Grp78 heterozygosity promotes adaptive unfolded protein response and attenuates diet-induced obesity and insulin resistance.
ABSTRACT To investigate the role of the endoplasmic reticulum (ER) chaperone glucose-regulated protein (GRP) 78/BiP in the pathogenesis of obesity, insulin resistance, and type 2 diabetes.
Male Grp78(+/-) mice and their wild-type littermates were subjected to a high-fat diet (HFD) regimen. Pathogenesis of obesity and type 2 diabetes was examined by multiple approaches of metabolic phenotyping. Tissue-specific insulin sensitivity was analyzed by hyperinsulinemic-euglycemic clamps. Molecular mechanism was explored via immunoblotting and tissue culture manipulation.
Grp78 heterozygosity increases energy expenditure and attenuates HFD-induced obesity. Grp78(+/-) mice are resistant to diet-induced hyperinsulinemia, liver steatosis, white adipose tissue (WAT) inflammation, and hyperglycemia. Hyperinsulinemic-euglycemic clamp studies revealed that Grp78 heterozygosity improves glucose metabolism independent of adiposity and following an HFD increases insulin sensitivity predominantly in WAT. As mechanistic explanations, Grp78 heterozygosity in WAT under HFD stress promotes adaptive unfolded protein response (UPR), attenuates translational block, and upregulates ER degradation-enhancing alpha-mannosidase-like protein (EDEM) and ER chaperones, thus improving ER quality control and folding capacity. Further, overexpression of the active form of ATF6 induces protective UPR and improves insulin signaling upon ER stress.
HFD-induced obesity and type 2 diabetes are improved in Grp78(+/-) mice. Adaptive UPR in WAT could contribute to this improvement, linking ER homeostasis to energy balance and glucose metabolism.
- SourceAvailable from: Hidde L Ploegh[Show abstract] [Hide abstract]
ABSTRACT: THE TOPOLOGICAL BARRIERS DEFINED BY BIOLOGICAL MEMBRANES ARE NOT IMPERMEABLE: from small solutes to intact proteins, specialized transport and translocation mechanisms adjust to the cell's needs. Here, we review the removal of unwanted proteins from the endoplasmic reticulum (ER) and emphasize the need to extend observations from tissue culture models and simple eukaryotes to studies in whole animals. The variation in protein production and composition that characterizes different cell types and tissues requires tailor-made solutions to exert proper control over both protein synthesis and breakdown. The ER is an organelle essential to achieve and maintain such homeostasis.F1000prime reports. 07/2014; 6:49.
- [Show abstract] [Hide abstract]
ABSTRACT: Sel1L is an essential adaptor protein for the E3 ligase Hrd1 in the endoplasmic reticulum (ER)-associated degradation (ERAD), a universal quality-control system in the cell; but its physiological role remains unclear. Here we show that mice with adipocyte-specific Sel1L deficiency are resistant to diet-induced obesity and exhibit postprandial hypertriglyceridemia. Further analyses reveal that Sel1L is indispensable for the secretion of lipoprotein lipase (LPL), independent of its role in Hrd1-mediated ERAD and ER homeostasis. Sel1L physically interacts with and stabilizes the LPL maturation complex consisting of LPL and lipase maturation factor 1 (LMF1). In the absence of Sel1L, LPL is retained in the ER and forms protein aggregates, which are degraded primarily by autophagy. The Sel1L-mediated control of LPL secretion is also seen in other LPL-expressing cell types including cardiac myocytes and macrophages. Thus, our study reports a role of Sel1L in LPL secretion and systemic lipid metabolism.Cell metabolism. 07/2014;
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ABSTRACT: As an adipokine in circulation, adiponectin has been extensively studied for its beneficial metabolic effects. While many important functions have been attributed to adiponectin under high-fat diet conditions, little is known about its essential role under regular chow. Employing a mouse model with inducible, acute β-cell ablation, we uncovered an essential role of adiponectin under insulinopenic conditions to maintain minimal lipid homeostasis. When insulin levels are marginal, adiponectin is critical for insulin signaling, endocytosis and lipid uptake in subcutaneous white adipose tissue. In the absence of both insulin and adiponectin, severe lipoatrophy and hyperlipidemia lead to lethality. In contrast, elevated adiponectin levels improve systemic lipid metabolism in the near absence of insulin. Moreover, adiponectin is sufficient to mitigate local lipotoxicity in pancreatic islets, and it promotes reconstitution of β-cell mass, eventually reinstating glycemic control. We uncovered an essential new role for adiponectin, with major implications for type 1 diabetes.eLife Sciences 10/2014; 3. · 8.52 Impact Factor
Grp78 Heterozygosity Promotes Adaptive Unfolded
Protein Response and Attenuates Diet-Induced Obesity
and Insulin Resistance
Risheng Ye,1Dae Young Jung,2John Y. Jun,2Jianze Li,1Shengzhan Luo,1Hwi Jin Ko,2Jason K. Kim,2
and Amy S. Lee1
OBJECTIVE—To investigate the role of the endoplasmic retic-
ulum (ER) chaperone glucose-regulated protein (GRP) 78/BiP in
the pathogenesis of obesity, insulin resistance, and type 2
RESEARCH DESIGN AND METHODS—Male Grp78?/?mice
and their wild-type littermates were subjected to a high-fat diet
(HFD) regimen. Pathogenesis of obesity and type 2 diabetes was
examined by multiple approaches of metabolic phenotyping.
Tissue-specific insulin sensitivity was analyzed by hyperinsuline-
mic-euglycemic clamps. Molecular mechanism was explored via
immunoblotting and tissue culture manipulation.
RESULTS—Grp78 heterozygosity increases energy expenditure
and attenuates HFD-induced obesity. Grp78?/?mice are resis-
tant to diet-induced hyperinsulinemia, liver steatosis, white
adipose tissue (WAT) inflammation, and hyperglycemia. Hyper-
insulinemic-euglycemic clamp studies revealed that Grp78 het-
adiposity and following an HFD increases insulin sensitivity
predominantly in WAT. As mechanistic explanations, Grp78
heterozygosity in WAT under HFD stress promotes adaptive
unfolded protein response (UPR), attenuates translational block,
and upregulates ER degradation-enhancing ?-mannosidase–like
protein (EDEM) and ER chaperones, thus improving ER quality
control and folding capacity. Further, overexpression of the
active form of ATF6 induces protective UPR and improves
insulin signaling upon ER stress.
CONCLUSIONS—HFD-induced obesity and type 2 diabetes are
improved in Grp78?/?mice. Adaptive UPR in WAT could con-
tribute to this improvement, linking ER homeostasis to energy
balance and glucose metabolism. Diabetes 59:6–16, 2010
ment for Ca2?, which regulates multiple pathways of
signal transduction. ER stress is defined as an imbalance
between protein load and folding capacity of the ER,
which triggers the evolutionarily conserved mechanism
referred to as the unfolded protein response (UPR) (1).
The UPR induces three major ER signaling pathways,
namely PKR-like endoplasmic reticulum kinase (PERK),
inositol requiring-1 (IRE-1), and activating transcription
factor (ATF) 6. As an acute response, autophosphorylation
of PERK leads to phosphorylation of eukaryotic initiation
factor (eIF) 2? and global inhibition of mRNA translation,
immediately reducing ER protein load. However, this early
protective process is unfavorable for long-term cellular
function and is reversible during the adaptive phase of
UPR. The adaptive UPR signaling, including activation of
transcription factors ATF4 (by eIF2? phosphorylation),
X-box binding protein (XBP)-1 (by active IRE-1), and
ATF6, promotes recovery from translational block, ER-
associated protein degradation (ERAD), and upregulation
of ER chaperones (2). Within the lumen of the ER, protein
chaperones and folding enzymes such as glucose-regu-
lated protein (GRP)78, GRP94, protein disulphide isomer-
ase (PDI), calnexin (CNX), and calreticulin (CRT) assist in
folding of newly synthesized polypeptides and prevent
aggregation of unfolded or misfolded protein (3). Differ-
entiated adipocytes are potent endocrine cells, secreting
large amounts of peptides and lipid mediators such as
leptin and adiponectin (4). Biosynthesis of these adipo-
kines requires chaperones and folding enzymes in the ER.
Thus, ER stress of adipocytes may play a significant role in
obesity-related pathogenesis, and understanding the con-
tribution of the UPR to adipocyte stress may identify new
targets toward the development of preventive and thera-
peutic strategies (5).
The molecular mechanism linking obesity to insulin
resistance in the peripheral tissues has recently been
elucidated in mouse models (6). Excessive fat storage
stimulates ER stress in liver and adipose tissue, which
subsequently activates IRE-1 and the downstream kinase,
c-Jun NH2-terminal kinase (JNK), through the ER stress
signaling pathway. Active JNK can phosphorylate the
insulin receptor substrate (IRS)-1 on Ser307, thus inhibit-
ing tyrosine phosphorylation of IRS-1 and the downstream
insulin signaling pathway (7). Administration of chemical
chaperones such as 4-phenyl butyric acid or tauroursode-
oxycholic acid was able to reduce ER stress, restore
he endoplasmic reticulum (ER) is a specialized
perinuclear organelle where secretory and mem-
brane proteins, as well as lipids, are synthesized.
It is also a major intracellular storage compart-
From the1Department of Biochemistry and Molecular Biology, University of
Southern California/Norris Comprehensive Cancer Center, University of
Southern California Keck School of Medicine, Los Angeles, California; and
the2Department of Cellular and Molecular Physiology, Pennsylvania State
University College of Medicine, Hershey, Pennsylvania.
D.Y.J., H.J.K., and J.K.K. are currently affiliated with the Department of
Molecular Medicine, University of Massachusetts Medical School, Worces-
ter, Massachusetts. S.L. is currently affiliated with the Department of
Biological Sciences, Stanford University, Stanford, California.
Corresponding author: Amy S. Lee, email@example.com.
Received 19 May 2009 and accepted 9 September 2009. Published ahead of
print at http://diabetes.diabetesjournals.org on 6 October 2009. DOI:
© 2010 by the American Diabetes Association. Readers may use this article as
long as the work is properly cited, the use is educational and not for profit,
and the work is not altered. See http://creativecommons.org/licenses/by
-nc-nd/3.0/ for details.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked “advertisement” in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
6 DIABETES, VOL. 59, JANUARY 2010diabetes.diabetesjournals.org
glucose homeostasis, and improve insulin sensitivity in the
peripheral tissues of leptin-deficient ob/ob mice (8), imply-
ing that ER homeostasis is key to improved glucose
The 78-kDa glucose regulated protein GRP78, also re-
ferred to as BiP (immunoglobulin heavy-chain binding
protein) or HSPA5, is a key rheostat in controlling ER
homeostasis. GRP78 regulates ER function due to its role
in protein folding and assembly, targeting misfolded pro-
tein for degradation, ER Ca2?binding, and controlling the
activation of transmembrane ER stress sensors (3,9,10).
Among the peripheral tissues, GRP78 expression is readily
detected in adult adipose tissues and liver but is very low
in muscle. To study its in vivo role in cell metabolism, we
created targeted mutation of the Grp78 allele in mouse
models. Homozygous deletion of Grp78 results in early
embryonic lethality; however, the Grp78?/?mice with
partial reduction in GRP78 expression level are viable and
fertile (11). Serendipitously, we discovered that the
Grp78?/?mice in the C57BL/6 genetic background sub-
stantially mitigates high-fat diet (HFD)-induced obesity
and insulin resistance. In addition to improved energy
expenditure, a striking increase in insulin-stimulated glu-
cose uptake was observed in the white adipose tissue
(WAT) of Grp78?/?mice. We further discovered that
Grp78 heterozygosity in WAT triggers adaptive UPR sig-
naling as well as compensatory increases in ER chaperone
levels, associating with attenuation of translational block
and improved insulin signaling. As proof of principle,
overexpression of active ATF6 in mouse embryo fibro-
blasts (MEFs) induces protective UPR and leads to im-
provement of insulin sensitivity under ER stress. Thus, the
Grp78?/?mouse model uncovers novel modulatory mech-
anisms in WAT linking ER integrity to energy balance,
glucose homeostasis, and adipocyte stress.
RESEARCH DESIGN AND METHODS
The Grp78?/?mice were generated as described (11) and were backcrossed
into the C57BL/6 genetic background for five to eight generations. Mice were
fed on regular diet (11% fat by calories; Harlan Teklad) continuously after
weaning (at ?3 weeks of age) or changed to HFD (45% fat by calories;
Research Diets) at 10 weeks of age. Only male mice were used in this study.
Mouse body weight was measured after overnight fasting. Food intake was
analyzed by daily food mass measurement for 5 successive days during the
third week of the HFD regimen. Mouse stool was processed to Oil-O-Red
staining for lipids as described (12). All protocols for animal use and
euthanasia were reviewed and approved by the University of Southern
California Institutional Animal Care and Use Committee.
Measurement of body composition and energy balance. Twenty-week-old
mice were fed an HFD for 10 weeks. Whole-body fat and lean mass were
noninvasively measured in conscious mice using proton magnetic resonance
1012 1416 18
20 22 24 26 28 30
Body Weight (g)
Body Composition by 1H-MRS (g)
FIG. 1. Attenuation of diet-induced obesity in Grp78?/?mice. A: Fasting body weight on regular diet (RD) or HFD from 10-week-old mice (n >
7 mice per condition). E,?/?HFD; F,?/?HFD; ‚,?/?regular diet; Œ,?/?regular diet. B: Body size after 20-week HFD. C: Body composition after
11-week HFD. n ? 9 (?/?, ?) or 6 (?/?, f). D: Food intake measurement. n ? 7 (?/?, ?) or 5 (?/?, f). E: Oil Red O staining of stool smear from
mice on HFD. Negative control: dH2O; positive control: white adipose extract. Data are presented as the means ? SE. *P < 0.05; **P < 0.01 for
?/?vs.?/?. (A high-quality color digital representation of this figure is available in the online issue.)
R. YE AND ASSOCIATES
diabetes.diabetesjournals.orgDIABETES, VOL. 59, JANUARY 20107
spectroscopy (1H-MRS) (Echo Medical Systems, Houston, TX). A 3-day
measurement of water intake, energy expenditure, and physical activity was
performed using the metabolic cages (TSE Systems, Bad Homburg, Germany).
All procedures were approved by the Pennsylvania State University Institu-
tional Animal Care and Use Committee.
Assay of blood glucose and insulin. Mouse tail blood was measured for
glucose by the OneTouch Ultra System (LifeScan, Milpitas, CA). For insulin,
plasma was prepared from blood by centrifugation and measured with an
enzyme-linked immunosorbent assay (ELISA) kit (Linco Research).
Tissue processing. After the mice were killed, mouse tissues were fixed in
10% formalin for histological analysis or immediately frozen in liquid nitrogen
and stored at ?80°C for immunoblotting.
Immunohistochemistry. Paraffin sections of formalin-fixed tissues were
stained with hematoxylin and eosin for morphological evaluation. For immu-
nohistochemistry, primary antibodies used included insulin (1:100; Signet)
and CD68 (1:50; Santa Cruz Biotechnology).
Insulin tolerance test. Mice were subjected to intraperitoneal injection of
insulin (0.5 mU/g body wt) after 6 h fasting, followed by blood glucose
Hyperinsulinemic-euglycemic clamp. After a 10-week HFD regimen, 20-
week-old mice were subjected to hyperinsulinemic-euglycemic clamp to
assess insulin sensitivity in vivo as described (13). Details of the clamp
methodology, biochemical assays, and the metabolic rate calculations were
described in online appendix (available at http://diabetes.diabetesjournals.
Insulin signaling analysis. Following clamps, WAT was prepared for lysates.
Total and phospho-Tyr IRS-1 levels were analyzed by immunoblotting. Insulin-
stimulated AKT activity was determined by immunoprecipitating tissue ly-
sates with a polyclonal AKT antibody (Upstate Biotechnology) that recognized
both AKT1 and AKT2, coupled with protein G–Sepharose beads (Amersham
Pharmacia Biotechnology, Piscataway, NJ) as previously described (13).
Cell culture and transfection. The Grp78?/?MEFs were isolated and
immortalized with SV40 large-tumor antigen as described (14). The similarly
transformed wild-type MEFs were provided by Dr. Stanley Korsmeyer (Har-
vard University). To overexpress an HA-tagged active nuclear form of ATF6,
cells were transfected with the plasmid pCGN-ATF6(373) (15) using PolyFect
(Qiagen) for 24 h, controlled by the vector transfection. To induce ER stress,
cells were treated with tunicamycin (1.5 ?g/ml; Sigma) for 14 h.
Insulin sensitivity analysis. After cultured in serum-free medium for 5 h,
cells were treated with insulin (100 nmol/l; Sigma) for 15 min, followed by
immediate lysate preparation (16). Insulin-stimulated phosphorylation of AKT
was determined by immunoblotting phosphor-Ser473 and total AKT.
Immunoblotting. Lysates of tissues from individual mice or cells were
extracted in ice-cold radioimmunoprecipitation assay buffer (50 mmol/l Tris-
Cl, 150 mmol/l NaCl, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS),
containing cocktails of proteinase inhibitors and phosphatase inhibitors
(Roche), by centrifugation (13,000g, 15 min) following homogenization and
three freeze-thaw cycles. Proteins were separated by 8% or 12% SDS-PAGE
and transferred to nitrocellulose membrane (Pall) and subjected to Western
blotting (15). Primary antibodies used included pSer473-AKT, AKT (1:1,000;
Cell Signaling or Upstate Biotechnology), pTyr–IRS-1, IRS-1 (Upstate Biotech-
nology), JNK1, glyceraldehyde-3-phosphate dehydrogenase, ATF4, CHOP,
GADD34, XBP-1, ER degradation-enhancing ?-mannosidase-like protein
(EDEM), GRP78, hemagglutinin (HA) tag (1:1,000; Santa Cruz Biotechnology),
pSer51-eIF2?, eIF2? (1:1,000; Cell Signaling), ATF6 (1:100; Abcam), GRP94,
PDI, CNX, and CRT (1:2000; Stressgen), peroxisome proliferator–activated
receptor ? coactivator (PGC)-1 (1:1,000, Calbiochem), and ?-actin (1:5,000;
Sigma). Western blotting was repeated two to six times and quantitated using
the Quantity One system (Bio-Rad).
Statistical analysis. A two-tailed Student’s t test was applied for all pairwise
Grp78 heterozygosity increases energy expenditure
and attenuates diet-induced obesity. To investigate the
role of GRP78 in obesity and type 2 diabetes, cohorts of
Grp78?/?mice and their wild-type (?/?mice, Grp78?/?)
littermates in a C57BL/6 background were subjected to the
HFD regimen beginning at the age of 10 weeks. Male mice
were used since hormonal cycle in females may affect
metabolism and confound the results. Surprisingly, we
discovered that following HFD, Grp78?/?mice showed
significantly lower body weight than the?/?siblings (Fig.
1A and B), as confirmed by body composition analysis by
1H-MRS showing primarily reduction in fat mass (Fig.
1C and supplementary Fig. 1A). The lean phenotype of the
Grp78?/?mice was not caused by alterations in food and
water intake (Fig. 1D and supplementary Fig. 1B) or
intestinal fat absorption (Fig. 1E). Rather, it is at least in
part due to increased energy expenditure in the Grp78?/?
mice, as measured by indirect calorimetry (Fig. 2A).
Significant enhancement of both O2consumption (P ?
0.006) and CO2production (P ? 0.0009) was observed in
observed in the respiratory exchange ratios (Fig. 2C) or
physical activities (Fig. 2D).
Grp78?/?mice are resistant to diet-induced hyperin-
sulinemia, liver steatosis, WAT inflammation, and
hyperglycemia. Diet-induced obesity causes insulin resis-
tance, pancreatic islet hyperplasia, hyperinsulinemia, and
hyperglycemia (17). After 20 weeks of HFD (30 weeks
old), while?/?mice developed hyperglycemia (159 ? 6
mg/dl) as expected,
lower fasting glucose level (118 ? 9 mg/dl) (Fig. 3A). The
fasting plasma insulin level was also lower in the?/?mice
(0.26 ? 0.06 vs. 0.55 ? 0.08 ng/ml in?/?mice, P ? 0.009)
(Fig. 3B). The
?-cell hyperplasia evident in the
contrasting with normal glucagon distribution staining
pattern for both?/?and?/?mice (supplementary Fig. 2A).
Transmission electron microscopy further showed that
?/?pancreatic ?-cells had normal number and distribution
of secretory granules (supplementary Fig. 2B). Hepatic
steatosis could be a complication of diet-induced type 2
diabetes (18). In agreement with the improved hypergly-
?/?mice (Fig. 2B). In contrast, no difference was
?/?mice maintained significantly
?/?mice were resistant to HFD-induced
?/?mice (Fig. 3C),
FIG. 2. Enhancement of energy expenditure in Grp78?/?mice. Meta-
bolic cage studies on mice after 10-week HFD (n > 3 mice per
condition). A: Energy expenditure. B: O2consumption and CO2produc-
tion. C: Respiratory exchange ratio. D: Total physical activity. Data are
presented as the means ? SE. **P < 0.01 for?/?(f) vs.?/?(?).
Grp78?/?LESSENS OBESITY AND INSULIN RESISTANCE
8 DIABETES, VOL. 59, JANUARY 2010 diabetes.diabetesjournals.org
Blood Insulin (ng/mL)
10 1214 16 18
20 2224 262830
Blood Glucose (mg/dL)
FIG. 3. Resistance to HFD-induced diabetic phenotypes in Grp78?/?mice. A: Fasting blood glucose (n > 7 mice per condition). E,?/?HFD; F,?/?
HFD; ‚,?/?regular diet; Œ,?/?regular diet. B: Fasting blood insulin after 19-week HFD. n ? 15 (?/?, ?) or 16 (?/?, f). Data are presented as
means ? SE. **P < 0.01 for?/?vs.?/?. C–E: Histochemical studies on 25-week-old mice on regular diet (RD) or after 15-week HFD (n > 3 mice
per condition). Numbers above scale bars indicate the represented object distance. C: Insulin immunostaining on pancreas. D: Hematoxylin and
eosin staining on liver (C and D: Lower panels exhibit the boxed areas within the corresponding upper panels.) E: Hematoxylin and eosin
and CD68 staining on WAT. Arrowheads indicate inflammation. (A high-quality color digital representation of this figure is available in the
R. YE AND ASSOCIATES
diabetes.diabetesjournals.orgDIABETES, VOL. 59, JANUARY 20109
cemia (Fig. 3A), the?/?mice showed reduced steatosis in
liver (Fig. 3D). Adipose inflammation has recently been
linked to obesity-associated insulin resistance (19). Corre-
spondingly, WAT from the?/?mice showed greatly re-
duced inflammation, as revealed by hematoxylin and eosin
and CD68 staining (Fig. 3E). Compared with the regular
diet–fed mice, the HFD regimen led to an increase in
adipocyte size in both Grp78?/?and?/?mice. However,
there is no apparent difference in adipocyte size or mor-
phology between the two genotypes, either regular diet fed
or HFD fed (Fig. 3E).
Grp78 heterozygosity improves glucose metabolism
independent of adiposity. To test whether Grp78 het-
erozygosity–mediated improvement on glucose metabolism
is dependent on adiposity, we performed a hyperinsuline-
mic-euglycemic clamp on regular diet–fed mice at the age
of 13 weeks, when there was no significant difference in
body weight and fat mass between the two genotypes (Fig.
4A). Steady-state glucose infusion rates (GINF) to main-
tain euglycemia were significantly elevated in the
mice, corresponding with increased insulin sensitivity
(Fig. 4B). Insulin-stimulated whole-body glucose turnover
and glycolysis both exhibited a trend toward enhancement
in the?/?mice (Fig. 4C). These data suggest that Grp78
heterozygosity improves glucose metabolism and insulin
sensitivity in mice, independent of reduced adiposity. As
supporting evidence, we established MEF cell lines from
stimulation following serum starvation in culture. We ob-
served increase of insulin-stimulated AKT phosphorylation in
Grp78?/?MEFs compared with wild-type MEFs (1.6-fold,
P ? 0.002) (Fig. 4D). Thus, Grp78 heterozygosity improves
insulin sensitivity in MEFs in culture.
?/?mice and subjected them to insulin
Toward further confirmation, we examined metabolic
parameters after 3 weeks of HFD when the body weights
were comparable between the?/?and?/?mice (Fig. 4E).
While both?/?and?/?mice were able to maintain normal
blood glucose levels at both fasting and fed states (Fig.
4F), HFD-fed?/?mice exhibited a 45% decrease in fed
insulin level (1.12 ? 0.11 vs. 2.01 ? 0.21 ng/ml in?/?mice,
P ? 0.002) (Fig. 4G). In addition, blood insulin level was
decreased ?25% in the age-matched 13-week-old regular
diet–fed?/?mice, at both fasting (P ? 0.008) and fed (P ?
0.2) states (Fig. 4G), consistent with the clamp studies
(Fig. 4B and C). This decrease was magnified by the HFD
challenge, since an insulin tolerance test performed at 3
weeks of HFD showed improved glucose clearance for the
?/?mice (Fig. 4H). Collectively, the results show that
Grp78 heterozygosity enhances insulin sensitivity in both
in vivo and in vitro systems.
Grp78?/?mice fed on HFD exhibit a striking increase
in insulin sensitivity in WAT. Toward understanding
how the HFD-fed Grp78?/?mice improve glucose metab-
olism, a hyperinsulinemic-euglycemic clamp was per-
formed after 10–11 weeks of HFD to examine the insulin
sensitivity of individual tissues (Fig. 5A–E). Whole-body
glucose metabolism was significantly elevated in the?/?
mice, both at steady state and upon insulin stimulation,
corresponding with increased insulin sensitivity (Fig. 5A).
Insulin-stimulated whole-body glucose flux (glycolysis and
glycogen plus lipid synthesis) was increased by ?25% in
the?/?mice (Fig. 5B). Organ-specific glucose metabolism
was assessed using 2-deoxy-D-[1-14C]glucose injection dur-
ing clamps. Strikingly, for the?/?mice, insulin-stimulated
glucose uptake was increased by twofold in WAT (P ?
0.001) (Fig. 5C) but was unaltered in skeletal muscle and
FIG. 4. Grp78 heterozygosity improves insulin sensitivity independently of adiposity. A–C: Hyperinsulinemic-euglycemic clamp studies on
13-week-old?/?(n ? 5) and?/?(n ? 4) mice on regular diet (RD). A: Body composition. B: GINF. C: Whole-body glucose turnover and glycolysis
during clamps. D: Immortalized Grp78?/?and?/?MEFs were treated with insulin (100 nmol/l, 15 min) following 5-h serum starvation. Whole cell
lysates were subjected to Western blot for phosphorylated (Ser473) and total AKT. Lanes were run on the same gel but noncontiguous. E–H: For
13-week-old mice on regular diet or after 3-week HFD. E: Fasting body weight (HFD). F: Blood glucose (regular diet and HFD). G: Blood insulin
(regular diet and HFD). (E–G: n > 6 mice per condition.) H: Insulin tolerance test (HFD). n ? 4 (?/?, E) or 5 (?/?, F). Data are presented as the
means ? SE. *P < 0.05; **P < 0.01 for?/?vs.?/?. ?,?/?; f,?/?.
Grp78?/?LESSENS OBESITY AND INSULIN RESISTANCE
10DIABETES, VOL. 59, JANUARY 2010 diabetes.diabetesjournals.org
brown adipose tissue (Fig. 5D). There was a trend toward
increased insulin sensitivity in
lower rate of hepatic glucose production at basal and
clamp states (Fig. 5E). We further observed increases in
insulin-stimulated phosphorylation of IRS-1 (2.1-fold, P ?
0.006) and AKT (1.5-fold, P ? 0.06) in the WAT of the?/?
mice (Fig. 5F), suggesting that Grp78 heterozygosity im-
proves IRS-associated insulin signaling. JNK1 was re-
ported to be upregulated by obesity and to impair insulin
signaling (6,7). A 38% decrease (P ? 0.004) in JNK1 level
was observed in the WAT of the
compared with the HFD-fed?/?mice (Fig. 5G). This is
consistent with the improved insulin signaling that re-
sulted from Grp78 heterozygosity and the recent findings
that JNK1 deletion in adipose tissue increased AKT signal-
ing in HFD-fed mice (20).
Grp78 heterozygosity promotes adaptive UPR and
improves ER homeostasis in WAT. Considering that
GRP78 is well established to protect against ER stress,
which activates inflammation and inhibits insulin signaling
in both genetic and diet-induced obese mouse models (6),
it is unanticipated that Grp78 heterozygosity confers
beneficial metabolic effects. However, chronic ER stress
could elicit adaptive survival responses that improve pro-
tein folding capacity of the ER, while destabilizing the
proapoptotic pathways (21). Partial loss of GRP78 due to
Grp78 heterozygosity could mimic low chronic ER stress
and promote adaptive UPR. To test this, protein lysates
were prepared from WAT of Grp78?/?and?/?mice and
?/?liver, indicated by a
?/?mice on HFD,
the UPR signaling molecules and their downstream targets
were examined by Western blot (supplementary Fig. 3)
and the expression levels were quantitated and summa-
rized (Fig. 6A). The level of Ser51 phosphorylation of the
eukaryotic translation initiation factor eIF2? is a marker
of ER stress and attenuation of global protein translation
(22). In agreement with previous reports that HFD induces
ER stress (6), eIF2? phosphorylation was dramatically
increased in WAT of HFD-fed
0.002), in comparison with the regular diet–fed?/?mice
(Fig. 6A). The regular diet–fed?/?mice showed no signif-
icant elevation of eIF2? phosphorylation, but a 40% de-
crease was observed in HFD-fed?/?mice, compared with
the HFD-fed?/?mice (P ? 0.03).
How might Grp78 heterozygosity reduce diet-induced
eIF2? phosphorylation? As downstream signaling of eIF2?
phosphorylation, upregulation of GADD34 by transcrip-
tion factors ATF4 and CHOP mediates dominant dephos-
phorylation of eIF2?, thus promoting protein synthesis
resumption and recovery from ER stress (23,24). We
observed that while HFD significantly reduced the expres-
sion level of ATF4 and GADD34 in WAT of the?/?mice,
this suppression was not observed in the WAT of the?/?
mice (Fig. 6A). Thus, the ability of the WAT of?/?mice to
maintain expression of the dominant regulator GADD34
under HFD stress could contribute in part to the attenu-
ated eIF2? phosphorylation observed.
The ATF6 and the IRE-1 branches of the UPR signaling
were also analyzed in WAT. We detected an increase in the
?/?mice (2.8-fold, P ?
FIG. 5. Grp78 heterozygosity improves insulin sensitivity predominantly in WAT. A–E: Hyperinsulinemic-euglycemic clamp studies on?/?(f, n ?
6) and?/?(?, n ? 9) mice after 10–11 weeks of HFD. A: Whole-body glucose metabolism indicated by GINF and clamp glucose turnover. B:
Whole-body glycolysis and glucose anabolism during clamps. C and D: Glucose uptake by white adipose (C), skeletal muscle, and brown adipose
(D) during clamps. E: Hepatic glucose production. F and G: Representative Western blots and quantitation of phosphorylation of IRS-1 (Tyr) and
AKT (Ser473) in WAT of mice after clamps (n ? 4 mice per genotype) (F) and JNK1 in WAT of 25-week-old mice on regular diet (RD) or after
15-week HFD (n ? 3–5 mice per condition) (G). ?,?/?; f,?/?. Data are presented as the means ? SE. *P < 0.05; **P < 0.01 for?/?vs.?/?; ##P <
0.01 for HFD vs. regular diet.
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active ATF6 cleaved form in WAT of HFD-fed?/?mice,
compared with the
(1.5-fold, P ? 0.003) and HFD (1.3-fold, P ? 0.02) condi-
tions and the?/?mice under regular diet (1.3-fold, P ?
0.03) conditions (Fig. 6A). As an essential prosurvival
transcription factor downstream of IRE-1 phosphorylation
(2), spliced XBP-1 was strikingly elevated in
either under regular diet–fed (2.2-fold, P ? 0.02) or HFD-
fed (3.6-fold, P ? 0.002) conditions (Fig. 6A and supple-
mentary Fig. 3). One of the protective roles of XBP-1
splicing during ER stress is to promote ERAD and improve
protein quality control by transcriptional activation of a
subset of genes, including EDEM (25). While the level of
EDEM was relatively unchanged among regular diet–fed
significant increase (1.8-fold, P ? 0.001) of EDEM was
observed in WAT of HFD-fed?/?mice in comparison to
suggesting that upon HFD stress, ERAD was induced in
WAT by Grp78 heterozygosity.
To monitor the ER protein folding capacity in WAT, the
levels of ER chaperones GRP78, GRP94, PDI, CNX, and
?/?mice under both regular diet
?/?mice, or HFD-fed
?/?mice, consistently, a
?/?mice (Fig. 6A and supplementary Fig. 3),
CRT, which are downstream targets of adaptive UPR, were
measured by Western blot. As expected, heterozygous loss
of the Grp78 allele in the regular diet–fed
resulted in a partial decrease in GRP78 protein level (37%,
P ? 0.02) (Fig. 6B). On the other hand, GRP94 was
increased by 1.4-fold (P ? 0.03) and PDI by 1.4-fold (P ?
0.009), and no change was detected for CNX and CRT
levels (Fig. 6B). Similar observations were reported for
primary Grp78?/?MEFs (11) and confirmed in the immor-
talized Grp78?/?MEF cell lines used in this study (sup-
plementary Fig. 4A). Therefore, both in MEFs and WAT,
Grp78 heterozygosity induces compensatory upregulation
of GRP94 and PDI in response to partial reduction of
GRP78. Correspondingly, Grp78?/?MEF cell lines exhib-
ited enhanced insulin sensitivity with or without tunica-
mycin-induced ER stress (supplementary Fig. 4B).
However, after 15 weeks of HFD, protein levels of
GRP78, GRP94, PDI, CNX, and CRT were all significantly
reduced (by ?30–65%) in the WAT of the?/?mice (Fig.
6B), despite increases in their mRNA levels (supplemen-
tary Fig. 5A), suggesting translational block or some other
posttranscriptional regulation. To confirm this unantici-
FIG. 6. Grp78 heterozygosity promotes adaptive UPR and improves ER homeostasis in WAT. Whole cell lysates were prepared from WAT of
25-week-old mice on regular diet (RD) or after 15-week HFD (n ? 4–6 mice per condition) and subjected to Western blotting. The protein loading
was normalized against ?-actin. Quantitation of relative protein levels of indicated UPR signaling molecules (A) and ER chaperones (B) are
presented as the means ? SE. *P < 0.05; **P < 0.01 for?/?vs.?/?; #P < 0.05; ##P < 0.01 for HFD vs. regular diet. ?,?/?regular diet; u,?/?regular
diet; p,?/?HFD; f,?/?HFD.
Grp78?/?LESSENS OBESITY AND INSULIN RESISTANCE
12DIABETES, VOL. 59, JANUARY 2010diabetes.diabetesjournals.org
pated finding, we expanded the observation on?/?mice
after 3, 7, 10, or 24 weeks of HFD. Compared with the
age-matched regular diet–fed?/?mice, protein levels of
ER chaperones were significantly reduced in WAT after 10
weeks of the HFD regimen (supplementary Fig. 5B).
Interestingly, for the HFD-fed?/?mice, GRP78 level was
nearly comparable to the?/?mice, and all the other ER
chaperone levels (GRP94, PDI, CNX, and CRT) were
upregulated by about twofold compared with the HFD-fed
?/?mice (Fig. 6B). These results suggest that HFD stress
could decrease the ER protein folding capacity in the WAT
of the?/?mice; however, Grp78 heterozygosity promotes
adaptive responses in WAT to maintain ER chaperone
synthesis, which may contribute in part to the metabolic
benefits. Consistent with this notion, in skeletal muscle of
the?/?mice, where insulin sensitivity was not improved
under HFD conditions (Fig. 5D), compensatory ER chap-
erone upregulation was not observed (supplementary Fig.
6A). In the?/?liver, showing a trend of enhanced insulin
sensitivity after HFD (Fig. 5E), the level of GRP94, but not
the other ER chaperones, was upregulated (supplemen-
tary Fig. 6B).
Recently, expression of metabolic genes was suggested
to directly respond to ER homeostasis (26). Correspond-
ing to the adaptive UPR and improved protein folding
capacity in Grp78?/?WAT, we observed that the expres-
sion level of PGC-1?, a transcriptional coactivator and
regulator of mitochondrial energy metabolism and biogen-
esis (27), was enhanced in the WAT of the regular diet–fed
Grp78?/?mice (30% increase, P ? 0.03) (Fig. 7A). Follow-
ing 15 weeks of HFD, while the?/?mice showed a 74%
decrease in PGC-1? level in WAT compared with the
showed a 90% increase compared with the HFD-fed?/?
mice (P ? 0.004) (Fig. 7A). Interestingly, GRP75, a mito-
chondrial chaperone mediating the coupling of ER and
mitochondrial Ca2?channels (28), was also upregulated in
WAT of regular diet–fed (1.2-fold, P ? 0.02) and HFD-fed
(1.5-fold, P ? 0.008) Grp78?/?mice (Fig. 7B).
As proof of principle that induction of protective UPR
leads to improved insulin sensitivity under ER stress, we
enforced expression of the active, nuclear form of ATF6
[ATF6(N)] in immortalized wild-type MEF cell line and
tested for insulin sensitivity. The expression of HA-tagged
ATF(N) was confirmed by Western blot in the transfected
cells. Compared with cells transfected with the empty
vector, cells expressing ATF(N) showed moderate upregu-
lation of GRP94, GRP78, and CRT, and upon treatment of
cells with ER stress inducer tunicamycin for 14 h, the
overall level of these chaperones was further enhanced
Insulin sensitivity of the cells was monitored by Ser473
phosphorylation of AKT upon insulin stimulation follow-
ing serum starvation. In nonstressed cells, no significant
difference between MEFs transfected with HA-ATF(N)
and the vector control was observed. ER stress induced by
tunicamycin treatment severely impaired insulin signaling
in the control cells (82%, P ? 0.0003) (Fig. 8B). However,
in tunicamycin-treated cells, ATF6(N) overexpression
showed significant improvement in insulin sensitivity (3.3-
fold, P ? 0.01) compared with the vector-transfected cells
(Fig. 8B). These in vitro experiments support our in vivo
observations that adaptive UPR triggered by Grp78 het-
erozygosity might contribute to the improved insulin sen-
sitivity under HFD-induced ER stress.
?/?mice (P ? 0.00005), the
While the role of ER stress in energy balance and meta-
bolic homeostasis is still emerging, the notion that adap-
tive UPR may confer beneficial effects is supported by the
discovery that expression of metabolic gene network is
directly responsive to ER homeostasis (26). ER chaper-
ones could also be important for energy and metabolic
homeostasis. Homozygous knockout of CRT in mice leads
to postnatal growth retardation, hypoglycemia, and in-
creased levels of serum triglycerides and cholesterol (29).
CRT and CNX modulate insulin signaling through associ-
ation and stabilization of the insulin receptor (30,31).
Thus, ER chaperones, either individually or collectively,
may be protective against metabolic stress, consistent
with the finding that chemical chaperones that modulate
ER stress and increase ER folding capacity improve sys-
temic insulin action (8). Here, we demonstrate that Grp78
heterozygosity triggers adaptive UPR, resulting in attenu-
ation of translational block, increase in GRP94 and PDI,
and improvement of insulin sensitivity, particularly in
WAT. Recent studies reported that the chemical chaper-
one 4-phenylbutyrate, in its role as modulator of the UPR,
reduces weight gain in HFD-fed C57BL/6 mice by inhibit-
ing adipogenesis (32). This provides a potential mecha-
nism whereby improvement of ER homeostasis could
result in reduced obesity. We observed that HFD led to
increase in adipocyte size in both Grp78?/?and?/?mice
and there is no apparent difference in adipocyte size or
FIG. 7. Grp78 heterozygosity upregulates PGC-1? and GRP75 in WAT.
Whole cell lysates from WAT of 25-week-old mice on regular diet (RD)
or after 15-week HFD (n ? 4–6 mice per condition) were subjected to
Western blotting for PGC-1? (A) and GRP75 (B). In the representative
blots, lanes that were run on the same gel but noncontiguous are
divided by lines. Quantitative protein levels are presented as the
means ? SE. *P < 0.05; **P < 0.01 for?/?vs.?/?; #P < 0.05; ##P < 0.01
for HFD vs. regular diet. ?,?/?; f,?/?.
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diabetes.diabetesjournals.orgDIABETES, VOL. 59, JANUARY 201013
morphology between the two genotypes, either regular
diet fed or HFD fed. Nonetheless, CHOP, a known inhibi-
tor of C/EBP? critical for adipogenesis (33), is induced in
the WAT of Grp78?/?mice. This, coupled with decreased
fat mass, suggests that adipogenesis might be inhibited in
Grp78?/?mice via CHOP induction in preadipocytes.
However, these remain to be established in future exper-
iments. ER stress also contributes to the activation of
inflammatory response and insulin/leptin resistance in
hypothalamus, which could be part of the underlying
mechanism of diet-induced obesity (34). Whether Grp78
heterozygosity improves ER homeostasis in hypothalamic
neurons and protects against HFD-induced obesity awaits
In summary, while the mechanisms for observed pheno-
types of the Grp78?/?mice are likely to be complex and
involve multiple pathways that await further investiga-
tions, based on our results, we propose a working model
as part of the contributing factors for the attenuation of
diet-induced obesity and insulin resistance for the
Grp78?/?mice (supplementary Fig. 7). We speculate that
because of the critical requirement of GRP78 in maintain-
ing ER homeostasis and the extra demand for protein
synthesis and trafficking in the ER of WAT magnified by
exposure to HFD, compensatory adaptive responses are
activated, including upregulation of ER chaperones and
ERAD, which improves ER homeostasis and attenuates
inflammation, consequentially improving insulin signaling
and glucose metabolism. The other beneficial effects from
the improved ER homeostasis may include increases in
mitochondrial activity and energy expenditure. These ef-
fects of Grp78 heterozygosity mediate, in part, the resis-
tance to diet-induced obesity, insulin resistance, and type
2 diabetes. Our studies also suggest that how UPR path-
ways are regulated in vivo in specific tissues under phys-
iologic stress is likely to be more complex than what has
been reported for tissue culture cells treated with phar-
macological reagents. Thus, in adipose tissues subjected
to diet-induced metabolic stress, we observed sustained
eIF2? phosphorylation in the WAT of the HFD-fed
Grp78?/?mice, correlating with translational block and
downregulation of UPR markers. In contrast, eIF2? phos-
phorylation was much reduced in the WAT of Grp78?/?
mice, consistent with upregulation of UPR markers from
resumption of protein synthesis following feedback inhi-
bition of eIF2? phosphorylation by GADD34. It is also
possible that other yet unknown pathways may contribute
to their upregulation.
Recently, it has been reported that GRP78 overexpres-
sion inhibits insulin and ER stress–induced sterol regula-
tory element–binding protein-1c activation and reduces
hepatic steatosis in the ob/ob mice (35). These new find-
ings support the notion that chaperone balance modulates
insulin sensitivity. In their study, increase in chaperone
activity is achieved through adenoviral GRP78 injection
into mice; in our study, Grp78 heterozygosity results in
compensatory increase of GRP94 and PDI, as well as other
adaptive UPRs. Their finding that GRP78 overexpression
attenuates hepatic insulin resistance is in agreement with
our observation of improved insulin signaling in MEFs,
where GRP78 and other ER chaperone levels were ele-
vated upon overexpression of active ATF6.
Potential exciting links between mitochondria function
and energy expenditure to chaperone proteins have re-
cently been reported. One explanation why GRP78 level
might affect energy expenditure may lie in its property as
p-Ser / total AKT (arbitrary unit)
-++ + +-- -
FIG. 8. Overexpression of active ATF6 improves insulin sensitivity in MEFs under ER stress. A HA-tagged nuclear form of ATF6 [ATF6(N)]
was overexpressed in immortalized wild-type MEFs via transient transfection 72 h prior to insulin stimulation, controlled by the empty
vector. Transfected MEFs were treated with tunicamycin (Tu, 1.5 ?g/ml) or DMSO for 14 h before insulin stimulation. Following 5 h serum
starvation, cells were treated with insulin (100 nmol/l, 15 min). A: Whole cell lysates were immunoblotted for indicated proteins. B:
Quantitation of AKT Ser473 phosphorylation is presented as the means ? SE. *P < 0.05 for?/?vs.?/?; ##P < 0.01 for HFD vs. regular diet.
?, vector; f, ATF6(N).
Grp78?/?LESSENS OBESITY AND INSULIN RESISTANCE
14DIABETES, VOL. 59, JANUARY 2010 diabetes.diabetesjournals.org
an ER Ca2?binding protein. Partial reduction in GRP78
level could lead to increased Ca2?efflux from the ER to
the cytosol. Modulation of cytosolic Ca2?levels has been
reported to affect energy metabolism (36). There is evi-
dence that the members of the GRP family are important
regulators of mitochondria function. For instance, GRP94,
which is upregulated by Grp78 heterozygosity, is reported
to molecularly interact with GRP75, an essential mito-
chondrial chaperone that imports mitochondrial proteins
into the matrix (37). GRP75 is in turn physically linked to
the voltage-dependent anion channel and mediates the
coupling of ER and mitochondrial Ca2?channels (28). Our
studies revealed that the level of GRP75, as well as
PGC-1?, is elevated in WAT of Grp78?/?mice. Increases
in mitochondrial biogenesis and function are associated
with elevated energy expenditure. We have recently
discovered that in cell cultures where endogenous
GRP78 was depleted by siRNA, the ER was expanded,
coupled with a substantial increase in mitochondria
quantity (38). In view of the emerging evidence indicat-
ing that ER and the mitochondria are linked physically
and functionally at least in part through chaperone
interaction, the Grp78?/?mouse model will provide a
novel experimental system for future investigations into
this exciting new area. Furthermore, with the recent
establishment of the floxed Grp78 mouse model (39),
future creation of tissue-specific deletion of GRP78 will
identify directly the target tissues of GRP78 function in
This work is supported in part by National Institutes of
Health (NIH) Grants CA027607, DK070582, and DK079999
to A.S.L.; NIH Grant TDK80756 to J.K.K.; American Diabe-
tes Association Grants 1-04-RA-47 and 7-07-RA-80 to J.K.K.;
and the Pennsylvania Department of Health Tobacco
Settlement Funds to J.K.K.
No potential conflicts of interest relevant to this article
We thank Drs. Richard Bergman and Hooman Allayee
and members of the Lee lab for helpful discussions and the
Bergman lab for assistance on insulin ELISA. We thank Dr.
Stanley Korsmeyer for providing the SV40-immortalized
wild-type MEFs. We also thank Ernesto Barron at the Cell
and Tissue Imaging Core facility of the University of
Southern California/Norris Comprehensive Cancer Center
for technical assistance on the electron microscopy. We
thank Dr. Guadalupe Sabio at Dr. Roger Davis lab (Uni-
versity of Massachusetts Medical School) for assistance on
the JNK1 measurement.
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