Sarco(endo)plasmic reticulum Ca2+-ATPase 2b is
a major regulator of endoplasmic reticulum
stress and glucose homeostasis in obesity
Sang Won Park, Yingjiang Zhou, Jaemin Lee, Justin Lee, and Umut Ozcan1
Division of Endocrinology, Children’s Hospital Boston, Harvard Medical School, Boston, MA 02115
Edited* by Michael Karin, University of California at San Diego, School of Medicine, La Jolla, CA, and approved September 22, 2010 (received for review
August 13, 2010)
Increased endoplasmic reticulum (ER) stress is one of the central
mechanisms that lead to dysregulated metabolic homeostasis in
obesity. It is thus crucial to understand the underpinnings of the
mechanisms that lead to the development of ER stress. A high level
of ER Ca2+ is imperative for maintenance of normal ER function and
this high Ca2+ concentration of ER is maintained by sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA). Here, we show that SERCA2b
protein and mRNA levels are dramatically reduced in the liver of
obese mice and restoration of SERCA2b in the liver of obese and
diabetic mice alleviates ER stress, increases glucose tolerance, and
significantly reduces the blood glucose levels. Furthermore, overexpression of SERCA2b in the liver of obese mice significantly reduces
the lipogenic gene expression and the triglyceride content in the
liver. Our results document the importance of SERCA2b in dysregulated glucose and lipid homeostasis in the liver of obese mice and
suggest development of drugs to increase SERCA2b activity for treatment of type 2 diabetes and nonalcoholic steatohepatitis.
|
obesity type 2 diabetes
calcium
| unfolded protein response | insulin resistance |
O
besity is one of the greatest public health concerns (1) and it
is a major underlying pathology for development of several
serious medical conditions, such as type 2 diabetes, nonalcoholic
steatohepatitis (NASH), and cardiovascular disease (2–4). Despite intense research efforts, the molecular links among obesity,
type 2 diabetes, and NASH are not well understood.
The endoplasmic reticulum (ER) is a cellular organelle where
secretory and membrane proteins are folded into their lowenergy, 3D structures. In addition to protein folding, the ER also
plays a central role in lipid and cholesterol biosynthesis (5–7).
Conditions that interfere with the folding capacity of the ER, or that
increase the folding demand beyond the levels that ER can cope
with, create a condition known as ER stress, which in turn leads to
an initiation of a complex signaling cascade called the unfolded protein response (UPR) (5–7). Three ER membrane proteins, PKR-like ER kinase (PERK), inositol-requiring enzyme-1
(IRE1), and activating transcription factor-6 (ATF6), are the main
players in the initiation of the main signaling arms of the UPR (5–8).
In recent years, we and others have shown that increased ER
stress is a key contributor to the development of glucose intolerance
and insulin resistance in obesity (9–14). ER stress and activated
UPR signaling pathways also create severe leptin resistance in the
hypothalamus and play a significant role in the development of
obesity (15). Considering the contribution of ER stress to obesityrelated pathologic processes and the development of obesity itself,
it is of crucial importance to understand the pathological mechanisms that create ER stress in obesity conditions. We also demonstrated that ER folding capacity is significantly reduced in obese
mice, preventing the ER from responding properly to metabolic
overload and resulting in activation of the UPR in the fed state (13).
Moreover, the chaperone response is completely blunted in obese
mice as a result of the inability of X-Box binding protein 1 (XBP1),
a master regulator of ER folding capacity, to move to the nucleus as
19320–19325 | PNAS | November 9, 2010 | vol. 107 | no. 45
a result of the loss of interaction between XBP1 and p85α and p85β,
the regulatory subunits of PI3K (13).
In addition to being the main organelle in which free calcium is
stored, operations in ER lumen depend heavily upon intraluminal
calcium concentrations (6, 16, 17): high calcium levels are critical
for optimum activity of different enzymes and chaperones that
have key roles in protein folding and ER homeostasis (6, 16, 17).
Perturbations in ER-luminal Ca2+ inhibit chaperone function and
agents that disturb calcium homeostasis in the ER create severe
stress in this organelle. For example, thapsigargin, an inhibitor of
sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA), blocks
reuptake of Ca2+ from the cytoplasm and creates a chaotic environment in the lumen of the ER, leading to ER stress and
subsequent initiation of the UPR (18). The same is true for Ca2+
ionophores, such as A23187, which is also known to disrupt ER
Ca2+ homeostasis and create severe ER stress (5–7, 17).
SERCA is a Ca2+-transport ATPase, whose only known function is the reuptake of Ca2+ from the cytosol into the ER lumen.
In mammals, three different SERCA genes (ATP2A1–3) lead to
generation of three different isoforms (SERCA1–3), each of
which has at least two further subisoforms. SERCA2 is by far the
most widespread of all SERCA isoforms and has two well known
subisoforms: SERCA2a and SERCA2b. The main isoform of
SERCA2 in the liver is SERCA2b (16, 17).
In the current report, we demonstrate that SERCA2b protein
and mRNA levels are dramatically reduced in the liver of ob/ob
mice and increasing the levels of SERCA2b greatly reduces ER
stress in the liver, increases glucose tolerance, and establishes
euglycemia in severely obese and diabetic mice.
Results
SERCA2b Levels Are Dramatically Reduced in the Liver of Obese Mice.
In light of the central role of Ca2+ in ER homeostasis and major
contribution of SERCA2b in keeping ER calcium levels at optimally high concentrations, we investigated whether perturbations in the protein and gene expression levels of SERCA2b can
be detected in obese states compared with lean conditions. For
this purpose, we first determined the SERCA2b protein in whole
liver lysates of lean and obese mice; we observed a significant
(P < 0.001) reduction in protein levels in the liver of ob/ob mice
relative to lean controls (Fig. 1 A and B). Next, we isolated ER
fractions from the liver of lean and ob/ob mice and determined
SERCA2b protein levels by immunoblotting. In parallel to the
results obtained from whole liver lysates, SERCA2b protein
levels were significantly (P < 0.01) reduced in the isolated liver
ER fractions of the ob/ob mice compared with those of lean
controls (Fig. 1 C and D). As a loading control, we analyzed the
level of IRE1α that resides in ER-membrane (Fig. 1C). We also
Author contributions: S.W.P. and U.O. designed research; S.W.P., Y.Z., Jaemin Lee, Justin
Lee, and U.O. performed research; S.W.P. and U.O. analyzed data; S.W.P. and U.O. wrote
the paper.
The authors declare no conflict of interest.
*This Direct Submission article had a prearranged editor.
1
To whom correspondence should be addressed. E-mail: [email protected].
edu.
www.pnas.org/cgi/doi/10.1073/pnas.1012044107
Regulation of SERCA2b Levels During Metabolic Overload Is Blunted
in Obese Mice. We have previously shown that components of ER
machinery are up-regulated in the liver of WT mice during
refeeding after a fasting period to cope with the increasing demand (13). In the mean time, this response was completely lost
in the obese mice (13). To investigate whether SERCA2b, which
is one of the most crucial elements for ER homeostasis in the
liver, is also regulated during refeeding, we fasted 8-wk-old lean
WT mice for 24 h and refed for 1, 3, and 5 h. SERCA2b levels in
the liver of WT lean mice were significantly increased during
refeeding (Fig. 1I). We next analyzed SERCA2b levels at 24 h
fasting and after 1, 3, and 5 h of refeeding in the liver of ob/ob
mice. Of interest, up-regulation of SERCA2b levels during refeeding was lost in the liver of the ob/ob mice (Fig. 1J). These
results prompted us to test whether SERCA2b is regulated by
XBP1s, as we have previously shown that XBP1s nuclear translocation is defective in obesity conditions and restoration of the
ability of XBP1s to translocate to the nucleus has reinstated the
chaperone response (13). For this purpose, we overexpressed
XBP1s in the liver of ob/ob mice by tail vein injection of XBP1sexpressing adenovirus (Ad-XBP1s). LacZ-expressing adenovirus
(Ad-LacZ) were used as a control. Injection of Ad-XBP1s into
the tail vein of ob/ob mice led to a significant increase in the expression of XBP1s in the liver (Fig. 1K). Analysis of protein and
mRNA levels of SERCA2b revealed a dramatic augmentation in
the XBP1s-overexpressed livers, indicating that XBP1s is one of
the regulators of SERCA2b in the liver (Fig. 1 L and M).
SERCA2b Increases ER Folding Capacity. It is very well known that
a reduction in SERCA function leads to development of ER stress
(6, 16, 17). We thus sought to investigate whether an increase in
SERCA function leads to a corresponding up-regulation of ER
folding capacity and makes the ER more resistant to various stresscausing stimuli. To test this, we created an adenovirus that encodes
mouse SERCA2b (Ad-SERCA2b). Infection of mouse embryonic
fibroblasts (MEFs) with Ad-SERCA2b increased levels of
mRNA and protein for SERCA2b, relative to infection with
LacZ-expressing adenovirus (Ad-LacZ) controls (Fig. 2 A and B).
To explore whether overexpression of SERCA2b increases resistance of the MEFs to ER stress, we infected the cells with AdLacZ or Ad-SERCA2b; following overnight starvation in 0.5%
FBS-containing medium, we stimulated the MEFs with DMSO,
thapsigargin (0.5 nM), or tunicamycin (0.0625 μg/mL) for various
amounts of time and analyzed phosphorylation of PERK as an
indicator of ER stress. SERCA2b overexpression dramatically
reduced the thapsigargin- (Fig. 2C) and tunicamycin-induced ER
stress (Fig. 2D) as evinced by delayed PERK phosphorylation.
We had expected that overexpression of SERCA2b would make
the cells more resistant to thapsigargin-induced ER stress because
this agent is a direct inhibitor of SERCA. However, the ability
of SERCA2b to increase the resistance of the cells even to
tunicamycin-induced ER stress indicated that SERCA2b can increase ER folding capacity, as tunicamycin creates ER stress by
blocking N-glycosylation and increasing the amount of unfolded
proteins in the ER lumen.
Overexpression of SERCA2b in the Liver of Obese Mice Reduces ER
Stress and Improves Glucose Homeostasis. Considering the central
role of ER stress in glucose homeostasis and the role of SERCA in
keeping ER capacity intact and the observation that SERCA2b
levels were dramatically reduced in the liver of ob/ob mice, we asked
whether increasing the levels of SERCA2b in the liver of obese mice
would have an influence on glucose homeostasis. For this purpose, 8-wk-old male ob/ob mice were injected with 5.8 × 107 pfu/g of
Ad-SERCA2b or Ad-LacZ. Analysis of the expression levels for
Fig. 1. SERCA2b protein and mRNA
1.2
levels are decreased in the liver of
1.2
**
***
Lean
ob/ob
Lean
ob/ob
1
obese mice. (A) SERCA2b and tubulin
1
0.8
immunoblotting from the whole
0.8
SERCA2b
SERCA2b
liver lysates of lean WT and ob/ob
0.6
0.6
Tubulin
IRE1α
mice. (B) Quantification of the West0.4
0.4
ern blot showing the ratio of SER0.2
0.2
CA2b to tubulin. (C) SERCA2b and
0
0
Lean
ob/ob
Lean
ob/ob
IRE1α immunoblotting in the isolated
liver ER fractions of WT and ob/ob
1.2
1.2
1.4
mice. (D) Quantification of the blot
*
**
*
1.2
1
1
in the ratio of SERCA2b to IRE1α. (E)
Diet
High
Fat
Normal
1
mRNA levels of SERCA2b in the liver
0.8
0.8
0.8
of WT and ob/ob mice. 18S was used
SERCA2b
0.6
0.6
0.6
as a control. (F) Western blot for
0.4
0.4
Tubulin
0.4
SERCA2b and tubulin in the whole
0.2
0.2
liver lysates of mice that were kept
0.2
0
0
on normal or high fat diet for 16 wk.
0
Lean
ob/ob
ND
HFD
ND
HFD
(G) Quantification of the blot showing the ratio of SERCA2b/tubulin. (H)
XBP1s
LacZ
4.5
***
10
10
***
Quantitative PCR for SERCA2b in the
4
***
liver of normal diet and high-fat diet
XBP1s
8
8
3.5
mice. (I) Fold increase of SERCA2b
*
Akt
3
6
6
mRNA expression during 1, 3, and 5 h
2.5
of refeeding after 24 h of fasting in
2
4
4
lean WT mice. (J) Fold increase of
1.5
LacZ
XBP1s
1
SERCA2b mRNA expression during
2
2
SERCA2b
0.5
refeedings after 24 h of fasting in ob/
0
0
0
Tubulin
ob mice. (K–M) Seven-week-old male
0
1
3
5
0
1
3
5
LacZ
XBP1s
Time (h)
Time (h)
ob/ob mice were injected with Ad-LacZ
or Ad-XBP1s (4 × 108 pfu/g) through
a tail vein. (K) Western blots for the levels of XBP1s and Akt as a control in the liver of adenovirus-injected ob/ob mice on day 7 after injection. (L) SERCA2b and
tubulin levels in the liver of Ad-LacZ or Ad-XBP1s injected mice on day 7 after injection. (M) mRNA levels of SERCA2b in the liver of Ad-LacZ and Ad-XBP1s
injected mice with 18S level as a control. Error bars are SEMs; P values were determined by Student t test (*P < 0.05, **P < 0.01, ***P < 0.001).
SERCA2b/IRE1α
SERCA2b/Tubulin
L
H
M
Total Lysate
Fold Increase
Fold Increase
K
Total Lysate
Total Lysate
SERCA2b/18S
J
I
G
SERCA2b/18S
F
E
D
SERCA2b/18S
Total Lysate
C
ER
B
SERCA2b/Tubulin
A
Park et al.
PNAS | November 9, 2010 | vol. 107 | no. 45 | 19321
CELL BIOLOGY
determined levels of SERCA2b mRNA levels in the liver of lean
and ob/ob mice; SERCA2b mRNA was significantly (P < 0.05)
down-regulated in the liver of ob/ob mice (Fig. 1E). Next, we
analyzed the SERCA2b protein and mRNA levels in the liver of
normal diet-fed lean and high-fat diet-fed obese mice. In parallel
to the results obtained from ob/ob mice, protein and mRNA
levels of SERCA2b were also markedly reduced in the liver of
the diet-induced obesity model (Fig. 1 F–H).
SERCA2b on postinjection day 8 confirmed the successful upregulation of protein as well as mRNA in the liver of ob/ob mice
(Fig. 3 A and 3B). Consistent with our hypothesis, overexpression of
SERCA2b in the liver of ob/ob mice significantly lowered blood
glucose levels as early as 3 d after injection (Fig. 3C) and analysis at
8 d after injection revealed a significant decrease in circulating insulin levels in the Ad-SERCA2b-injected ob/ob mice relative to the
Ad-LacZ controls (Fig. 3D). We next performed a glucose tolerance test (GTT) on postinjection day 4 and showed that the hyperglycemic response to the i.p. glucose challenge was significantly
reduced in SERCA2b-overexpressing ob/ob mice compared with
the Ad-LacZ–injected group (Fig. 3E). Also, an insulin tolerance
test (ITT) done on day 7 demonstrated that insulin-stimulated
disposal of glucose was enhanced in the Ad-SERCA2b-injected ob/
ob mice compared with control levels (Fig. 3F). The body weight of
the Ad-LacZ– and Ad-SERCA2b–injected mice remained unchanged throughout the course of the experiment (Fig. 3G).
Our working hypothesis that up-regulation of SERCA2b function
increases ER folding capacity by increasing the efficiency of chaperones led us to anticipate that, after overexpression of SERCA2b,
ER stress in the liver of ob/ob mice would resolve without up-regulation of the chaperones. This was indeed the case, as shown in Fig. 3
H and I; IRE1αSer724 phosphorylation was significantly reduced, but
no accompanying increase was noted in the mRNA levels for chaperones such as protein disulphide isomerase (PDI), ER-localized
DnaJ homologue 4 (ERDJ4), calnexin, calreticulin (Calr), ER degradation enhancing α-mannosidase-like protein (EDEM), ER oxidoreductase (ERO1α), homocysteine-inducible ER stress protein
(HERP), glucose-regulated protein, 58-kDa (GRP58), and GRP78.
In fact, we observed a slight decrease in the mRNA levels of some of
these chaperones (Fig. 3J). These results indicate that up-regulation
of SERCA2b levels and consequent increased Ca2+ levels in the
lumen of ER can establish the homeostasis with already available
chaperones and reduce the activation of UPR. Furthermore, we
demonstrated that genes that are involved in regulation of glucose
homeostasis, such as glucose 6 phosphatase (G6PAse), phosphoenolpyruvate carboxykinase (PEPCK), and peroxisome proliferatoractivated receptor γ coactivated-1 α (PGC1α), were significantly
reduced in the SERCA2b-overexpressing ob/ob mice (Fig. 3K).
Overexpression of SERCA2b Reduces Steatohepatitis in Obese Mice.
Next, to investigate whether overexpression of SERCA2b would
reduce triglyceride (TG) levels in the liver of ob/ob mice, we examined H&E-stained sections taken from the liver of ob/ob mice that
A
SERCA2b/18S
12
B
***
10
LacZ
SERCA2b
8
SERCA2b
6
Tubulin
4
2
0
LacZ
C
LacZ
SERCA2b
SERCA2b
0 1.5 3 0 1.5 3
Tg (h)
D
LacZ
SERCA2b
0 0.5 1 1.5 2 2.5 3 0 0.5 1 1.5 2 2.5 3
Tm (h)
P-PERK
P-PERK
SERCA2b
SERCA2b
Tubulin
Tubulin
Fig. 2. SERCA2b increases resistance of cells to ER stress. (A) qPCR analysis of
the mRNA levels of SERCA2b in the Ad-LacZ and Ad-SERCA2b-infected cells. (B)
SERCA2b immunoblotting in the whole lysates of MEFs infected with Ad-LacZ
or Ad-SERCA2b. Tubulin was used as a control. (C) MEFs was infected with AdLacZ or Ad-SERCA2b for 12 h and treated with thapsigargin (0.5 nM) for 0, 1.5,
and 3 h. Immunoblotting was performed for phospho-PERKThr980, SERCA2b,
and tubulin levels. (D) Ad-LacZ or Ad-SERCA2b-infected MEFs were stimulated
with tunicamycin (0.0625 μg/mL) for 0, 0.5, 1, 1.5, 2, 2.5, and 3 h, and total
lysates were blotted against phospho-PERKThr980, SERCA2b, and tubulin.
19322 | www.pnas.org/cgi/doi/10.1073/pnas.1012044107
were injected with Ad-LacZ or Ad-SERCA2b. We noted a marked
decrease in the amount of lipid droplets in the liver of SERCA2b
overexpressing ob/ob mice compared with the controls (Fig. 4A).
Evaluation of TG content in the liver of Ad-LacZ and Ad-SERCA2b-injected ob/ob mice revealed a significant reduction in TG
levels (in mg/g) associated with the Ad-SERCA2b overexpression
(Fig. 4B). Finally, the expression profile of lipogenic genes showed
that mRNA levels for stearoyl-CoA desaturase-1 (SCD1), diacylglycerol acyltransferase 2 (DGAT2), fatty acid synthase (FASn),
and acetyl co-A carboxylase 2 (ACC2) were dramatically reduced,
without significant alterations in ACC1 and sterol regulatory element binding protein 1c (SREBP1c) levels (Fig. 4 C–H).
Overexpression of SERCA2b Does Not Alter Glucose Homeostasis in
Lean Mice. One of the remaining questions was whether over-
expression of SERCA2b in lean and healthy mice could lead to
development of hypoglycemia, which would be an obstacle in the
approaches to activate SERCA2b as a therapeutic option for the
treatment of type 2 diabetes. For this purpose, 8-wk-old male WT
mice were injected with 5.8 × 107 pfu/g of Ad-SERCA2b or AdLacZ through a tail vein. Blood glucose levels were measured on
postinjection days 3, 7, and 9 (Fig. 5A). No differences were observed in the blood glucose levels of the LacZ and SERCA2bexpressing WT mice. Performance of ITT on postinjection day 6
(Fig. 5B) demonstrated that insulin sensitivity was not altered.
Body weight of both groups during the course of experiment displayed no difference (Fig. 5C).
Discussion
In 2005, the World Health Organization reported that 1.6 billion
adults worldwide were overweight and 400 million were obese.
Obesity is the leading cause of several debilitating diseases such as
type 2 diabetes, NASH, cardiovascular disease, and stroke (2–4). In
particular, type 2 diabetes and NASH are most prominently associated with obesity (1). The World Health Organization estimated
that 171 million people worldwide had type 2 diabetes in 2000, and
this number is projected to increase to 366 million by 2030 as
a result of an uncontrolled increase in the incidence of obesity. To
develop new and effective therapeutic agents to treat these diseases, it is of crucial importance to understand the pathological
mechanisms that link obesity to type 2 diabetes and NASH.
During the past decade, we have witnessed the emerging role of
ER stress in the pathology of several obesity-related diseases:
increased ER stress in obesity is linked to glucose intolerance,
insulin resistance, and ultimately to type 2 diabetes, and chemical
reduction of ER stress greatly enhances metabolic homeostasis in
severely obese and diabetic mice (10–12, 14, 15). These observations provide support for the view that the ER system is an attractive target for the treatment of obesity and related pathologic
processes such as insulin resistance and type 2 diabetes. However,
despite increasing efforts in this area of research, the pathogenesis
of ER stress in obesity conditions is not well understood.
Our current work indicates that level of SERCA2b, which is one of
the key players in the maintenance of ER homeostasis, is severely
reduced in the liver of obese mice. Furthermore, we document that,
when WT lean mice were challenged with refeeding after a fasting
period, the liver SERCA2b level was significantly increased. These
results indicate that SERCA2b is also dynamically regulated to
enhance the efficiency of ER system to cope with demanding conditions. We have previously shown that obese mice cannot up-regulate the ER chaperones during refeeding compared to lean mice
(13). In our current work, we document that SERCA2b up-regulation
is also blunted in the liver of obese mice during refeeding. In addition
to blunted chaperone response, lack of proper SERCA2b regulation
during refeeding could be playing a role in the development of ER
stress in obese mice. Indeed, we showed that restoration of SERCA2b
abundance in the liver of ob/ob mice alleviates ER stress, significantly
improves glucose tolerance, and establishes euglycemia.
One of the important questions that need to be answered is how
SERCA2b is regulated in obesity conditions. Our results indicate
that XBP1s is one of the factors that regulate SERCA2b expresPark et al.
C
SERCA2b
Total Lysate
LacZ
SERCA2b/18S
7
SERCA2b
Tubulin
6
5
4
3
2
1
250
200
150
100
50
SERCA2b
***
** ***
*
0
15
30
60
******
SERCA2b
*
* ***
0
90 120
15
Total Lysate
SERCA2b
Arbitary Units
P-IRE1
IRE1α
Phospho IRE1α/Total IRE1α
I
H
J
60
*
4
2
LacZ
SERCA2b
LacZ
SERCA2b
50
40
30
20
10
0
90 120
0
3
8
Time (day)
K
**
1.4
6
SERCA2b
Time (min)
Time (min)
LacZ
30
8
G 60
LacZ
1.2
Arbitary Units
*
450
400
350
300
250
200
150
100
50
0
Body Weight (g)
LacZ
Blood Glucose (mg/dl)
Blood Glucose (mg/dl)
F
***
10
0
LacZ
SERCA2b
**
12
0
LacZ
500
450
400
350
300
250
200
150
100
50
0
14
**
300
0
E
D
350
1.0
0.8
0.6
0.4
2
*
LacZ
SERCA2b
*
1.6
1.2
*
0.8
0.4
0.2
0
LacZ
SERCA2b
0
G6pase
1.6
PEPCK
PGC1α
LacZ
SERCA2b
1.2
0.8
0.4
0
PDI
ERDJ4
Calnex
Calr
EDEM
ERO1α
sion; XBP1s overexpression in the liver of ob/ob mice significantly
increased the levels of SERCA2b. Furthermore, reduced SERCA2b levels also correlate with loss of XBP1s activity in obesity
conditions. However, further investigation is necessary to completely understand the regulatory factors for SERCA2b.
Our current results suggest an interesting possibility that ER
calcium levels in the liver cannot be maintained at the desired high
concentrations under obesity conditions because the “gatekeeper”
for high ER calcium concentration, SERCA2b, is dramatically reduced in the liver and cannot be up-regulated during metabolic
overload such as refeeding. Considering the “addictive” nature of
ER chaperones for the high calcium level for their optimal activity,
we suggest that loss of SERCA2b function/regulation and a consequent decrease in ER calcium levels in the liver reduce chaperone
function and ER folding capacity. Indeed, when SERCA2b levels
are increased in the liver of ob/ob mice, ER stress is reduced without
a corresponding increase in the levels of chaperones: in fact, some
chaperones are expressed at slightly lower levels.
In addition, overexpression of SERCA2b increases the resistance of MEFs to stimuli that increase the level of unfolded
proteins in the ER lumen. For example, stimulation of SERCA2boverexpressing MEFs with tunicamycin blocks initiation of the
UPR. These data provide further evidence that ER function is
enhanced by SERCA2b overexpression.
Our results also indicate that the ER calcium homeostasis is
closely related with accumulation of TGs in the liver, as evidenced by
the marked reduction in the liver TG content of the ob/ob mice that
were injected with Ad-SERCA2b. It is interesting to note that there
is also a marked decrease in most lipogenic genes after SERCA2b
Park et al.
HERP
GRP58
GRP78
Fig. 3. Overexpression of SERCA2b in
the liver of ob/ob mice increases glucose
tolerance and establishes euglycemia.
(A–J) Eight-week-old male ob/ob mice
were injected with Ad-LacZ or Ad-SERCA2b (5.8 × 107 pfu/g) through a tail
vein. (A) SERCA2b immunoblotting in
the liver of Ad-LacZ or Ad-SERCA2binjected ob/ob mice. (B) mRNA levels of
SERCA2b in the liver of ob/ob mice
that were injected with Ad-LacZ or
Ad-SERCA2b. (C) Blood glucose level
(mg/dL) on day 3 after injection at 6 h
of fasting. (D) Serum insulin level (ng/
mL) on postinjection day 8. (E) GTT
was performed with i.p. injection of
0.5 g/kg of glucose on day 4 after
injections. (F) ITT was performed with
i.p. injection of insulin (2 IU/kg) on
postinjection day 7 after 6 h fasting.
(G) Body weight (in g) at postinjection
days 3 and 5. (H) Phospho-IRE1αSer724
and total IRE1 protein levels in the liver
of Ad-LacZ and Ad-SERCA2b-injected
ob/ob mice. (I) Quantification of phospho/total IRE1 ratio. (J) qPCR analysis
of the mRNA levels of PDI, ERDJ4, calnexin, calreticulin, EDEM, ERO1α, HERP,
GRP58, and GRP78. (K) Gene expression
level of G6Pase, PEPCK, and PGC1α was
analyzed by qPCR in the liver of Ad-LacZ
or Ad-SERCA2b-injected ob/ob mice. Error bars are means SEM; P values were
determined by Student t test (*P < 0.05,
**P < 0.01, ***P < 0.001).
expression. It is possible that increased efficiency of chaperones after
SERCA2b expression leads to retention of SREBP1c in the ER
membrane and reduces nuclear abundance of this lipogenic transcription factor; it was previously shown that GRP78 interacts with
SREBP1c in the ER membrane and decreases lipogenic gene expression by blocking its cleavage and nuclear translocation (19).
The findings we present here highlight that the level of SERCA2b protein in the liver are critically important in the maintenance of metabolic homeostasis in obese mice. We have previously
reported that obese mice are unable to up-regulate the expression
levels of chaperone during metabolic overloading (13). Taken together with our previous observations regarding the loss of chaperone response in obesity conditions, the current results led us to
propose that the synergy between diminished chaperone response
and reduction in ER Ca2+ levels may be the factors most critical in
the development of ER stress in obesity conditions. Possible reduced Ca2+ levels in the ER caused by down-regulated SERCA2b
levels and the resulting decrease in chaperone function provide
further insight into how and why ER stress might develop in obesity.
Furthermore, overexpression of SERCA2b in the liver of
euglycemic lean mice did not lead to hypoglycemia, indicating that
activation of SERCA2b will be a safe approach that does not alter
the glucose homeostasis. Hence, a significant outcome from the
current findings could be development of therapeutic agents targeted at effectively enhancing SERCA2b activity, which would in
turn decrease ER stress, enhance glucose tolerance, and reduce
hepatosteatosis.
PNAS | November 9, 2010 | vol. 107 | no. 45 | 19323
CELL BIOLOGY
***
8
Blood Insulin (ng/ml)
B
Blood Glucose (mg/dl)
A
SERCA2b
B
Triglyceride (mg/g)
LacZ
300
**
A
250
200
150
100
50
0
LacZ
LacZ
SERCA2b
180
Blood Glucose (mg/dl)
A
SERCA2b
160
140
120
100
80
60
40
20
0
0.6
1.4
1
0.8
0.6
1
0.8
0.6
0.4
0.4
0.2
0.2
0
0
0
SERCA2b
LacZ
F
1.4
1.2
G 1.2
ACC1/18S
LacZ
1
1
ACC2/18S
1.4
0.8
0.6
0.4
SERCA2b
***
LacZ
H
0.8
0.6
0.4
C
1
0.6
0.4
0
0
0
SERCA2b
120
LacZ
100
SERCA2b
80
60
40
20
0
0
15
30
60
90
120
Time (min)
LacZ
SERCA2b
LacZ
SERCA2b
30
0.8
0.2
LacZ
Day9
1.2
0.2
SERCA2b
SERCA2b
Day7
140
1.4
0.2
LacZ
B
1.2
0.2
0.4
*
1.4
Blood Glucose (mg/dl)
0.8
1.2
E
*
Body Weight (g)
1
1.6
SREBP1c/18S
SCD1/18S
1.2
D
FASn/18S
*
DGAT2/18S
Day3
C 1.6
1.4
25
20
15
10
5
Fig. 4. SERCA2b reduces lipogenic gene expression and TG levels in the liver
of ob/ob mice. Eight-week-old male ob/ob mice were injected with Ad-LacZ
or Ad-SERCA2b (5.8 × 107 pfu/g) through a tail vein. (A) Sections of the liver
from Ad-LacZ or Ad-SERCA2b-injected ob/ob mice were stained with H&E.
(B) TG content (mg/g) in the liver of Ad-LacZ or Ad-SERCA2b-injected ob/ob
mice. mRNA levels of (C) SCD1, (D) DGAT2, (E) FASn, (F) ACC1, (G) ACC2, and
(H) SREBP1c were quantified by qPCR. Error bars represent SEM; P values
were determined by Student t test (*P < 0.05, **P < 0.01, ***P < 0.001).
Materials and Methods
Biochemical Reagents. SERCA2b (ATPA2/SERCA2), phospho-PERKThr980, Akt,
and lamin A/C-specific antibodies were purchased from Cell Signaling Technology. XBP-1 and tubulin-specific antibodies were from Santa Cruz Biotechnology. Antibodies for phospho-IRE1αSer724 and total IRE1α were
purchased from Novus Biologicals. DMEM, FBS, penicillin, and streptomycin
were purchased from Gibco. Leupeptin, aproptonin, and PMSF were from
Sigma-Aldrich. Nuclear extraction kit was purchased from Thermo Scientific.
cDNA synthesis kit was from BioRad and SYBR quantitative PCR (qPCR) Supermix was from Fermentas. TRIzol reagent was from Invitrogen. Free glycerol
reagent, triglycerol reagent, glycerol, and ER isolation kit were purchased
from Sigma-Aldrich. Chemiluminescence substrates were from Roche. ELISA
kit was from Crystal Chem. Tunicamycin and thapsigargin were from Calbiochem. High-fat diet (45 kcal%) was purchased from Research Diets.
Production of Adenovirus. Mouse SERCA2b cDNA (GenBank ID no. AJ131821)
was synthesized and cloned into pENTR3C vector to create pENTR3C-SERCA2b.
pAd-SERCA2b was created from pENTR3C-SERCA2b and pAd/CMV/V5-DEST
(Invitrogen) by homologous recombination using the manufacturer’s gateway
system protocol. pAd-SERCA2b was linearized by restriction endonuclease
digestion with PacI and transfected to 293A cells. The media was replaced
with new media every 3 d until the cytopathic effect was observed. When the
cytopathic effect reached 80%, cells were collected and the virus was harvested
by repeating freezing and thawing cycles at –80 °C and 37 °C four times. Viral
supernatant was obtained by centrifuging at 4,000 × g for 20 min. Ad-XBP1s
and Ad-LacZ were created in the same manner as previously reported (15).
Isolation of ER Fractions from Liver Tissue. Fresh liver tissue (100 mg) was
washed twice with PBS solution and then cut into small pieces. Isotonic extraction buffer from ER isolation kit (800 μL, 1×) was added. The sample was
homogenized in a glass tube homogenizer and ER fractions were isolated
according to the manufacturer’s protocol. After ultracentrifuging to obtain
ER fraction, the pellet was resuspended in 300 μL of 1× isotonic extraction
buffer and homogenized in a glass tube homogenizer.
19324 | www.pnas.org/cgi/doi/10.1073/pnas.1012044107
0
Day3
Day5
Fig. 5. Overexpression of SERCA2b in the liver of WT lean mice does not alter
the glucose levels and insulin sensitivity. Eight-week-old male mice were
injected with Ad-LacZ or Ad-SERCA2b (5.8 × 107 pfu/g) through a tail vein. (A)
Blood glucose level at days 3, 7, and 9 after injection. (B) ITT was performed
with i.p. injection of insulin (1 IU/kg) on postinjection day 6 after 6 h of fasting.
(C) Body weight was measured on days 3 and 5 after injection.
TG Measurements. Liver tissue (100 mg) was homogenized in buffer containing 0.8 mL of H2O and 1.5 mL of chloroform/methanol (2:1 vol/vol) for
1 min. One milliliter of H2O and 0.5 mL of chloroform were added to homogenized sample and the tubes were vortexed, followed by centrifugation
at 500 × g for 20 min at 4 °C. The lower chloroform phase was collected in
a new Eppendorf tube and 500 μL of collected liquid was dried in a hood
for overnight. The dried pellet was dissolved in 200 μL of isopropanol/Triton
X-100 (90:10 vol/vol) and a small amount was further diluted 20 times in the
same solution. Two microliters of diluted sample was added to 200 μL of free
glycerol reagent in a 96-well plate. After 15 min of incubation, the absorbance at a wavelength of 490 nm was recorded for free glycerol content. TG
reagent (40 μL) was added and incubated for 15 min. The total glycerol level
was measured at a wavelength of 490 nm. Triglycerol level was calculated by
subtracting free glycerol level from total glycerol. A standard curve was created with series of dilution of a known concentration of glycerol.
Insulin ELISA. Blood was collected into a heparinized tube from the retroorbital space and centrifuged at 12,000 × g for 20 min at 4 °C. The serum was
collected and diluted three times with sample diluent provided with an ultrasensitive mouse insulin ELISA kit. The insulin level in the serum was
measured with a protocol provided by the manufacturer.
RNA Isolation and Real-Time qPCR. RNA was extracted from the liver of mice by
using TRIzol reagent according to the manufacturer’s instructions. cDNA was
synthesized from 1 μg of RNA using a cDNA synthesis kit with the following
conditions: 25 °C for 10 min, 42 °C for 30 min, 85 °C for 5 min. qPCR was performed with SYBR Green qPCR Supermix from Invitrogen according to the
manufacturer’s instructions. The following primer sets were used for qPCR:
18S (forward), 5′-AGTCCCTGCCCTTTGTACACA-3′; 18S (reverse), 5′-CGATCCGAGGGCCTCACTA-3′; mSERCA2b (forward), 5′-ATGAGCAAGATGTTTGTGAAGG-3′; mSERCA2b (reverse), PDI (forward), 5′-CAAGATCAAGCCCCACCTGAT-3′;
PDI (reverse), 5′-AGTTCGCCCCAACCAGTACTT-3′; ERDJ4 (forward), 5′-CCCCAGTGTCAAACTGTACCAG-3′; ERDJ4 (reverse), 5′AGCGTTTCCAATTTTCCATA-
Park et al.
Total Protein Extraction from Tissue. Liver tissues (100 mg) were homogenized
with a bench-top homogenizer from Polytron (PT2100) in 3 mL of ice-cold
tissue lysis buffer (25 mM Tris-HCl, pH 7.4, 10 mM Na3VO4, 100 mM NaF, 50 mM
Na4P2O7, 10 mM EGTA, 10 mM EDTA, 1% Nonidet P-40, 10 mg/mL leupeptin,
10 g/mL aproptonin, 2 mM PMSF, and 20 nM okadaic acid) in 50-mL roundbottom tubes. The homogenized samples were centrifuged at 8,000 × g for
20 min at 4 °C. The lipid layer was removed and the supernatant was transferred into Eppendorf tubes. After centrifuging at 16,000 × g for 60 min at
4 °C, the supernatants were normalized to the same concentration and boiled
at 100 °C in 1× Laemmli buffer for 5 min. The lysate was cooled to room
temperature before loading for Western blot analysis.
Cell Culture. MEFs were cultured in DMEM with 10% FBS, 10 U/mL penicillin,
and 1 mg/mL streptomycin. Cells were maintained at 37 °C with 5% CO2.
secondary antibody in TBST/10% blocking reagent for 1 h. The membrane was
washed three times for 20 min and developed using a chemiluminescence
assay system. To strip a membrane for another primary antibody, the membrane
was agitated at 50 °C for 20 min in a box with stripping buffer (2% SDS and 100
mM 2-mercaptoethanol in TBS, pH 7.5). The membrane was washed three times
for 20 min before blocking and an incubation with primary antibody.
Tail Vein Infection. Adenovirus was thawed at 25 °C and diluted in saline
solution to a final volume of 100 μL. Mice were restrained in a restrainer and
adenovirus was injected into a tail vein with a 30-gauge needle. Mild pressure was applied to the spot of injection to prevent the backflow of virus.
Blood Glucose Level Measurement. Blood was taken from mice by tail clipping
and blood glucose level was measured with a portable glucose meter
(Contour; Bayer).
GTT. For GTT, mice were fasted overnight and 0.5 g/kg of D-glucose at a final
volume of 100 μL was administrated intraperitoneally. The blood glucose
level was measured 0, 15, 30, 60, 90, and 120 min after glucose administration
through a tail vein with use of a portable glucose meter (Contour; Bayer).
ITT. Mice were fasted for 6 h and 2.0 IU/kg of insulin for ob/ob mice and 1.0
IU/kg of insulin for WT lean mice at a final volume of 100 μL were administrated intraperitoneally. The blood glucose level was measured 0, 15, 30,
60, 90, and 120 min after insulin administration through a tail vein with
a portable glucose meter (Contour; Bayer).
Adenovirus Transduction. MEFs were plated at a density of 3 × 106 cells per
10-cm plate. The following day, the media were replaced with 3 mL of 1%
FBS containing media and adenovirus was added. The dish was rotated every
15 min for 1 h and 7 mL of media with 1% FBS was added. Cells were incubated with adenovirus-containing media for 16 h.
High-Fat Diet Experiments. C57BL/6 mice were started on high-fat diet feeding
at the age of 3.5 wk, right after weaning. Following 16 wk of high-fat diet
feeding, mice were starved for 4 h and the liver was extracted following
establishment of anesthesia.
Total Protein Extraction from MEFs. MEFs were lysed in lysis buffer (25 mM Tris,
pH 7.4, 2 mM NaVo4, 10 mM NaF, 10 mM Na4P2O7, 1 mM EGTA, 1 mM EDTA,
1% Nonidet P-40, 10 μg/mL leupeptin, 10 μg/mL aproptonin, 2 mM PMSF,
and 20 nM okadaic acid). The concentrations of protein were normalized
with the lysis buffer to have equivalent amounts of protein (200–400 μg) and
volume (300 μL). After adding Laemmli buffer, protein was boiled at 100 °C
for 5 min and kept at 25 °C for 15 min.
Statistical Analyses. We used two-tailed Student t tests to determine P values
for statistical significance. Error bars in the figures represent SEM.
Western Blotting. Protein lysate was resolved on SDS polyacrylamide gel and
transferred onto PVDF membrane at a voltage of 100 V for 2 h at 4 °C. The
membrane was blocked in 10% blocking reagent and incubated overnight
with primary antibody in Tris-buffered saline solution/Tween (TBST)/10%
blocking reagent at 4 °C. After the incubation, the membrane was washed
three times in TBST for 20 min and incubated at room temperature with
ACKNOWLEDGMENTS. We thank the members of the U.O. laboratory for
their support during the execution of the experiments and useful discussions.
This study was supported by Junior Faculty Start Up Funds, National Institute
of Diabetes and Digestive and Kidney Diseases/National Institutes of Health
Grant R01DK081009 (to U.O.), and Timothy Murphy funds provided to the
Division of Endocrinology, Children’s Hospital Boston.
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Animal Experiments. All animal experiments were approved by the institutional animal care and use committee at Children’s Hospital Boston.
PNAS | November 9, 2010 | vol. 107 | no. 45 | 19325
CELL BIOLOGY
AATT-3′; Calnexin (forward), 5′-ATGGAAGGGAAGTGGTTACTGT-3′; Calnexin
(reverse), 5′-GCTTTGTAGGTGACCTTTGGAG-3′; Calreticulin (forward), 5′-CCTGCCATCTATTTCAAAGAGCA-3′; Calreticulin (reverse), 5′-GCATCTTGGCTTGTCTGCAA-3′; EDEM (forward), 5′-AAGCCCTCTGGAACTTGCG -3′; EDEM (reverse), 5′AACCCAATGGCCTGTCTGG-3′; ERO1α (forward), 5′-TCAGTGGACCAAGCATGATGA-3′; ERO1α (reverse), 5′-TCCACATACTCAGCATCGGG3′; HERP (forward),
5′-CATGTACCTGCACCACGTCG-3′; HERP (reverse), 5′-GAGGACCACCATCATCCGG-3′; GRP58 (forward), 5′-GAGGCTTGCCCCTGAGTATG-3′; GRP58 (reverse), 5′GTTGGCAGTGCAATCCACC-3′; GRP78 (forward), 5′-TCATCGGACGCACTT GGAA3′; GRP78 (reverse), 5′-CAACCACCTTGAATGGCAAGA-3′; G6Pase (reverse), 5′CAATGCCTGACAAGACTCCA-3′; PEPCK (forward), 5′-ATCATCTTTGGTGGCCGTAG-3′; PEPCK (reverse), 5′-ATCTTGCCCTTGTGTTCTGC-3′; PGC1α (forward), 5′TGATGTGAATGACTTGGATACAGACA-3′; PGC1α (reverse), 5′-CAATGCCTGACAAGACTCCA-3′; SCD-1 (forward), 3′-AGATCTCCAGTTCTTACACGACCAC-3′; SCD-1
(reverse), 3′-GACGGATGTCTTCTTCCAGGTG-3′; DGAT2 (forward), 5′-TTCCTGGCATAAGGCCCTATT-3′; DGAT2 (reverse), 5′-AGTCTATGGTGTCTCGGTTGAC3′; FASn (forward), 5′-GGAGGTGGTGATAGCCGGTAT-3′; FASn (reverse), 5′-TGGGTAATCCATAGAGCCCAG-3′; ACC1 (forward), 5′-ATTGGGCACCCCAGAGCT A3′; ACC1 (reverse), 5′-CCCGCTCCTTCAACTTGCT-3′; ACC2 (forward), 5′-GGGCTCCCTGGATGACAA C-3′; ACC2 (reverse), 5′-TTCCGGGAGGAGTTCTGGA-3′;
SREBP1c (forward), 5′-GCGGTTGGCACAGAGCTT-3′; and SREBP1c (reverse), 5′GGACTTGCTCCTGCCATCAG-3′.