall, the severity of the phenotype, including
hyperglycemia, insulin secretion defect, and islet mass loss,
were dependent on the transgene expression level.
The islets and b-cells from TgRIP-SREBP-2 mice wereabnormal in many different ways. Islet mass reduction

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Fig. 5. Insulin content and ATP/ADP levels in islets
from TgRIP-SREBP-2 mice. A: Insulin secretion fromisolated islets of line A homo (TgBP2 homo), line A
hemi (TgBP2 hemi), and WT. Ten islets of similar size
from each group (6–10 batches) were incubated in
0.5% BSA KRBH buffer containing 2.8 mM glucose,
20 mM glucose, or 2.8 mM glucose with 30 mM KCl,and insulin secretion per islet was measured. B:
Stimulation of inslin secretion by GLP-1 (100 nM)
or glibenclamide (0.3 mM) from isolated islet of line
A hemi (TgBP-2 hemi) and WT. C: DNA content per
islet from line A homo (homo), line A hemi (hemi),and WT (7–11 batches). D: Insulin content per islet
from line A homo (homo), line A hemi (hemi), andWT (8–12 batches). E: ATP and ADP levels per islet
from line A homo (TgBP2 homo), line A hemi
(TgBP2 hemi), and WT (8–12 batches). *P , 0.05
and **P , 0.01, compared with WT of each group.

was characterized by decreased islet number, decreased
b-cell number per islet, and decreased insulin content
per islet. The smaller and deformed islets were connected
to or located close to connective tissues surrounding
pancreatic ducts. These changes in islet construction weresimilar to those noted in PDX1 and IRS-1 double heterozygote knockout mice, or E2F1 knockout mice (24–26).
Expression of PDX1 and BETA2 was suppressed in isletsof TgRIP-SREBP-2. Because both transcription factors are
intimately involved in islet genesis, especially b-cell genesis(27–29), suppression of these factors could explain theb-cell mass reduction that was observed even at an early
age in these mice. Mice with heterozygous PDX1 gene disruption have been reported to exhibit enhanced b-cell
apoptosis (30), but apoptosis of b-cells was barely observedin both TgRIP-SREBP-2 and control islets assessed by
TUNEL staining (data not shown). These data suggest thatb-cell reduction was due mainly to impaired b-cell genesis.
Our preliminary data indicate that repression of PDX1
and BETA2, as well as the insulin gene, by SREBP-2 could
be mediated by inhibiting promoters of these genes

Fig. 7. Expression of cholesterol-related genes in isolated islets
from TgRIP-SREBP-2 mice. A: mRNA levels of HMG-CoA synthase,
HMG-CoA reductase, and LDL receptor (LDLR) in islets from line
A homo (TgBP2 homo), line A hemi (TgBP2 hemi), and WT. B:
mRNA levels of FA synthase (FAS), endogenous SREBPs, liver X receptor (LXR)-b and -a, ABCA-1, ABCG-1, and uncoupling protein
2 (UCP-2) in islets from line A homo (TgBP2 homo), line A hemi
(TgBP2 hemi), and WT. All results were normalized to mRNA levelsof cyclophilin. Experiments were performed in triplicate wells on
sets of islets from three to six mice per replicate. *P , 0.05 and**P , 0.01, compared with WT of each group.

(Fig. 8D, E). The precise molecular mechanism is currently under investigation.
TgRIP-SREBP-2 mice accumulate only cholesteryl esters
and not triglycerides in the islets, contrasting with islets
from TgRIP-SREBP-1c mice, which exhibit accumulationof triglycerides but not cholesteryl esters. Consistently,
TgRIP-SREBP-1c islets induced genes for FA synthesis
and not genes for cholesterol synthesis, whereas elevationof gene expression of cholesterogenic genes and LDL receptor gene were prominent in TgRIP-SREBP-2 mice. In
addition to this distinction in lipid synthesis, the expression pattern of genes known to affect b-cell function was
different between mice expressing islet-specific SREBP-1c
and those expressing SREBP-2. IRS-2, reported to be important for growth and survival of b-cells via insulin signaling (31–33), was decreased in islets of TgRIP-SREBP-1c
mice, partially contributing to the decreased b-cell mass,
especially when mice were fed a high-fat diet. In contrast,
IRS-2 expression was sustained in TgRIP-SREBP-2 b-cells.
Similarly, UCP-2, an important contributor to lipotoxicity(34–37) was markedly upregulated in TgRIP-SREBP-1c islets
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Fig. 6. Lipid content in isolated islets from TgRIP-SREBP-2 mice.
A: Cholesteryl ester and free cholesterol of 100 islets isolated fromhigh expression line hemi (TgBP2 Line A hemi), low expression
line hemi (TgBP2 Line B hemi), and WT (6–8 batches) were extracted and measured as described in the Methods section. B: Triglycerides of 100 islets isolated from high-expression line A hemi(Line A hemi), low-expression line B hemi (Line B hemi), WT, andb-cell-specific nuclear SREBP-1c transgenic mice (TgRIP-SREBP-1c)were extracted and measured as described in the Methods section(5 batches for each group). *P , 0.05 and **P , 0.01, comparedwith WT of each group.

(10), whereas it was increased only slightly in TgRIPSREBP-2 islets. These data indicate that the mechanism
for decreased b-cell mass was not identical between
SREBP-2 and SREBP-1c transgenic mice. Downregulationof IRS-2 and upregulation of UCP-2 were observed in islets
of diet-induced obese mice and isolated islets treated withsaturated FAs. This is consistent with the fact that TgRIPSREBP-1c mice required consumption of a high-fat dietto induce diabetes. Thus, IRS-2 and UCP-2 are FA-related
mediators of b-cell lipotoxicity, but are not involved in
the development of diabetes caused by islet SREBP-2 overexpression. b-cell failure and diabetes were more severe in
TgRIP-SREBP-2 than in TgRIP-SREBP-1c mice, althougha precise comparison of nuclear protein level or stoichiometric estimation has not been performed. It is currently
unknown whether this is due to potential toxicity of cholesterol accumulation or whether there are other genes specific to SREBP-2 and not SREBP-1 that cause b-cell failure(such as PDX1 and BETA2), or both.
One important aspect of this study is the potential effectof disturbed cholesterol metabolism on b-cell function. Accumulation of cholesterol could be a cellular stress or
could be toxic, and lead to b-cell failure through a different mechanism than accumulation of triglycerides as wasobserved in TgRIP-SREBP-1c and other lipotoxic models(38). Accumulation of cholesterol could also contribute
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to loss of b-cell mass. It has been recently reported thattreatment with a high dose of statins, inhibitors of HMGCoA reductase, caused disturbed insulin secretion in ab-cell line (39), indicating that disturbed cholesterol metabolism could affect insulin secretion. Inhibition of cholesterol synthesis by HMG-CoA reductase inhibitorscaused initial intracellular cholesterol depletion, but secondarily, adaptic SREBP-2 activation, as was induced invivo in our current study. Our data imply that in addition
to well-established FAs, cholesterol could be added to thelist of lipotoxic agents and that SREBP-2, a key transcription factor for cholesterol synthesis, could be a direct
and/or indirect mediator of this new type of lipotoxicity.
A recent paper reported, from studies on ABCA-1-null
mice, that ABCA-1, a cellular cholesterol transporter, wasshown to regulate cholesterol homeostasis and insulin secretion in pancreatic b-cells (40, 41). Their data and ourcurrent data consistently demonstrate that alterations ofislet cholesterol levels through cholesterol efflux and cholesterol synthesis, respectively, could contribute to islet dysfunction and loss of insulin secretion. In addition, a direct
link between elevated serum cholesterol and reduced insulin secretion has been reported (42). Using a combination of apolipoprotein E knockout and ob/ob mice, they
showed a correlation between islet cholesterol content
and impaired GSIS. The impaired GSIS was restored by

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Fig. 8. b-Cell-specific factors and insulin in isolated
islets from TgRIP-SREBP-2 mice. A: mRNA levels of
PDX1, BETA2/NeuroD, neurogenin3 (ngn3), and insulin receptor substrate 2 (IRS-2) in isolated islets
from line A homo (TgBP2 homo), line A hemi
(TgBP2 hemi), and WT mice, age 16 to 24 weeks,
by real-time PCR. All results were normalized tomRNA levels of cyclophilin. Experiments were performed in triplicate wells on sets of islets from three
to six mice per replicate. *P , 0.05 and **P , 0.01,
compared with WT of each group. B: PDX1 protein in
islets of TgRIP-SREBP-2 (line A hemi) mice with
Lamin A/C as a loading control as estimated by immunoblot analysis. C: Formation of islets during differentiation of b-cell in TgRIP-SREBP-2 mice. Representative
islets from pancreas sections of line A hemi (TgBP2hemi) and WT at the age of 2 weeks stained with immunofluorescent antibodies for insulin (green) andglucagon (red) as described in the Methods section.
Original magnification, 3200. D: Insulin gene expression and its quantification in isolated islets from highexpression line hemi (Line A hemi), low-expression
line hemi (Line B hemi), and WT as estimated by
Northern blot analysis. E: Activation of the insulin
gene promoter by SREBP-2 in MIN6 cells. MIN6 cells
were transfected with an RIP-luciferase reporter plasmid (Insulin-luc) or an SRE-luciferase reporter plasmid (SRE-luc), and 0, 10, 50, 100 ng of expression
vector for nuclear SREBP-2 (SREBP-2) for 48 h. The
cells were subjected to reporter assays for measurement of firefly luciferase activity. Experiments were
performed in triplicate. *P , 0.05, compared with control (SREBP-2 expression vector 5 0 ng) for each group.

cholesterol depletion with TbCD or statin. We also observed increased cholesterol content in islets of db/db
mice (data not shown). In contrast to the pathological process caused by elevated plasma cholesterol level, our model
also implies that endogenous cholesterol accumulation
could lead to b-cell dysfunction.
Collectively, current data indicate that chronic activation of SREBP-2 impairs b-cell mass and function throughits own transcription effect and cholesterol accumulation.
Elucidation of the clinical relevance of this artificial system
to human diabetes requires precise estimation of nuclear
SREBP-2 protein in b-cells from patients with diabetes.
Further studies are needed to clarify the details and theprecise mechanisms of a link between cholesterol metabolism and insulin secretion.

REFERENCES
1. Kahn, S. E. 2003. The relative contributions of insulin resistanceand beta-cell dysfunction to the pathophysiology of Type 2 diabetes.
Diabetologia. 46: 3–19.
2. Unger, R. H. 1995. Lipotoxicity in the pathogenesis of obesitydependent NIDDM. Genetic and clinical implications. Diabetes.
44: 863–870.
3. Shimano, H., N. Yahagi, M. Amemiya-Kudo, A. H. Hasty, J. Osuga, Y.
Tamura, F. Shionoiri, Y. Iizuka, K. Ohashi, K. Harada, et al. 1999.
Sterol regulatory element-binding protein-1 as a key transcription
factor for nutritional induction of lipogenic enzyme genes. J. Biol.
Chem. 274: 35832–35839.
4. Shimano, H. 2002. Sterol regulatory element-binding protein family
as global regulators of lipid synthetic genes in energy metabolism.
Vitam. Horm. 65: 167–194.
5. Horton, J. D., Y. Bashmakov, I. Shimomura, and H. Shimano.
1998. Regulation of sterol regulatory element binding proteins inlivers of fasted and refed mice. Proc. Natl. Acad. Sci. USA. 95:
5987–5992.
6. Yahagi, N., H. Shimano, A. H. Hasty, M. Amemiya-Kudo, H. Okazaki,
Y. Tamura, Y. Iizuka, F. Shionoiri, K. Ohashi, J. Osuga, et al. 1999. A
crucial role of sterol regulatory element-binding protein-1 in theregulation of lipogenic gene expression by polyunsaturated fatty
acids. J. Biol. Chem. 274: 35840–35844.
7. Kato, T., H. Shimano, T. Yamamoto, M. Ishikawa, S. Kumadaki, T.
Matsuzaka, Y. Nakagawa, N. Yahagi, M. Nakakuki, A. H. Hasty, et al.
2008. Palmitate impairs and eicosapentaenoate restores insulin
secretion through regulation of SREBP-1c in pancreatic islets. Diabetes. 57: 2382–2392.
8. Shimano, H., M. Amemiya-Kudo, A. Takahashi, T. Kato, M. Ishikawa,and N. Yamada. 2007. Sterol regulatory element-binding protein-1c
and pancreatic beta-cell dysfunction. Diabetes Obes. Metab. 9 (Suppl.):133–139.
9. Ide, T., H. Shimano, N. Yahagi, T. Matsuzaka, M. Nakakuki, T.
Yamamoto, Y. Nakagawa, A. Takahashi, H. Suzuki, H. Sone, et al.
2004. SREBPs suppress IRS-2-mediated insulin signalling in theliver. Nat. Cell Biol. 6: 351–357.
10. Takahashi, A., K. Motomura, T. Kato, T. Yoshikawa, Y. Nakagawa, N.
Yahagi, H. Sone, H. Suzuki, H. Toyoshima, N. Yamada, et al. 2005.
Transgenic mice overexpressing nuclear SREBP-1c in pancreaticbeta-cells. Diabetes. 54: 492–499.
11. Kato, T., H. Shimano, T. Yamamoto, T. Yokoo, Y. Endo, M. Ishikawa,T. Matsuzaka, Y. Nakagawa, S. Kumadaki, N. Yahagi, et al. 2006.
Granuphilin is activated by SREBP-1c and involved in impaired insulin secretion in diabetic mice. Cell Metab. 4: 143–154.
12. Horton, J. D., I. Shimomura, M. S. Brown, R. E. Hammer, J. L.
Goldstein, and H. Shimano. 1998. Activation of cholesterol synthesis in preference to fatty acid synthesis in liver and adipose tissue

Diabetes in b-cell SREBP-2 transgenic mice

2533

Downloaded from www.jlr.org at Univ of Tokyo Medical Library on November 18, 2008

The authors are grateful to Dr. Alyssa H. Hasty for careful reading of this manuscript.

of transgenic mice overproducing sterol regulatory elementbinding protein-2. J. Clin. Invest. 101: 2331–2339.
13. Brown, M. S., and J. L. Goldstein. 1997. The SREBP pathway: regulation of cholesterol metabolism by proteolysis of a membranebound transcription factor. Cell. 89: 331–340.
14. Brown, M. S., and J. L. Goldstein. 1999. A proteolytic pathway that
controls the cholesterol content of membranes, cells, and blood.
Proc. Natl. Acad. Sci. USA. 96: 11041–11048.
15. Brown, M. S., J. Ye, R. B. Rawson, and J. L. Goldstein. 2000. Regulated intramembrane proteolysis: a control mechanism conserved
from bacteria to humans. Cell. 100: 391–398.
16. Hua, X., C. Yokoyama, J. Wu, M. R. Briggs, M. S. Brown, J. L.
Goldstein, and X. Wang. 1993. SREBP-2, a second basic-helixloop-helix-leucine zipper protein that stimulates transcription bybinding to a sterol regulatory element. Proc. Natl. Acad. Sci. USA.
90: 11603–11607.
17. Shimano, H., J. D. Horton, R. E. Hammer, I. Shimomura, M. S.
Brown, and J. L. Goldstein. 1996. Overproduction of cholesteroland fatty acids causes massive liver enlargement in transgenic miceexpressing truncated SREBP-1a. J. Clin. Invest. 98: 1575–1584.
18. Scharp, D. W., C. B. Kemp, M. J. Knight, W. F. Ballinger, and P. E.
Lacy. 1973. The use of ficoll in the preparation of viable islets oflangerhans from the rat pancreas. Transplantation. 16: 686–689.
19. Detimary, P., G. Van den Berghe, and J. C. Henquin. 1996. Concentration dependence and time course of the effects of glucose on
adenine and guanine nucleotides in mouse pancreatic islets. J. Biol.
Chem. 271: 20559–20565.
20. Folch, J., M. Lees, and G. H. Sloane Stanley. 1957. A simple method
for the isolation and purification of total lipides from animal tissues.
J. Biol. Chem. 226: 497–509.
21. Heider, J. G., and R. L. Boyett. 1978. The picomole determination of free and total cholesterol in cells in culture. J. Lipid Res.
19: 514–518.
22. Amemiya-Kudo, M., J. Oka, T. Ide, T. Matsuzaka, H. Sone, T.
Yoshikawa, N. Yahagi, S. Ishibashi, J. Osuga, N. Yamada, et al.
2005. Sterol regulatory element-binding proteins activate insulin
gene promoter directly and indirectly through synergy with
BETA2/E47. J. Biol. Chem. 280: 34577–34589.
23. Hua, X., A. Nohturfft, J. L. Goldstein, and M. S. Brown. 1996. Sterol
resistance in CHO cells traced to point mutation in SREBP cleavageactivating protein. Cell. 87: 415–426.
24. Dutta, S., S. Bonner-Weir, M. Montminy, and C. Wright. 1998. Regulatory factor linked to late-onset diabetes? Nature. 392: 560.
25. Kulkarni, R. N., U. S. Jhala, J. N. Winnay, S. Krajewski, M. Montminy,
and C. R. Kahn. 2004. PDX-1 haploinsufficiency limits the compensatory islet hyperplasia that occurs in response to insulin resistance.
J. Clin. Invest. 114: 828–836.
26. Fajas, L., J. S. Annicotte, S. Miard, D. Sarruf, M. Watanabe, and J.
Auwerx. 2004. Impaired pancreatic growth, beta cell mass, and beta
cell function in E2F1 (2/2)mice. J. Clin. Invest. 113: 1288–1295.
27. Ahlgren, U., J. Jonsson, and H. Edlund. 1996. The morphogenesisof the pancreatic mesenchyme is uncoupled from that of the pancreatic epithelium in IPF1/PDX1-deficient mice. Development. 122:
1409–1416.
28. Offield, M. F., T. L. Jetton, P. A. Labosky, M. Ray, R. W. Stein, M. A.
Magnuson, B. L. Hogan, and C. V. Wright. 1996. PDX-1 is requiredfor pancreatic outgrowth and differentiation of the rostral duodenum. Development. 122: 983–995.
29. Naya, F. J., H. P. Huang, Y. Qiu, H. Mutoh, F. J. DeMayo, A. B. Leiter,and M. J. Tsai. 1997. Diabetes, defective pancreatic morphogenesis,
and abnormal enteroendocrine differentiation in BETA2/neuroDdeficient mice. Genes Dev. 11: 2323–2334.
30. Johnson, J. D., N. T. Ahmed, D. S. Luciani, Z. Han, H. Tran, J.
Fujita, S. Misler, H. Edlund, and K. S. Polonsky. 2003. Increased islet
apoptosis in Pdx11/2 mice. J. Clin. Invest. 111: 1147–1160.
31. Withers, D. J., J. S. Gutierrez, H. Towery, D. J. Burks, J. M. Ren, S.
Previs, Y. Zhang, D. Bernal, S. Pons, G. I. Shulman, et al. 1998.
Disruption of IRS-2 causes type 2 diabetes in mice. Nature. 391:
900–904.
32. Withers, D. J., D. J. Burks, H. H. Towery, S. L. Altamuro, C. L. Flint,
and M. F. White. 1999. Irs-2 coordinates Igf-1 receptor-mediatedbeta-cell development and peripheral insulin signalling. Nat. Genet.
23: 32–40.
33. Kushner, J. A., J. Ye, M. Schubert, D. J. Burks, M. A. Dow, C. L. Flint,
S. Dutta, C. V. Wright, M. R. Montminy, and M. F. White. 2002. Pdx1
restores beta cell function in Irs2 knockout mice. J. Clin. Invest. 109:
1193–1201.

34. Zhang, C. Y., G. Baffy, P. Perret, S. Krauss, O. Peroni, D. Grujic, T.
Hagen, A. J. Vidal-Puig, O. Boss, Y. B. Kim, et al. 2001. Uncoupling
protein-2 negatively regulates insulin secretion and is a major link
between obesity, beta cell dysfunction, and type 2 diabetes. Cell. 105:745–755.
35. Chan, C. B., D. De Leo, J. W. Joseph, T. S. McQuaid, X. F. Ha, F. Xu,
R. G. Tsushima, P. S. Pennefather, A. M. Salapatek, and M. B.
Wheeler. 2001. Increased uncoupling protein-2 levels in beta-cells
are associated with impaired glucose-stimulated insulin secretion:mechanism of action. Diabetes. 50: 1302–1310.
36. Lameloise, N., P. Muzzin, M. Prentki, and F. AssimacopoulosJeannet. 2001. Uncoupling protein 2: a possible link between
fatty acid excess and impaired glucose-induced insulin secretion?
Diabetes. 50: 803–809.
37. Joseph, J. W., V. Koshkin, C. Y. Zhang, J. Wang, B. B. Lowell, C. B.
Chan, and M. B. Wheeler. 2002. Uncoupling protein 2 knockout
mice have enhanced insulin secretory capacity after a high-fat diet.
Diabetes. 51: 3211–3219.
38. Lee, Y., H. Hirose, M. Ohneda, J. H. Johnson, J. D. McGarry, andR. H. Unger. 1994. Beta-cell lipotoxicity in the pathogenesis of

non-insulin-dependent diabetes mellitus of obese rats: impairment
in adipocyte-beta-cell relationships. Proc. Natl. Acad. Sci. USA. 91:
10878–10882.
39. Ishikawa, M., F. Okajima, N. Inoue, K. Motomura, T. Kato, A.
Takahashi, S. Oikawa, N. Yamada, and H. Shimano. 2006. Distinct
effects of pravastatin, atorvastatin, and simvastatin on insulin secretion from a beta-cell line, MIN6 cells. J. Atheroscler. Thromb. 13:
329–335.
40. Brunham, L. R., J. K. Kruit, T. D. Pape, J. M. Timmins, A. Q. Reuwer,
Z. Vasanji, B. J. Marsh, B. Rodrigues, J. D. Johnson, J. S. Parks, et al.
2007. Beta-cell ABCA1 influences insulin secretion, glucose homeostasis and response to thiazolidinedione treatment. Nat. Med. 13:
340–347.
41. Brunham, L. R., J. K. Kruit, C. B. Verchere, and M. R. Hayden. 2008.
Cholesterol in islet dysfunction and type 2 diabetes. J. Clin. Invest.
118: 403–408.
42. Hao, M., W. S. Head, S. C. Gunawardana, A. H. Hasty, and D. W.
Piston. 2007. Direct effect of cholesterol on insulin secretion: anovel mechanism for pancreatic beta-cell dysfunction. Diabetes. 56:
2328–2338.

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