Endoplasmic ReticulumStress Links

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Endoplasmic Reticulum Stress Links

Obesity, Insulin Action, and

Type 2 DiabetesUmut Ozcan,1* Qiong Cao,1* Erkan Yilmaz,1 Ann-Hwee Lee,2

Neal N. Iwakoshi,2 Esra Ozdelen,1 Gurol Tuncman,1 Cem Gorgun,1

Laurie H. Glimcher,2,3 Gokhan S. Hotamisligil1.

Obesity contributes to the development of type 2 diabetes, but theunderlying mechanisms are poorly understood. Using cell culture and mousemodels, we show that obesity causes endoplasmic reticulum (ER) stress. Thisstress in turn leads to suppression of insulin receptor signaling throughhyperactivation of c-Jun N-terminal kinase (JNK) and subsequent serinephosphorylation of insulin receptor substrate–1 (IRS-1). Mice deficient in X-box–binding protein–1 (XBP-1), a transcription factor that modulates the ERstress response, develop insulin resistance. These findings demonstrate thatER stress is a central feature of peripheral insulin resistance and type 2diabetes at the molecular, cellular, and organismal levels. Pharmacologicmanipulation of this pathway may offer novel opportunities for treating thesecommon diseases.

The cluster of pathologies known as meta-

bolic syndrome, including obesity, insulin

resistance, type 2 diabetes, and cardiovascu-

lar disease, has become one of the most

serious threats to human health. The dramat-

ic increase in the incidence of obesity in

most parts of the world has contributed to the

emergence of this disease cluster, particular-

ly insulin resistance and type 2 diabetes.

However, understanding the molecular me-

chanisms underlying these individual dis-

orders and their links with each other has been challenging.

Over the past decade, it has become clear

that obesity is associated with the activation of

cellular stress signaling and inflammatory

pathways (1–4). However, the origin of this

stress is not known. A key player in the

cellular stress response is the ER, a membra-

nous network that functions in the synthesis

and processing of secretory and membrane

proteins. Certain pathological stress condi-

tions disrupt ER homeostasis and lead to

accumulation of unfolded or misfolded pro-

teins in the ER lumen (5–7 ). To cope with this

stress, cells activate a signal transductionsystem linking the ER lumen with the

cytoplasm and nucleus, called the unfolded

protein response (UPR) (5–7 ). Among the

conditions that trigger ER stress are glucose

or nutrient deprivation, viral infections,

lipids, increased synthesis of secretory pro-

teins, and expression of mutant or misfolded

proteins (8–10).

Several of these conditions occur in

obesity. Specifically, obesity increases the

demand on the synthetic machinery of the

cells in many secretory organ systems.

Obesity is also associated with mechanical

stress, excess lipid accumulation, abnormal-

ities in intracellular energy fluxes, and nutrient availability. In light of these obser-

vations, we postulated that obesity may be a

chronic stimulus for ER stress in peripheral

tissues and that perhaps ER stress is a core

mechanism involved in triggering insulin

resistance and type 2 diabetes.

Induction of ER stress in obesity. To

examine whether ER stress is increased in

obesity, we investigated the expression

patterns of several molecular indicators of

ER stress in dietary [high-fat diet (HFD)–

induced] and genetic (ob/ob) models of

murine obesity. The pancreatic ER kinase

or P KR-like kinase ( PERK) is an ER transmembrane protein kinase that phospho-

rylates the " subunit of translation initiation

factor 2 (eIF2") in response to ER stress.

The phosphorylation status of PERK and

eIF2" is therefore a key indicator of the

presence of ER stress (11–13). We deter-

mined the phosphorylation status of PERK

(Thr 980) and eIF2" (Ser 51) using phospho-

specific antibodies. These experiments dem-

onstr ated increased P ERK and eI F2"

phosphorylation in liver extracts of obese

mice compared with lean controls (Fig. 1

and B). The activity of c-Jun N-termi

kinase (JNK) is also increased by ER str

(14). Consistent with earlier observatio

(3), total JNK activity, indicated by c-J

phosphorylation, was also dramatically e

vated in the obese mice (Fig. 1, A and B)

The 78-kD glucose-regulated/binding i

munoglobulin protein (GRP78) is an chaperone whose expression is increa

upon ER stress (7 ). The GRP78 mRN

levels were elevated in the liver tissue

obese mice compared with matched le

controls (Fig. 1, C and D). Because GRP

expression is responsive to glucose (15),

tested whether this up-regulation mi

simply be due to increasing glucose leve

Treatment of cultured rat Fao liver cells w

high levels of glucose resulted in reduc

GRP78 expression (fig. S1A). Similar

GRP78 levels were not increased in a mou

model of hyperglycemia (fig. S1B), wh

indicates that regulation in obesity is unlikly to be related to glycemia alone.

We also tested adipose and mus

tissues, important sites for metabolic h

RESEARCH ARTICLE

1Department of Genetics and Complex Diseases,2Department of Immunology and Infectious Diseases,Harvard School of Public Health, 3Department of Medicine, Harvard Medical School, Boston, MA02115, USA.

*These authors contributed equally to this work..To whom correspondence should be addressed.E-mail: [email protected]

p-eIF2α p-eI

GRP78 GR

28s

18s

eIF2α eIF2

p-PERK p-P

PERK PER

p-c-Jun p-c-

JNK JNK

RD HFD B

C RD HFD Lean ob/obD

28s

18s

A Lean ob/ob

Fig. 1. Increased ER stress in obesity. Diet(HFD-induced) and genetic (ob/ob) modelsmouse obesity were used to examine markof ER stress in liver tissue compared with aand sex-matched lean controls. (A) ER strmarkers including eIF2" phosphorylation

eIF2"), PERK phosphorylation (p-PERK), a JNK activity (p-c-Jun) were examined in liver samples of the male mice (C57BL/6) twere kept either on regular diet (RD) or higfat diet (HFD) for 16 weeks. (B) Examinationthe same ER stress markers in the liversmale ob/ob and wild-type (WT) lean micethe age of 12 to 14 weeks. (C) Northern banalysis of GRP78 mRNA in the livers of mwith dietary-induced obesity and lean ctrols. (D) Northern blot analysis of GRPmRNA in the livers of ob/ob and WT lemice. Ethidium bromide staining is shown acontrol for loading and integrity of RNA.

www.sciencemag.org SCIENCE VOL 306 15 OCTOBER 2004



meostasis, for indications of ER stress in

obesity. As in liver, PERK phosphorylation,

JNK activity, and GRP78 expression were all

significantly increased in adipose tissue of

obese animals compared with lean controls

(fig. S2, A to C). However, no indication for

ER stress was evident in the muscle tissue of

obese animals (16 ). Taken together, these

results indicate that obesity is associated

with induction of ER stress predominantly

in liver and adipose tissues.

ER stress inhibits insulin action in liver cells. To investigate whether ER stress

interferes with insulin action, we pretreated

Fao liver cells with tunicamycin or thapsi-

gargin, agents commonly used to induce ER

stress. Tunicamycin significantly decreased

insulin-stimulated tyrosine phosphorylation

of insulin receptor substrate 1 (IRS-1) (Fig. 2,

A and B), and it also produced an increase in

the molecular weight of IRS-1 (Fig. 2A). IRS-

1 is a substrate for insulin receptor tyrosine

kinase, and serine phosphorylation of IRS-1,

particularly mediated by JNK, reduces insulin

receptor signaling (3). Pretreatment of Fao

cells with tunicamycin produced a significant

increase in serine phosphorylation of IRS-1

(Fig. 2, A and B). Tunicamycin pretreatment

also suppressed insulin-induced Akt phos-

phorylation, a more distal event in the insulin

receptor signaling pathway (Fig. 2, A and B).

Similar results were also obtained after

treatment with thapsigargin (fig. S3A), which

was independent of alterations in cellular

calcium levels (fig. S3B). Hence, experimen-

tal ER stress inhibits insulin action.

We next examined the role of JNK in

stress–induced IRS-1 serine phosphoryla

and inhibition of insulin-stimulated IR

tyrosine phosphorylation. Inhibition of J

activity with the synthetic inhibi

SP600125 (17 ), reversed the ER str

induced serine phosphorylation of IR

(Fig. 2, C and D). Pretreatment of Fao c

with a highly specific inhibitory pep

derived from the JNK-binding protein,

(18), also completely preserved insulin

ceptor signaling in cells exposed to tunica

cin (Fig. 2, E and F). Similar results w

obtained with the synthetic JNK inhib

SP600125 (16 ). These results indicate that

stress promotes a JNK-dependent serine p

phorylation of IRS-1, which in turn inh

insulin receptor signaling.

IP: IRS-1IB: pY

IP: IRS-1

Thap: - - + +

JNKi: - + - +

Tun: - - +Ins: - + +

JNKi: - - - +Tun: - - + +Ins: - + + +

I R S

- 1 - p

S

200

120

40

A B C D

E F

0 100 200Phospho/Total

Thap: - - + +JNKi: - + - +

JNKi: - - - +Tun: - - + +Ins: - + + +

I R S

- 1 - p

Y

200

120

40

IP: IRS-1IB: IRS-1-pS

IP: IRS-1

Akt-pS

Akt

IP: IR

IP: IRIB: IR

IB: IRS-1

IP: IRS-1

IP: pYIB: IRS-1

IP: pY

GTun: - + + +

Time(hr): 0 1 2 4

IB: IRS-1-pS

IP: IRS-1IB: pY

IP: IRS-1

IB: IRS-1

H IRE-1α +/+ IRE-1α -/- Tun(hr): 0 0 1 2 3 4 0 0 1 2 3 4

Ins: - + + + + + - + + + + +

Tun(hr): 0 0 1 2 3 4Ins: - + + + + +

500

300

100 I R S

- 1 - p

Y

p-c-Jun

IP: IRS-1

IP: IRS-1IB: IRS-1

IB: IRS-1-pS

p-c-Jun

IP: IRS-1

IP: IRS-1IB: IRS-1

I R E - 1

α

+ / +

I R E - 1

α

- / -

IB: IRS-1-pS

IB: IRS-1

IB: pYIB: pY I R

- p Y

A K T - p S

I R S - 1 - p

S

I R S - 1 - p Y

Fig. 2. Induction of ER stress impairs insulin action through JNK-mediated phosphorylation of IRS-1. (A) ER stress was induced in Faoliver cells by a 3-hour treatment with 5 6g/ml tunicamycin (Tun).Cells were subsequently stimulated with insulin (Ins). IRS-1 tyrosine(pY) and Ser307 (pS) phosphorylation, Akt Ser473 (Akt-pS) phospho-rylation, insulin receptor (IR) tyrosine phosphorylation, and their totalprotein levels were examined either with immunoprecipitation (IP)followed by immunoblotting (IB) or by direct immunoblotting. (B)Quantification of IRS-1 (tyrosine and Ser307), Akt (Ser473), and IR(tyrosine) phosphorylation under the experimental conditions describedin (A) with normalization to protein levels for each molecule. (C) Inhibitionof ER stress–induced (300 nM thapsigargin for 4 hours) Ser307 phospho-rylation of IRS-1 by JNK-1 inhibitor, SP600125 (JNKi, 25 6M). (D)Quantification of IRS-1 Ser307 phosphorylation under the conditions

described in (C). (E) Reversal of ER stress–induced inhibition of insustimulated tyrosine phosphorylation (pY) of IRS-1 by a peptide inhibitor. (F) Quantification of insulin-induced IRS-1 tyrosine pphorylation levels described in (E). (G) JNK activity (p-c-Jun), Sephosphorylation of IRS-1, and total IRS-1 levels at indicated tiafter tunicamycin treatment (Tun, 10 6g/ml) in IRE-1"þ/þ and 1"j/j fibroblasts. (H) Insulin-stimulated IRS-1 tyrosine phosprylation and total IRS-1 levels after tunicamycin treatment (Tun, 10ml ) in IRE-1"þ/þ and IRE-1"j/j fibroblasts. Quantification of insinduced IRS-1 tyrosine phosphorylation levels in IRE-1"þ/þ and 1"j/j cells is displayed in the bottom of the panel. All graphs smeans T SEM from at least two independent experiments, and tistical significance (P G 0.005) from the controls is indicated byasterisk (*).

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15 OCTOBER 2004 VOL 306 SCIENCE www.sciencemag.org458



Inositol-requiring kinase–1! (IRE-1! )plays a crucial role in insulin receptor signaling. In the presence of ER stress,

increased phosphorylation of IRE-1" leads

to recruitment of tumor necrosis factor

receptor–associated factor 2 (TRAF2) pro-

tein and activation of JNK (14). To address

whether ER stress–induced insulin resistance

is dependent on intact IRE-1", we measured

JNK activation, IRS-1 serine phosphoryla-

tion, and insulin receptor signaling after

exposure of IRE-1"j/j and wild-type fibro-

blasts to tunicamycin. In the wild-type, but

not IRE-1"j/j cells, induction of ER stress

by tunicamycin resulted in strong activation

of JNK (Fig. 2G). Tunicamycin also stimu-

lated phosphorylation of IRS-1 at the Ser 307

residue in wild-type, but not IRE-1"j/j,

fibroblasts (Fig. 2G). It is noteworthy that

tunicamycin inhibited insulin-stimulated

tyrosine phosphorylation of IRS-1 in the

wild-type cells, whereas no such effect was

detected in the IRE-1"j/j cells (Fig. 2H).

The level of insulin-induced tyrosine phos-

phorylation of IRS-1 was dramatically

higher in IRE-1"j/j cells, despite lower

total IRS-1 protein levels (Fig. 2H). Th

results demonstrate that ER stress–induc

inhibition of insulin action is mediated by

IRE-1" – and JNK–dependent protein kin

cascade.

Manipulation of X-box–binding prote1 (XBP-1) levels alters insulin recepsignaling. The transcription factor XBP-1

a bZIP protein. The spliced or proces

form of XBP-1 (XBP-1s) is a key factor

ER stress through transcriptional regulat

of an array of genes, including molecu

chaperones (19–22). We therefore reason

that modulation of XBP-1s levels in ce

should alter insulin action via its poten

impact on the magnitude of the ER str

responses. To test this possibility, we esta

lished XBP-1 gain- and loss-of-funct

cellular models. First, we established

inducible gene expression system wh

exogenous XBP-1s is expressed only in

absence of tetracycline/doxycycline (F

3A). In parallel, we also studied mo

embryo fibroblasts (MEFs) derived fr

XBP-1j/j mice (Fig. 3B). In fibrobla

without exogenous XBP-1s expression, tucamycin treatment (2 6g/ml) resulted

PERK phosphorylation starting at 30 m

and peaking at 3 to 4 hours, associated wit

mobility shift characteristic of PERK ph

phorylation (Fig. 3C). In these cells, th

was also a rapid and robust activation

JNK in response to ER stress (Fig. 3

When XBP-1s expression was induced, th

was a dramatic reduction in both PER

phosphorylation and JNK activation a

tunicamycin treatment (Fig. 3C). Hen

overexpression of XBP-1s rendered wi

type cells refractory to ER stress. Simi

experiments performed in XBP-1j/j ME

revealed an opposite pattern (Fig. 3D

XBP-1j/j MEFs mounted strong ER str

responses even when treated with a l

dose of tunicamycin (0.5 6g/ml), wh

failed to stimulate significant ER stress

wild-type cells (Fig. 3D). Under these co

ditions, PERK phosphorylation and JN

activation levels in XBP-1j/j MEFs w

significantly higher than those seen in wi

type controls (Fig. 3D), which indicates t

XBP-1j/j cells are prone to ER stress. Th

alterations in the levels of cellular XBP

protein result in alterations in the ER str

responses. Next, we examined whether these dif

ences in the ER stress responses produc

alterations in insulin action as assessed

IRS-1 serine phosphorylation and insul

stimulated IRS-1 tyrosine phosphorylati

Tunicamycin-induced IRS-1 serine phosph

rylation was significantly reduced in fib

blasts exogenously expressing XBP

compared with that of control cells (Fig. 3

On insulin stimulation, the extent of IR

tyrosine phosphorylation was significan

A B

C D

E F

G H

p-PERK

PERK

p-c-Jun

JNK

Tun(2µg/ml): - + + + + + + - + + + + + +

p-PERK

Tun(0.5µg/ml): - + + + + + + - + + + + + +

Time(hr): 0 0.5 1 2 3 4 5 0 0.5 1 2 3 4 5 Time(hr): 0 0.5 1 2 3 4 5 0 0.5 1 2 3 4 5

+Dox -Dox

Tun(2µg/ml): - + + + + + - + + + + +

Time (hr): 0 1 2 3 4 5 0 1 2 3 4 5

+Dox -Dox

XBP-1s

PERK

p-c-Jun

JNK

XBP-1 -/- XBP-1 +/+

XBP-1 -/- XBP-1 +/+

9.4 kb

6.5 kb

Tun(2µg/ml): - + + - + +

Time(hr): 0 4 5 0 4 5

+Dox -Dox

IB: IRS-1-pS

Time(hr):0 4 5

800

400

1200

I R S

- 1 - p

S

Time(hr): 0 0 4 5Tun: - - + +Ins: - + + +

200

400

600

I R S - 1 - p Y

Time(hr): 0 0 4 5Tun: - - + +Ins: - + + +

Tun(0.5µg/ml): - + + - + +

Time(hr): 0 4 5 0 4 5

XBP-1-/- XBP-1+/+

Time(hr):0 4 5

I R S - 1 - p S

1000

2000

3000

I R S - 1 - p

Y

300

200

100

IP: IRS-1

IP: IRS-1

IB: IRS-1-pSIP: IRS-1

IP: IRS-1IB: IRS-1

IB: IRS-1

Fig. 3. Alteration of the ER stress response by manipulation of XBP-1 levels modulates insulinreceptor signaling. ER stress responses in cells overexpressing XBP-1s, XBP-1j/j cells, and theircontrols. (A) Induction of exogenous XBP-1s expression on removal of doxycycline in MEFs. (B)Southern blot analysis of XBP-1j/j MEFs and their WT controls (9.4 kb) and targeted (6.5 kb)alleles. (C) PERK phosphorylation (p-PERK) and JNK activity (p-c-Jun) in cells overexpressing XBP-1s and control cells (–Dox and þDox, respectively) after tunicamycin treatment (Tun, 2 6g/ml).(D) PERK phosphorylation and JNK activity after low-dose tunicamycin treatment (Tun, 0.5 6g/ml)in XBP-1j/j MEFs and their WT controls. (E) IRS-1 Ser307 phosphorylation (pS) after tunicamycintreatment (Tun, 2 6g/ml) in cells overexpressing XBP-1s and control cells (–Dox and þDox,

respectively), detected by using immunoprecipitation (IP) of IRS-1 followed by immunoblotting(IB) with an IRS-1 phosphoserine 307–specific antibody. The graph next to the blots shows thequantification of IRS-1 Ser307 phosphorylation under the conditions described in (E). (F) Insulin-stimulated tyrosine phosphorylation of IRS-1 in cells overexpressing XBP-1s and control cells, withor without tunicamycin treatment (Tun, 2 6g/ml). The ratio of IRS-1 tyrosine phosphorylation tototal IRS-1 level was summarized from independent experiments and presented in the graph. (G)IRS-1 Ser307 phosphorylation after tunicamycin treatment (Tun, 0.5 6g/ml) in XBP-1j/j cells andWT controls was detected as described in (C). The graph next to the blots shows the quantificationof IRS-1 Ser307 phosphorylation under conditions described in (G). (H) Insulin-stimulated tyrosinephosphorylation of IRS-1 in XBP-1j/j and WT control cells with or without tunicamycin treat-ment (Tun, 0.5 6g/ml). The ratio of IRS-1 tyrosine phosphorylation to total IRS-1 level was sum-marized from independent experiments and presented in the graph. All graphs show means T

SEM from at least two independent experiments, and statistical significance from the controls isindicated by * with P G 0.005.

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higher in cells overexpressing XBP-1s, com-

pared with controls (Fig. 3F). In contrast,

IRS-1 serine phosphorylation was strongly

induced in XBP-1j/j MEFs compared with

XBP-1þ/þ controls even at low doses of

tunicamycin treatment (0.5 6g/ml) (Fig. 3G).

After insulin stimulation, the amount of

IRS-1 tyrosine phosphorylation was signif-

icantly decreased in tunicamycin-treated

XBP-1j/j cells compared with tunicamycin-

treated wild-type controls (Fig. 3H). Insulin-

stimulated tyrosine phosphorylation of the

insulin receptor was normal in these cells

(fig. S4).XBP-1+/-- mice show impaired glucose

homeostasis. Complete XBP-1 deficiency

results in embryonic lethality (23) . To

investigate the role of XBP-1 in ER stress,

insulin sensitivity, and systemic glucose

metabolism in vivo, we studied BALB/c-

XBP-1þ/j mice with a null mutation in

one XBP-1 allele. We chose mice on the

BALB/c genetic background, because this

strain exhibits strong resistance to obesity-

induced alterations in systemic glucose

metabolism. Based on our results with cel-

lular systems, we hypothesized that XBP-1

deficiency would predispose mice to thedevelopment of insulin resistance and type

2 diabetes.

We fed XBP-1þ/j mice and their wild-

type littermates a HFD at 3 weeks of age. In

parallel, control mice of both genotypes

were placed on laboratory feed, a regular

diet. The total body weights of both geno-

types were similar with regular diet and until

12 weeks of age when fed HFD. After this

period, the XBP-1þ/janimals fed HFD

exhibited a small, but significant, increase

in body weight (Fig. 4A). Serum levels of

leptin, adiponectin, and triglycerides did not

exhibit any statistically significant differ-

ences between the genotypes measured after

16 weeks of HFD (fig. S5).

Fed HFD, XBP-1þ/j mice developed

continuous and progressive hyperinsulinemia

evident as early as 4 weeks (Fig. 4B). Insulin

levels continued to increase in XBP-1þ/j

mice for the duration of the experiment.

Blood insulin levels in XBP-1þ/þ mice were

significantly lower than those in XBP-1þ/j

littermates (Fig. 4B). C-peptide levels were

also significantly higher in XBP-1þ/j ani-mals than in wild-type controls (Fig. 4C).

Blood glucose levels also began to rise in

the XBP-1þ/j mice fed HFD starting at

8 weeks and remained high until the con-

clusion of the experiment at 20 weeks (Fig.

4D). This pattern was the same in both fasted

(Fig. 4D) and fed (16 ) states. The rise in

blood glucose in the face of hyperinsuline-

mia in the mice fed HFD is a strong indicator

of the development of peripheral insulin

resistance.

To investigate systemic insulin sensitiv-

ity, we performed glucose tolerance tests

(GTT) and insulin tolerance tests (ITT) inXBP-1þ/j mice and XBP-1þ/þ controls.

Exposure to HFD resulted in significant

glucose intolerance in XBP-1þ/j mice.

After 7 weeks of HFD, XBP-1þ/j mice

showed significantly higher glucose levels

on glucose challenge than XBP-1þ/þ mice

(Fig. 4E). This glucose intolerance contin-

u ed t o b e e vi de nt i n X BP -1þ/j mice

compared with wild-type mice after 16

weeks of HFD (Fig. 4F). During ITT, the

hypoglycemic response to insulin was also

significantly lower in XBP-1þ/j mice c

pared with XBP-1þ/þ littermates at 8 w

of HFD (Fig. 4G), and this reduced resp

siveness continued to be evident after

weeks of HFD (Fig. 4H). Examination

islets morphology and function did not

veal significant differences between ge

types (fig. S6). Hence, loss of an XB

allele predisposes mice to diet-indu

peripheral insulin resistance and typ

diabetes.

Increased ER stress and impainsulin signaling in XBP-1+/-- mice.

experiments with cultured cells demstrated an increase in ER stress an

decrease in insulin signaling capacity

XBP-1–deficient cells, as well as reversa

these phenotypes on expression of h

levels of XBP-1s. If this mechanism is

basis of the insulin resistance seen in X

1þ/j mice, these animals should exhibit h

levels of ER stress coupled with impa

insulin receptor signaling. To test this,

first examined PERK phosphorylation

JNK activity in the livers of obese XBP-1

and wild-type mice. These experim

revealed an increase in PERK levels

seemingly an increase in liver PERK ph phorylation in obese XBP-1þ/j mice c

pared with wild-type controls fed H

(Fig. 5A). There was a significant incre

in JNK activity in XBP-1þ/j mice c

pared with wild-type controls (Fig.

Consistent with these results, Ser 307 p

phorylation of IRS-1 was also increase

XBP-1þ/j mice compared with wild-

controls fed HFD (Fig. 5C). Finally,

studied in vivo insulin-stimulated, ins

receptor–signaling capacity in these m

Fig. 4. Glucose homeostasisin XBP-1þ/j mice fed HFD.The XBP-1þ/j (>) and XBP-1þ/þ (h) mice were fed HFDimmediately after weaningat 3 weeks of age. Totalbody weight (A), fastingblood insulin (B), C-peptide(C), and glucose (D) levelswere measured in the XBP-1þ/j and XBP-1þ/þ miceduring the course of HFD.

GTT were performed after 7(E) and 16 (F) weeks of HFDin XBP-1þ/j and XBP-1þ/þ

mice. ITT were performedaf ter 8 (G) a n d 1 7 (H)weeks of HFD in XBP-1þ/j

(n 0 11) and XBP-1þ/þ (n 0

8) mice. Data are shown asmeans T SEM. Statistical sig-nificance in two-tailed Stu-dent’s t test is indicated by*P e 0.05, **P e 0.005, and***P e 0.0005. XBP-1þ/j

and XBP-1þ/þ groups arealso compared by ANOVA(A to H).

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15 OCTOBER 2004 VOL 306 SCIENCE www.sciencemag.org460



There was no detectable difference in any of

the insulin receptor–signaling components

in liver and adipose tissues between geno-

types taking regular diet (fig. S7). However,

after exposure to HFD, major components

of insulin receptor signaling in the liver,

including insulin-stimulated insulin recep-

tor, IRS-1, and IRS-2 tyrosine- and Akt

serine-phosphorylation, were all decreased

in XBP-1þ/j mice compared with wild-type

c on tr ol s ( Fi g. 5 , D t o G ). A s im il ar

suppression of insulin receptor signaling

was also evident in the adipose tissues of

XBP-1þ/j mice compared with XBP-1þ/þ

mice fed HFD (fig. S8). The suppression of

IR tyrosine phosphorylation in XBP-1þ/j

mice differs from the observations made in

XBP-1j/j cells, where ER stress inhibited

insulin action after the receptor signal in

the pathway. It is likely that this difference

reflects the effects of chronic hyperinsuline-

mia in vivo on insulin receptors. Hence, our

data demonstrate the link between ER stress

and insulin action in vivo but are not

conclusive in determining the exact locus

in insulin receptor signaling pathway that is

targeted through this mechanism.

Discussion. In this study, we identifyER stress as a molecular link between

obesity, the deterioration of insulin action,

and the development of type 2 diabetes. In-

duction of ER stress or reduction in the com-

pensatory capacity through down-regulation

of XBP-1 leads to suppression of insulin

receptor signaling in intact cells via IRE-1" –

dependent activation of JNK. Experiments

with mouse models also yielded data consist-

ent with the link between ER stress and

systemic insulin action. Deletion of an XBP-1

allele in mice leads to enhanced ER stress,

hyperactivation of JNK, reduced insulin

receptor signaling, systemic insulin resist-

ance, and type 2 diabetes.

Our findings point to a fundamental

mechanism underlying the molecular sensing

of obesity-induced metabolic stress by the

ER and inhibition of insulin action that

ultimately leads to insulin resistance and

type 2 diabetes. We therefore postulate that

ER stress underlies the emergence of the

stress and inflammatory responses in obesity

and the integrated deterioration of systemic

glucose homeostasis.Although our results in this study pre-

dominantly point to a role for ER stress in

peripheral insulin resistance, earlier studies

have linked ER stress with islet function

and survival. For example, PERK j/j mice

exhibit a phenotype resembling type 1

diabetes resulting from pancreatic islet

destruction soon after birth (24). PERK

mutations also cause a rare inherited form

of type 1 diabetes in humans (25). Loss of

eIF2" phosphorylation by targeted mutation

of serine 51 residue of eIF2" to alanine also

leads to alterations in pancreatic beta cell

function, in addition to its impact on liver gluconeogenesis (11, 26 ). Therefore, we

propose that the effect of chronic ER

stress on glucose homeostasis in obesity

could represent a central and integrating

mechanism underlying both peripheral

insulin resistance and impaired insulin

secretion.

The critical role of ER stress responses in

insulin action may represent a mechanism

conserved by evolution, whereby stress

signals are integrated with metabolic regula-

tory pathways through the ER. This integ

tion could have been advantageous, becau

proper regulation of energy fluxes and

suppression of major anabolic pathwa

might have been favorable during ac

stress, pathogen invasion, and immu

responses. Hence, the trait would propag

through natural selection. However, in

presence of chronic ER stress, such as we

in obesity, the eff ect of ER str ess

metabolic regulation would lead to

development of insulin resistance and, ev

tually, type 2 diabetes. In terms of therape

tics, our findings suggest that interventi

that regulate the ER stress response of

new opportunities for preventing and treat

type 2 diabetes.

References and Notes1. G. S. Hotamisligil, in Diabetes Mellitus, D. LeRo

S. I. Taylor, J. M. Olefsky, Eds. (Lippincott Willi& Wilkins, Philadelphia, 2003), pp. 953–962.

2. K. T. Uysal, S. M. Wiesbrock, M. W. Marino, GHotamisligil, Nature 389, 610 (1997).

3. J. Hirosumi et al., Nature 420, 333 (2002).4. M. Yuan et al., Science 293, 1673 (2001).

5. R. Y. Hampton, Curr. Biol. 10, R518 (2000).6. K. Mori, Cell 101, 451 (2000).7. H. P. Harding, M. Calfon, F. Urano, I. Novoa, D. R

Annu. Rev. Cell Dev. Biol. 18, 575 (2002).8. Y. Ma, L. M. Hendershot, Cell 107, 827 (2001).9. R. J. Kaufman et al., Nature Rev. Mol. Cell Biol

411 (2002).10. I. Kharroubi et al., Endocrinology (2004); publis

online 5 August 2004 (10.1210/en.2004-0478).11. Y. Shi, S. I. Taylor, S. L. Tan, N. Sonenberg, End

Rev. 24, 91 (2003).12. Y. Shi et al., Mol. Cell. Biol. 18, 7499 (1998).13. H. P. Harding, Y. Zhang, D. Ron, Nature 397,

(1999).14. F. Urano et al., Science 287, 664 (2000).15. R. P. Shiu, J. Pouyssegur, I. Pastan, Proc. Natl. Ac

Sci. U.S.A. 74, 3840 (1977).16. U. Ozcan, G. S. Hotamisligil, unpublished observati17. B. L. Bennett et al., Proc. Natl. Acad. Sci. U.S.A.

13681 (2001).18. R. K. Barr, T. S. Kendrick, M. A. Bogoyevitch, J. B

Chem. 277, 10987 (2002).19. M. Calfon et al., Nature 415, 92 (2002).20. X. Shen et al., Cell 107, 893 (2001).21. H. Yoshida, T. Matsui, A. Yamamoto, T. Oka

K. Mori, Cell 107 , 881 (2001).22. A. H. Lee, N. N. Iwakoshi, L. H. Glimcher, Mol. C

Biol. 23, 7448 (2003).23. A. M. Reimold et al., Genes Dev. 14, 152 (2000)24. H. P. Harding et al., Mol. Cell 7, 1153 (2001).25. M. Delepine et al., Nature Genet. 25, 406 (200026. D. Scheuner et al., Mol. Cell 7, 1165 (2001).27. We thank the Hotamisligil laboratory for t

contributions and J. Gound and L. Beppu technical assistance. Supported in part by grants AI32412 (L.H.G.), DK52539 (G.S.H.), AmerDiabetes Association (G.S.H.), PO5-CA100

(L.H.G., A.H.L.), an Irvington Institute PostdoctFellowship Award (N.I.), an NIH training grant TDK07703 (Q.C.), and a postdoctoral fellowship fthe Iaccoca Foundation (G.T.). L.H.G. holds equityMannKind Corporation, which has licensed the X1 technology.

Supporting Online Materialwww.sciencemag.org/cgi/content/full/306/5695/45DC1Materials and MethodsFigs. S1 to S8References

23 July 2004; accepted 9 September 2004

Fig. 5. ER stress andinsulin receptor sig-naling in XBP-1þ/j

mice. PERK phospho-rylation (p-PERK) (A), JNK activity (p-c-Jun)(B), and Ser307 phos-phorylation (pS) of IRS-1 (C) were exam-ined in the livers of XBP-1þ/j and XBP-1þ/þ

mice after 16 weeks of

HFD. After infusion of insulin (1 U/kg) throughthe portal vein, insulinreceptor (IR) tyrosinephosphorylation (pY)(D), IRS-1 tyrosinephosphorylation (E),IRS-2 tyrosine phos-phorylation (F), andAkt Ser473 phosphoryl-

A

B

C

D

E

F

G

IP: IR

XBP-1+/+ XBP-1+/-

Ins: - + + - + +XBP-1+/+ XBP-1+/-

XBP-1+/+ XBP-1+/-

XBP-1+/+ XBP-1+/-

p-PERK

XBP-1+/+ XBP-1+/-

Ins: - + + - + +

XBP-1+/+ XBP-1+/-

Ins: - + + - + +

XBP-1+/+ XBP-1+/-

Ins: - + + - + +

p-c-Jun

PERK

JNK


IP: IRS-1IB: IRS-1

IP: IRIB: IR

IP: IRS-1IB: pY

IP: IRS-1

IB: IRS-1

IP: IRS-2IB: pY

IP: IRS-2IB: IRS-2

AKT-pS

AKT

IB: pY

ation (pS) (G) were examined in livers of XBP-1þ/j andXBP-1þ/þ mice after 16 weeks of HFD. Total protein levelsare shown in the lower point of each panel.

R E S E A R C H A R T I C




Science Supporting Online MaterialÖzcan et al., p. 1

Science Supporting Online Material

Endoplasmic Reticulum Stress Links Obesity, Insulin Action, and

Type 2 Diabetes

Umut Özcan, Qiong Cao, Erkan Yilmaz, Ann-Hwee Lee, Neal N. Iwakoshi, EsraÖzdelen, Gürol Tuncman, Cem Görgün, Laurie H. Glimcher, Gökhan S. Hotamisligil

ContentsMaterials and MethodsFigs. S1 and S8References and Notes

Materials and Methods

Biochemical reagents. Anti-IRS-1, anti-phospho-IRS-1 (Ser 307) and anti-IRS-2 antibodieswere from Upstate Biotechnology (Charlottesville, VA). Antibodies against phosphotyrosine, eIF2!, insulin receptor " subunit, and XBP-1 were from Santa Cruz

Biotechnology (Santa Cruz, CA). Anti-phospho-PERK, anti-Akt, and anti-phospho-Aktantibodies and c-Jun protein were from Cell Signaling Technology (Beverly, MA). Anti- phospho-eIF2! antibody was purchased from Stressgen (Victoria, British Columbia,Canada). Anti-insulin antibody and C-peptide RIA kit were purchased from LincoResearch (St. Charles, MO). Anti-glucagon antibody was from Zymed (San Francisco,CA). PERK antiserum was kindly provided by Dr. David Ron (New York UniversitySchool of Medicine). Texas red conjugated donkey anti-guinea pig IgG and fluorescein-conjugated (FITC-conjugated) goat anti-rabbit IgG were from Jackson Immuno ResearchLaboratories (West Grove, PA). Thapsigargin, tunicamycin, and JNK inhibitors werefrom Calbiochem (San Diego, CA). Insulin, glucose, and sulindac sulfide were fromSigma (St. Louis, MO). The Ultra Sensitive Rat Insulin ELISA kit was from Crystal

Chem Inc. (Downers Grove, IL).Cells. Rat Fao liver cells were cultured with RPMI 1640 (Gibco, Grand Island, NY)containing 10% fetal bovine serum (FBS). At 70-80% confluency, cells were serumdepleted for 12 hours prior to the experiments. Reagents including tunicamycin,thapsigargin, and JNK inhibitors were gently added to the culture dishes in the incubator to prevent any environmental stress due to vibration, temperature changes and lightexposure. JNK inhibitors were added 1 hour before tunicamycin/thapsigargin treatment.The XBP-1

–/– mouse embryonic fibroblasts (MEF) (S1), IRE-1! –/– MEF cells (kindly

provided by Dr. David Ron, New York University School of Medicine), and their WTcontrols were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, GrandIsland, NY) containing 10% FBS. A similar protocol was followed for experiments inMEF cells, except that the cells were serum depleted for only 6 hours

Overexpression of XBP-1s in MEFs. MEF-tet-off cells (BD Biosciences Clontech, PaloAlto, CA) were cultured in DMEM with 100 µg/ml G418 and 1 µg/ml doxycycline. TheMEF-tet-off cells express exogenous tTA (tetracycline-controlled transactivator) protein,which binds to TRE (tetracycline response element) and activates transcription only inthe absence of tetracycline or doxycycline. The cDNA of the spliced form of XBP-1 wasligated into pTRE2hyg2 plasmid (BD Biosciences Clontech, Palo Alto, CA). The MEF-




tet-off cells were transfected with the TRE2hyg2-XBP-1s plasmid, followed by selectionin the presence of 400 µg/ml hygromycin B. Individual clones of stable transfectantswere isolated and doxycycline-dependent XBP-1s expression was confirmed byimmunoblotting in each experiment.

Northern blot analysis. Total RNA was isolated from mouse liver using Trizol reagent

(Invitrogen, Carlsbad, CA), separated by 1% agarose gel, and then transferred ontoBrightStar Plus nylon membrane (Ambion, Austin, TX). GRP78 cDNA probe was prepared from mouse liver total cDNAs by RT-PCR using the following primers: 5’-TGGAGTTCCCCAGATTGAAG-3’ and 5’- CCTGACCCACCTTTTTCTCA-3’. TheDNA probes were purified and labeled with 32P-dCTP using random primed DNAlabeling kit (Roche, Indianapolis, IN). Hybridization was performed according to themanufacturer’s protocol (Ambion, Austin, TX) and visualized by Versa Doc ImagingSystem 3000 (BioRad, Hercules, CA).

Protein extracts from cells . At the end of each treatment, cells were immediately frozenin liquid nitrogen and kept at –80oC. Protein extracts were prepared with a lysis buffer

containing 25 mM Tris-HCl (pH 7.4), 2 mM Na3VO4, 10 mM NaF, 10 mM Na4P2O7,1 mM EGTA, 1 mM EDTA, 1% NP-40, 5 µg/ml leupeptin, 5 µg/ml aprotinin, 10 nMokadaic acid, and 1 mM phenylmethylsulfonyl fluoride (PMSF). Immunoprecipitationsand immunoblotting experiments were performed with 750 µg and 75 !g total protein,respectively without any freeze-thaw cycles from individual aliquots.

Animal studies and obesity models. Adult (10-12 weeks of age) male ob/ob mice andtheir WT littermates were purchased from Jackson Labs. Mice used in the diet-inducedobesity model were male C57BL/6. All mice were placed on high fat diet (HFD: 35.5%fat, 20% protein, 32.7% carbohydrates, Bio-Serve) immediately after weaning (at ~3weeks of age). The XBP-1+/– and XBP-1+/+ mice were on BALB/c genetic background.Insulin and glucose tolerance tests were performed as previously described (S3). Insulin

and C-peptide ELISA were performed according to manufacturer’s instructions usingmouse standards (Crystal Chem Inc., Downers Grove, IL). Pancreas isolated from 16-week-old mice was fixed in Bouin's fluid and formalin, and paraffin sections weredouble-stained with guinea pig anti-insulin and rabbit anti-glucagon antibodies. Texas reddye conjugated donkey anti-guinea pig IgG and FITC conjugated Goat anti-rabbit IgGwere used as secondary antibodies.

Insulin infusion and tissue protein extraction. Insulin was injected through the portal veinas previously described (S2, S3). Three minutes after insulin infusion, liver was removedand frozen in liquid nitrogen and kept at –80°C until processing. For protein extraction,liver tissue (~0.3g) was placed in 10 ml of lysis buffer containing 25 mM Tris-HCl

(pH7.4), 10 mM Na3VO4, 100 mM NaF, 50 mM Na4P2O7, 10 mM EGTA, 10 mM EDTA,1% NP-40, 5 µg/ml leupeptin, 5 µg/ml aprotinin, 10 nM okadaic acid, and 2 mM PMSF.After homogenization on ice, the tissue lysate was centrifuged at 4,000 rpm for 15minutes at 4°C followed by 55,000 rpm for 1 hour at 4°C. One milligram of total tissue protein was used for immunoprecipitation and subsequent immunoblotting, whereas 100-150 µg total tissue protein was used for direct immunoblotting (S3).




Fig. S1. Regulation of GRP78 expression by glucose in vitro and hyperglycemia in vivo.(A) Fao cells were treated with various doses of glucose (0, 5, 10, 25, and 75 mM) for 24hours. The GRP78 mRNA level was examined by Northern blot using the total RNAsisolated from these cells. Ethidium bromide staining is shown as a control for loading andintegrity of RNA. (B) Streptozotocin (STZ, 200 mg/kg) was injected intaperitoneally into8-week-old male mice. Three days after injection, blood glucose levels were measured toconfirm STZ-induced hyperglycemia (140.5±13.6 and 484.5±50.9 in controls and STZinjected diabetic animals, respectively). Livers were collected 10 days after injection andGRP78 expression was examined by Northern blot analysis using the liver total RNA.

A

0 5 10 25 75

Glu mM

GRP78

28s18s

B

GRP78

STZ: - +

28s

18s




Fig. S2. ER stress indicators in adipose tissues of obese mice. Dietary (high fat diet-

induced) and genetic (ob/ob) models of mouse obesity were used to examine markers of ER stress in adipose tissue compared with age and sex matched lean controls. (A) PERK phosphorylation (p-PERK) and JNK activity were examined in the adipose samples of themale mice (C57BL/6) that were kept either on regular chow diet (RD) or high fat diet(HFD) for 16 weeks. (B) PERK phosphorylation and JNK activity in the adipose tissuesof male ob/ob and WT lean mice at the ages of 12-14 weeks. (C) The GRP78 mRNAlevel was examined by Northern blot analysis in the adipose tissues of WT lean andob/ob animals. Ethidium bromide staining is shown as a control for loading and integrityof RNA.

RD HFD Lean ob/ob

A B

p-PERK

PERK

p-c-Jun

JNK

Lean ob/ob

C

GRP78

28s

18s




Fig. S3. Inhibition of insulin receptor signaling by thapsigargin-induced ER stress and therole of calcium levels in IRS-1 serine phosphorylation. (A) ER stress was induced in Faocells by 1-hour treatment with 300 nM thapsigargin (Thap), and cells were subsequentlystimulated with insulin (Ins). IRS-1 tyrosine phosphorylation (pY) and serine phosphorylation (pSer307), insulin receptor (IR) tyrosine phosphorylation, and total protein levels were examined using either immunoprecipitation (IP) followed byimmunoblotting (IB) or direct immunobloting. (B) Fao cells were treated with sulindac

sulfide (SS: 0, 7.5, 30, and 60 µM) for 45 minutes with or without an additional hour of exposure to 300 nM thapsigargin (Thap). IRS-1 serine phosphorylation and total IRS-1 protein levels were examined as described above.

These results demonstrate that treatment of cells with thapsigargin, an agent that inducesER stress by inhibiting calcium ATPase, also increased IRS-1 serine phosphorylation andsuppressed insulin receptor signaling. The use of sulindac sulfide to block calcium influxto the cytosol from the extracellular compartment addresses whether thapsigargin-induced inhibition of insulin action was simply due to alterations in cellular calciumlevels. Treatment with sulindac sulfide alone had no effect on Ser307 phosphorylation of IRS-1. Furthermore, in the presence of thapsigargin, sulindac sulfide did not interferewith IRS-1 serine phosphorylation indicating that the effect of thapsigargin on Ser307

phosphorylation of IRS-1 and inhibition of insulin receptor signaling is mediated throughER stress.


IP: IRS-1IB: IRS-1

SS (µM): - 7.5 30 60 - 7.5 30Thap: - - - - + + +

Thap: - - +Ins: - + +

BA

IP: IRS-1

IB: pY

IP: IRS-1

IB: IRS-1


IP: IRIB: pY

IP: IRIB: IR




Tun(0.5µg/ml): 0 0 0.5 1 2 3 4 0 0 0.5 1 2 3 4 (hr)

Ins: - + + + + + + - + + + + + +

Fig. S4. Insulin-induced insulin receptor autophosphorylation in XBP-1 overexpressingand XBP-1-deficient cells. (A) XBP-1 overexpressing fibroblasts (-Dox) and their controls (+Dox) were treated with 2 µg/ml tunicamycin (Tun) for indicated periods (0,0.5, 1, 2, 3, and 4 hours). Insulin-induced insulin receptor (IR) tyrosine phosphorylation(pY) and total IR levels were examined in those cells using immunoprecipitation (IP)with IR antibody followed by immnunoblotting (IB) with antibodies against IR or phospho tyrosine (pY). (B) XBP-1 –/– MEF cells and their WT controls were treated with0.5 µg/ml tunicamycin for indicated periods (0, 0.5, 1, 2, 3, and 4 hours). Insulin-inducedinsulin receptor (IR) tyrosine phosphorylation (pY) and total IR levels were examined as

in panel A.

IP: IR

IB: pY

IP: IRIB: IR

IP: IRIB: pY

IP: IR

IB: IR

Tun(2µg/ml): 0 0 0.5 1 2 3 4 0 0 0.5 1 2 3 4 (hr)

Ins: - + + + + + + - + + + + + +

+Dox -Dox

A

XBP-1-/- XBP-1+/+

B




1

1.5

2

2.5

1 2 i n s u l i n l e v e l ( n g / m l )

XBP-1 +/+

XBP-1 +/-

0

70

140

210

280

0 15 30 60 90 120

Time After Glucose (min)

B l o o d

G l u c o s e ( m g / d l )

XBP-1 +/+

XBP-1 +/-

Fig. S5. Insulin sensitivity in lean XBP-1+/– and XBP-1+/+ mice. In XBP-1+/– and XBP-1+/+ mice placed on regular chow diet, blood insulin (A) and c-peptide (B) levels weremeasured, and glucose (C) and insulin tolerance tests (D) were performed at 16 weeks of age. On chow diet, there was no difference in blood glucose levels, C-peptide levels, andinsulin sensitivity measured by glucose and insulin tolerance tests between genotypesfollowed up to 18 weeks of age.

At the onset of the HFD experiment, there was also no difference in glucosemetabolism between XBP-1+/– and XBP-1+/+ mice as determined by fasting and fed bloodglucose, insulin and C-peptide levels, and by intraperitoneal glucose and insulin tolerancetests. Serum levels of leptin (26.2±2.5 and 25.2±1.8 ng/ml in XBP-1+/+ and XBP-1+/– ,respectively), adiponectin (15.5±1.8 and 16.6±1.6 ng/dl in XBP-1+/+ and XBP-1+/– ,respectively) and triglycerides (67.7±5.5 and 62.8±3.4 mg/dl in XBP-1+/+ and XBP-1+/– ,respectively) did not exhibit any statistically significant differences between thegenotypes measured after 16 weeks of HFD. Blood insulin levels in XBP-1+/+ mice weresignificantly lower than those in XBP-1+/– littermates (0.89±0.25 and 2.27±0.32 ng/ml inXBP-1+/+ and XBP-1+/– after 20 weeks on HFD, respectively, p<0.05). C-peptide levelswere also significantly higher in XBP-1+/– animals than in WT controls (772.91±132.24

and 1374.11±241.8 ng/ml in XBP-1+/+ and XBP-1+/– ( p<0.05).

A B

C D

0

700

1400

2100

1 2

c - p e p t i d e l e v e l ( p

M ) XBP-1 +/+

XBP-1 +/-

0

35

70

105

140

1 2 3 4 5 6

Time After Insulin (min)

B l o o d G

l u c o s e ( m g / d l )

XBP-1 +/+

XBP-1 +/-




Fig. S6. Characterization of pancreatic islets in XBP-1

+/–

and XBP-1

+/+

mice. (A-H) Isletmorphology, size and immuno-histochemical staining for insulin (red) and glucagon(green) in pancreatic sections obtained from XBP-1+/– and XBP+/+ mice on either regular chow (A-D) or high fat (E-H) diet. (I). Glucose-stimulated insulin secretion in XBP-1+/–

and WT mice. Glucose was administered introperitoneally to mice in each genotypefollowing 16 weeks of high fat diet and blood samples are collected at the indicated timesfor insulin measurements.

In these experiments, there was no detectable abnormality in the XBP+/– islets and nodifference was evident between genotypes under standard conditions. On HFD, both theXBP-1+/– and XBP-1+/+ mice exhibited islet hyperplasia. This anticipated response toHFD was similar between genotypes and the hyperplastic component (islet size >150!M) comprised 40% of all islets in XBP-1+/– and 43% of all islets in WT mice on HFD.In experiments examining glucose-stimulated insulin secretion in XBP-1+/– and WT miceon HFD, the XBP-1+/– mice responded to glucose with even a stronger insulin secretoryresponse, which effectively eliminates the possibility of an isolated islet defectunderlying their phenotype. Hence, these data indicate that the phenotype of the XBP-1+/–

mice cannot be attributed to defective islets and even after 16 weeks on HFD, the isletsappear indistinguishable between genotypes. However, our data does not rule outinvolvement of XBP-1 in islet function during obesity and diabetes.

RD

HFD

XBP-1+/+ XBP-1+/– XBP-1+/+ XBP-1+/–

0

0.6

1.2

1.8

0 2 5 10 1 5 20 3 0 60

Time after glucose (min)

I

I n s u l i n ( n g / m l )




Fig. S7. Intact insulin receptor signaling in liver and adipose tissues of XBP-1+/– and

XBP-1

+/+

mice on regular chow diet. After infusion of insulin (1U/kg) through portalvein, insulin receptor (IR) tyrosine phosphorylation (pY), IRS-1 tyrosine phosphorylation, IRS-2 tyrosine phosphorylation, Akt Ser473 phosphorylation, and their total protein levels were examined in livers (A) and adipose tissues (B) of XBP-1+/– andXBP+/+ mice on regular chow diet.

Ins: - + + - + + - + + - + +

XBP-1+/+ XBP-1+/– XBP-1+/+ XBP-1+/– Liver FAT

A B

IP: IRIB: pY

IP: IRIB: IR

IP: IRS-1IB: pY

IP: IRS-1IB: IRS-1

IP: IRS-2IB: pY

IP: IRS-2IB: IRS-2

Akt

Akt-pS




Fig. S8. Reduced insulin receptor signaling in adipose tissues of XBP-1+/– and XBP-1+/+

mice on high fat diet. (A) After infusion of insulin (1U/kg) through portal vein, insulinreceptor (IR) tyrosine phosphorylation (pY), IRS-1 tyrosine phosphorylation, IRS-2tyrosine phosphorylation, Akt Ser473 phosphorylation, and their total protein levels wereexamined in adipose tissues of XBP-1+/– and XBP+/+ mice on high fat diet for 16 weeks.(B) JNK kinase assay was performed in adipose tissues of XBP-1+/– and XBP+/+ mice onhigh fat diet for 16 weeks.

IP: IR

IB: pY

IP: IR

IB: IR

IP: IRS-1

IB: pY

IP: IRS-1

IB: IRS-1

IP: IRS-2

IB: pYIP: IRS-2

IB: IRS-2

Akt

Akt-pS

Ins: - + + - + +

XBP-1+/+ XBP-1+/–A

B XBP-1+/+ XBP-1+/–

p-c-Jun




References and Notes

X. Shen et al ., Cell 107, 893 (2001).

S1. A. H. Lee, N. N. Iwakoshi, L. H. Glimcher, Mol. Cell Biol. 23, 7448 (2003).

S2. J. Hirosumi et al., Nature 420, 333 (2002).S3. K. T. Uysal, S. M. Wiesbrock, M. W. Marino, G. S. Hotamisligil, Nature 389, 610

(1997).

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