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.
[show abstract] [hide abstract]
ABSTRACT: Eukaryotic cells coordinate protein-folding reactions in the endoplasmic reticulum with gene expression in the nucleus and messenger RNA translation in the cytoplasm. As the rate of protein synthesis increases, protein folding can be compromised, so cells have evolved signal-transduction pathways that control transcription and translation — the 'unfolded protein response'. Recent studies indicate that these pathways also coordinate rates of protein synthesis with nutrient and energy stores, and regulate cell differentiation to survive nutrient-limiting conditions or to produce large amounts of secreted products such as hormones, antibodies or growth factors.Nature Reviews Molecular Cell Biology 05/2002; 3(6):411-421. · 39.12 Impact Factor
[show abstract] [hide abstract]
ABSTRACT: Cells respond to the accumulation of unfolded proteins by activating signal transduction cascades that improve protein folding. One example of such a cascade is the unfolded protein response (UPR), which senses protein folding stress in the endoplasmic reticulum (ER) and leads to improvement in the protein folding and processing capacity of the organelle. A central paradox of the UPR, and indeed of all such stress pathways, is that the response is designed to facilitate both adaptation to stress and apoptosis, depending upon the nature and severity of the stressor. Understanding how the UPR can allow for adaptation, instead of apoptosis, is of tremendous physiological importance. Recent advances have improved our understanding of ER stress and the vertebrate UPR, which suggest possible mechanisms by which cells adapt to chronic stress.Trends in Biochemical Sciences 11/2007; 32(10):469-76. · 10.85 Impact Factor
[show abstract] [hide abstract]
ABSTRACT: The field of endoplasmic reticulum (ER) stress in mammalian cells has expanded rapidly during the past decade, contributing to understanding of the molecular pathways that allow cells to adapt to perturbations in ER homeostasis. One major mechanism is mediated by molecular ER chaperones which are critical not only for quality control of proteins processed in the ER, but also for regulation of ER signaling in response to ER stress. Here, we summarized the properties and functions of GRP78/BiP, GRP94/gp96, GRP170/ORP150, GRP58/ERp57, PDI, ERp72, calnexin, calreticulin, EDEM, Herp and co-chaperones SIL1 and P58(IPK) and their role in development and diseases. Many of the new insights are derived from recently constructed mouse models where the genes encoding the chaperones are genetically altered, providing invaluable tools for examining the physiological involvement of the ER chaperones in vivo.FEBS Letters 08/2007; 581(19):3641-51. · 3.54 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 2224 2628 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
8DIABETES, VOL. 59, JANUARY 2010diabetes.diabetesjournals.org
Blood Insulin (ng/mL)
10 121416 18
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 2010diabetes.diabetesjournals.org