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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: ghotamis@hsph.harvard.edu
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.
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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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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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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-pS
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
www.sciencemag.org SCIENCE VOL 306 15 OCTOBER 2004
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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-
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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).
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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
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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
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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-pS
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: IRS-1IB: IRS-1-pS
IP: IRIB: pY
IP: IRIB: IR
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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
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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 +/-
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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 )
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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
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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
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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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