Xbp1s in Pomc Neurons Connects ER Stress
with Energy Balance and Glucose Homeostasis
Kevin W. Williams,1,2,14,* Tiemin Liu,1,14Xingxing Kong,7,14Makoto Fukuda,1,8,14Yingfeng Deng,3Eric D. Berglund,1,4,5
Zhuo Deng,1,9Yong Gao,1,10Tianya Liu,1,11Jong-Woo Sohn,1Lin Jia,1Teppei Fujikawa,1Daisuke Kohno,1,12
Michael M. Scott,13Syann Lee,1Charlotte E. Lee,1Kai Sun,3Yongsheng Chang,10Philipp E. Scherer,3,6,15
and Joel K. Elmquist1,5,15
1Division of Hypothalamic Research, Department of Internal Medicine
2Department of Neuroscience
3Touchstone Diabetes Center, Department of Internal Medicine
4Advanced Imaging Research Center
5Department of Pharmacology
6Department of Cell Biology
The University of Texas Southwestern Medical Center at Dallas, Dallas, TX 75390, USA
7Division of Endocrinology, Beth Israel Deaconess Medical Center and Harvard Medical School, Harvard University, Boston, MA 02115, USA
8Children’s Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, Houston, TX 77030, USA
9Department of Obstetrics and Gynecology, The First Affiliated Hospital, Medical School of Xi’an Jiaotong University, Xi’an 710061,
10National Laboratory of Medical Molecular Biology, Institute of Basic Medical Science, Chinese Academy of Medical Science and Peking
Union Medical College, Beijing 100005, P.R. China
11College of Pharmaceutical Sciences, Soochow University, Suzhou, Jiangsu 215021, P.R. China
12Advanced Scientific Research Leaders Development Unit, Gunma University, Maebashi, Gunma 371-8511, Japan
13Department of Pharmacology, University of Virginia School of Medicine, Charlottesville, VA 22908, USA
The molecular mechanisms underlying neuronal lep-
tin and insulin resistance in obesity and diabetes
remain unclear. Here we show that induction of
the unfolded protein response transcription factor
spliced X-box binding protein 1 (Xbp1s) in pro-opio-
melanocortin (Pomc) neurons alone is sufficient to
protect against diet-induced obesity as well as
improve leptin and insulin sensitivity, even in the
presence of strong activators of ER stress. We also
demonstrate that constitutive expression of Xbp1s
in Pomc neurons contributes to improved hepatic in-
sulin sensitivity and suppression of endogenous
glucose production. Notably, elevated Xbp1s levels
in Pomc neurons also resulted in activation of the
Xbp1s axis in the liver via a cell-nonautonomous
mechanism. Together our results identify critical
molecular mechanisms linking ER stress in arcuate
Pomc neurons to acute leptin and insulin resistance
as well as liver metabolism in diet-induced obesity
Obesity is associated with leptin resistance (Considine et al.,
1996; Frederich et al., 1995; Friedman, 2000; Morton et al.,
2006; Myers et al., 2008, 2012), while type 2 diabetes is charac-
terized by insulin resistance in multiple tissues (Guilherme et al.,
2008; Kahn et al., 2006; Ko ¨nner and Bru ¨ning, 2012; Weyer et al.,
1999). Hypothalamic Pomc neurons are direct targets of both
leptin and insulin, contributing to their role in regulating energy
expenditure, body weight, and glucose homeostasis (Belgardt
and Bru ¨ning, 2010; Myers and Olson, 2012; Schwartz and Porte,
2005; Spiegelman and Flier, 2001; Williams and Elmquist, 2012;
Yeo and Heisler, 2012). Recently, endoplasmic reticulum (ER)
stress and the unfolded protein response (UPR) have emerged
as a unifying and critical link in the development of cellular leptin
and insulin resistance (Ozcan et al., 2006, 2009; Shoelson et al.,
2006; Wellen and Hotamisligil, 2005; Zhang et al., 2008). In
particular, obesemice and mice fed high-fat diets(HFDs) display
ER stress in peripheral tissues as well as Pomc neurons within
the hypothalamus, suggesting that metabolic disorders associ-
ated with obesity and HFDs induce ER stress in vivo (Schnee-
berger et al., 2013; Thaler et al., 2012; Xu et al., 2005). Notably,
induction of ER stress or deficiency of the X-box-binding protein
1 (Xbp1) in neurons results in hyperleptinemia, obesity, hyper-
phagia, and reduced metabolic rate associated with severe
hypothalamic leptin resistance (Ozcan et al., 2009). Additionally,
ER stresssuppresses leptin and insulin signalingin the periphery
as well as the CNS via classical inhibitors of cytokine signaling
such as the suppressor of cytokine signaling-3 (Socs3) and pro-
tein tyrosine phosphatase 1b (Ptp1b) (Howard and Flier, 2006;
Myers et al., 2008; Ozcan et al., 2004; White et al., 2009; Zabo-
lotny et al., 2008). Importantly, the neuronal cell type(s) involved
in this response remains undefined. To address this issue, we
assessed the role of Xbp1s in Pomc neurons to regulate glucose
Cell Metabolism 20, 471–482, September 2, 2014 ª2014 Elsevier Inc. 471
metabolism and HFD-induced obesity. Additionally, we exam-
ined the cellular mechanisms of Ptp1b, Socs3, and Xbp1s in
the ER stress-induced acute leptin and insulin resistance of
arcuate Pomc neurons.
Constituitive Activation of Xbp1s in Pomc Neurons
Protects against Diet-Induced Obesity
Xbp1s improves leptin and insulin signaling along with meta-
bolism in the periphery as well as the CNS (Deng et al., 2013;
Ozcan et al., 2004, 2006, 2009). We recently developed a mouse
model that expresses an inducible ‘‘dominant active’’ Xbp1s
transgene via a conventional Tet-On system (Deng et al.,
2013). The Xbp1s transgene under the control of a tetracy-
cline-responsive element (TRE) supports inducible expression
by the tetracycline reverse transcriptional activator (rtTA) in the
presence of doxycycline (Dox). The rtTA transgene is driven by
the Rosa26promoter withatranscriptional stopcassette flanked
by two loxP sites upstream of rtTA (Belteki et al., 2005). Com-
bined with a Pomc promoter-driven Cre transgene (Balthasar
et al., 2004), we obtained a mouse model with Pomc-specific
inducible expression of Xbp1s (PIXs).
When fed HFD-Dox, male PIXs mice displayed an age-depen-
dent lean body weight compared to wild-type (WT) mice (Fig-
ure 1A), which was reflected by decreases in fat mass (t(11)=
3.965, p < 0.05; Figure 1B). The lean phenotype of PIXs was
concomitant with significantly lower visceral (t(11)= 3.395, p <
0.05) and subcutaneous fat (t(11)= 4.090, p < 0.05) distribution
than controls (Figures 1C and 1D). PIXs mice fed HFD-Dox
also displayed decreased snout-anus length (t(11)= 4.928, p <
0.05; Figure S1A) and decreased hepatic triglyceride (t(6) =
2.60, p < 0.05) and cholesterol (t(6)= 2.571, p < 0.05) levels
Age- and weight-matched PIXs males were hypermetabolic
independent of altered food intake, as demonstrated by sig-
nificant increases in energy expenditure (Figures 1F–1I and Fig-
ure S1B). Components of total energy expenditure include
energy required for physical activities and basal metabolism.
In particular, PIXs mice exhibited increased heat production
suggestive of higher metabolic rate (Figure 1I). PIXs mice also
showed increased ambulatory movements independent of
rearing activity (Figure 1J and Figure S1C). Although we did not
Figure 1. Body Weight and Metabolic Assessment of Male WT and PIXs Mice on HFD
(A) Body weight curve of male PIXs mice (*p < 0.05).
(B–D) Body fat composition: whole body volume (B), visceral (C), and subcutaneous (D).
(E–J) Male PIXs mice display increased hepatic triglyceride and cholesterol (E), increased VO2(F), increased VCO2(G), decreased RER (H), increased heat
production (I), and increased ambulatory activity (J). Error bars indicate SEM. Mice used in (E)–(I) were age-matched male littermates (8 weeks of age) and had
comparable body weight and lean mass. For (F)–(J), n = 14–16 per group; *p < 0.05.
(K) Leptin-induced hypophagia was observed at 1, 2, 4, and 6 hr after refeeding. PIXs mice exhibited increased hypophagia in response to pharmacological
administration of leptin at 4 and 6 hr after refeeding. n = 10 per group; *p < 0.05.
ER Stress in Obesity and Diabetes
472 Cell Metabolism 20, 471–482, September 2, 2014 ª2014 Elsevier Inc.
observe changes in ad libitum food intake, PIXs mice were more
sensitive to acute leptin-induced hypophagia when compared to
littermate controls at 4 and 6 hr after refeeding (Figure 1K).
In support of the hypermetabolic phenotype, PIXs mice
displayed increased expression of genes associated with heat
production in both brown adipose tissue (BAT) (for Ppargc1a:
t(9)= 2.957, p < 0.05; for Prdm16: t(9)= 3.691, p < 0.05; for
UCP1: t(9)= 2.527, p < 0.05; for Cidea: t(9)= 2.547, p < 0.05; for
Dio2: t(9)= 1.413, p > 0.05; for Elovl6: t(9)= 1.480, p > 0.05; Fig-
ure 2A) and inguinal white adipose tissue (iWAT) (for Ppargc1a:
t(9)= 3.289, p < 0.05; for Prdm16: t(9)= 4.158, p < 0.05; for
UCP1: t(9)= 4.573, p < 0.05; for Cidea: t(9)= 4.270, p < 0.05; for
Dio2: t(9)= 4.004, p < 0.05; for Elovl6: t(9)= 1.918, p > 0.05;
Figure 2B). These data are also supported by the apparent
decreased multilocular cells in BAT from PIXs mice (Figures 2C
and 2D) and increased expression of the browning marker
UCP1 in iWAT of PIXs mice (Figures 2E and 2F). Collectively,
these results indicate that constitutive expression of Xbp1s in
Pomc neurons is sufficient to improve body weight homeostasis
in the context of diet-induced obesity. Moreover, Xbp1s in Pomc
neurons is sufficient to regulate metabolic rate and locomotor
activity and to mediate thermogenesis (both BAT and iWAT).
Constitutive Activation of Xbp1s in Pomc Neurons
Improves Insulin Sensitivity and Glycemia
Along with the systemic effects on whole-body energy expendi-
ture and body weight, Xbp1s induction in Pomc neurons also
leads to profound changes in glucose metabolism. PIXs mice
fed a chow-Dox diet showed improved blood glucose levels in
the fed and fasted state when compared to littermate controls
(for chow-Dox-fed PIXs mice: t(15)= 2.763, p < 0.05; for chow-
Dox-fasted PIXs mice: t(15)= 2.250, p < 0.05; Figure 3). Serum
insulin levels were also decreased in PIXs mice during both fed
and fasted conditions (for chow-Dox-fed PIXs mice: t(15) =
2.217, p < 0.05; for chow-Dox-fasted PIXs mice: t(14)= 2.515,
p < 0.05; Figure 3).
We next performed hyperinsulinemic-euglycemic clamps to
assess whether insulin sensitivity was improved in chow-fed,
Dox-enriched PIXs mice compared with their littermates. Blood
glucose was successfully clamped at target levels (150 mg/dl;
Figure 4A), and the exogenous glucose infusion rate (GIR) was
higher in PIXs mice, indicating improved insulin sensitivity (Fig-
ated suppression of endogenous glucose appearance (endo Ra;
Figure 4C) and not glucose disappearance (Rd; Figure 4D).
Together these data suggest that constitutive expression of
Xbp1s in Pomc neurons is sufficient to mimic a postprandial
state in the liver, suppressing glucose production and ultimately
lowering blood glucose levels.
Xbp1s Is a Cell-Nonautonomous Feeding Sensor
Upregulation of Xbp1s and the UDP-galactose-4-epimerase
(GalE) in the liver may be indicative of a fed state and contribute
to metabolism (Deng et al., 2013). In support of these data, we
demonstrated that both Xbp1s and GalE are upregulated in the
liver after refeeding (2 hr) following an 18 hr fast (for Xbp1s:
t(8)= 2.852, p < 0.05; for GalE: t(8)= 2.464, p < 0.05; Figure 5A).
Refeeding (2 hr) following an 18 hr fast also readily elevated
mRNA for both Xbp1s and GalE in the arcuate nucleus from
WT mice, supporting an association of Xbp1s-GalE with feeding
or caloric intake (for Xbp1s: t(8)= 3.203, p < 0.05; for GalE: t(8)=
3.276, p < 0.05; Figure 5B). Similar to our observations in refeed-
ing after an overnight fast, Dox-containing diet induced Xbp1s
and GalE mRNA as well as Xbp1s target genes in the arcuate
nucleus from PIXs mice (for Xbp1s: t(9)= 3.094, p < 0.05; for
GalE: t(9)= 3.328, p < 0.05; for Edem1: t(9)= 4.779, p < 0.05;
for Erdj4: t(9)= 2.860, p < 0.05; for Bip: t(9)= 3.835, p < 0.05; Fig-
ure 5B). The enhanced expression of Xbp1s and target genes
was also apparent in FACS-isolated Pomc neurons subsequent
to refeeding or from FACS-isolated Pomc from PIXs mice fed
a Dox-containing diet (Figures 5C and 5D). Importantly,
increased expression of Xbp1s mRNA levels and Xbp1s target
genes in Pomc neurons from PIXs mice fed a Dox-containing
diet was analogous to the elevated Xbp1s levels observed in
Pomc neurons in the refed state, supporting an expression of
Xbp1s in PIXs mice that mimics a physiological postprandial
state. We also found that, similar to recent work in C. elegans
Figure2. PIXsMiceExpressIncreasedThermogenic MarkersinBAT
(A and B) Weight-matched PIXs mice were fed a Dox-enriched HFD for
2 weeks. qPCR was performed to examine the relative expression of
Ppargc1a, Prdm16, UCP1, Cidea, Dio2, and Elovl6, which are genes asso-
ciated with heat production in BAT (A) and iWAT (B). *p < 0.05.
(C and D) Brown adipose tissue (BAT) from WT (C) and PIXs (D) mice was
imaged by light microscopy after hematoxylin-eosin staining.
(E and F) Inguinal white adipose tissue from WT (E) and PIXs (F) mice was
imaged by light microscopy after UCP-1 immunohistochemistry. Bars,
100 mm. Note that (C) and (D) are the same scale as (E) and (F).
ER Stress in Obesity and Diabetes
Cell Metabolism 20, 471–482, September 2, 2014 ª2014 Elsevier Inc. 473
(Taylor and Dillin, 2013), constitutive expression of Xbp1s in mu-
rine Pomc neurons resulted in the transcriptional upregulation
of Xbp1s and Xbp1s target genes in the liver (for Xbp1s: t(13)=
2.284, p < 0.05; for erdj4: t(13)= 2.477, p < 0.05; for erdeml:
t(13)= 2.433, p < 0.05; for chop: t(13)= 2.545, p < 0.05; for bip:
t(13)= 1.556, p > 0.05; Figure 5E). Although we cannot exclude
the involvement of other arms of the UPR in this cell-nonautono-
mous regulation, the Pomc-dependent upregulation of Xbp1s in
the liver occurred independent of detectable Cre activity within
the liver. Together, these data demonstrate that Xbp1s and pre-
sumably activation of Xbp1s transcriptional targets is sufficient
to signal a fed state via a cell-nonautonomous regulation of the
homeostasis (Deng et al., 2013).
ER Stress Inhibits Leptin and Insulin Signaling
in the Arcuate Nucleus
In an effort to identify a cellular mechanism underlying the im-
provements in body weight and glucose homeostasis in PIXs
mice, we first utilized a model chronic culture system (organo-
typic slice preparation) recently developed in our lab (Fukuda
et al., 2011; Ga ¨hwiler and Llano, 1989). Recent evidence sug-
gests that overnutrition induces ER stress in arcuate Pomc neu-
may blunt leptin and insulin signaling directly in arcuate Pomc
neurons. Also, constitutive expression of Xbp1s in Pomc neu-
rons may improve leptin and insulin signaling in times of ER
stress. Organotypic slice cultures were exposed to tunicamycin
(tm), thapsigargin (tg), or dithiothreitol (dtt) in order to examine
the effects of ER stress. Similar to previous observations, tm
(15–30mM,6hr),tg (15mM,6hr),and dtt(1mM,6hr) suppressed
the leptin-induced phosphorylation of STAT3 in the arcuate
nucleus of our organotypic slice preparation (Figures 6A–6C).
Notably, at 6 hr, tm (30 mM) and dtt (1 mM) induced robust phos-
phorylation of the eukaryotic initiation factor 2 alpha (eif2a)
and increased the accumulation of the ER resident molecular
chaperone (Bip/Grp78), which are both known UPR target genes
Figure 3. Blood Glucose and Insulin Levels
of Male WT and PIXs Mice on Chow Diet
(A–D) Blood glucose and insulin levels were
measured from WT and PIXs mice fed a chow diet
without Dox (A), fed a chow diet without Dox and
then fasted overnight (B), fed a chow diet with
Dox for 1 week (C), and fed a chow diet with Dox
for 1 week and then fasted overnight (D). Blood
glucose and insulin levels were decreased in mice
fed or fasted after 1 week on a chow diet with Dox
(n = 10–15 per group; *p < 0.05).
and dtt (1 mM, 6 hr) potently increased
mRNA for Bip and CHOP/GADD153 (Fig-
ure S2B). In addition to leptin signaling,
stimulation of DMSO-treated cultures
with insulin led to an increase in AKT
phosphorylation (Figures 6D and 6E).
Pretreatment with tm suppressed the
insulin-induced phosphorylation of AKT
in the arcuate nucleus (Figures 6D and 6E). Thus, activators of
ER stress suppress the activation of multiple signaling cascades
activated by leptin and insulin in the arcuate nucleus of the
ER Stress Inhibits Acute Leptin and Insulin Signaling
in Arcuate Pomc Neurons
Leptin directly activates while insulin directly inhibits arcuate
Pomc neurons via PI3K-dependent mechanisms (Al-Qassab
et al., 2009; Hill et al., 2008; Morton et al., 2006; Williams
et al., 2010). We hypothesized that ER stress may blunt
the leptin-induced activation and insulin-induced inhibition of
arcuate Pomc neurons, which supports an ER stress-induced
cellular resistance to the acute effects of leptin and insulin on
Whole-cell recordings were performed on acute hypothalamic
slices containing Pomc-GFP neurons within the arcuate nucleus
the effects of ER stress on acute leptin and insulin signaling, we
Pomc neurons in an acute hypothalamic slice preparation
(Sohn et al., 2011). As expected, leptin failed to alter the mem-
brane potential of Pomc-hrGFP (green) neurons that did not
express Leprs. In current-clamp configuration, 75% of Pomc-
hrGFP::Lepr-cre::tdtomato (green/red) neurons from PLT mice
7A and 7B). Notably, none of the Pomc-hrGFP::Lepr-cre::
tdtomato (green/red) neurons from PLT mice responded to
insulin (Table S1). A subset (40%) of Pomc-hrGFP neurons that
did not express Leprs (green cells) were hyperpolarized in
response to insulin (Figures 7C and 7D), but were unresponsive
to leptin (Table S1). Together, these data support a model in
which the acute effects of leptin and insulin are functionally
segregated in distinct arcuate Pomc neurons. This model en-
riches the population of leptin-responsive neurons in arcuate
Pomc neurons, which allows for the rapid investigation of acute
ER stress-induced leptin and insulin resistance.
ER Stress in Obesity and Diabetes
474 Cell Metabolism 20, 471–482, September 2, 2014 ª2014 Elsevier Inc.
Pretreatment with either tm (30 mM, 6 hr; t(23)= 8.286, p <
0.0001) or tg (15 mM, 6 hr; t(21)= 9.894, p < 0.0001) blunted the
ability of leptin to depolarize Pomc-hrGFP::Lepr-cre::tdtomato
(green/red) neurons from PLT mice (Figures 7A and 7B). Similar
to the observed blunting of acute leptin action in arcuate Pomc
neurons, pretreatment with tm (30 mM, 6 hr; t(17)= 7.142, p <
0.0001; Figures 7C and 7D) or tg (15 mM, 6 hr; t(11)= 5.169, p <
0.001; Figure 7D) blunted the ability of insulin to hyperpolarize
Pomc-hrGFP (green) neurons from PLT mice. Similar results
were obtained in Pomc neurons from organotypic hypothalamic
slices (Figure S3).
Cellular Mechanism for ER Stress-Induced Acute Leptin
and Insulin Resistance in Arcuate Pomc Neurons
Includes Ptp1b, Socs3, and Xbp1s
Neuronal Ptp1b is required for body weight homeostasis and
leptin action (Bence et al., 2006). Mice lacking Ptp1b selectively
in Pomc neurons exhibited increased energy expenditure result-
ing in a resistance to HFD-induced obesity (Banno et al., 2010).
These mice were also more sensitive to the acute effect of leptin
to reduce both food intake and body weight, supportive of a role
for Ptp1b in the modulation of leptin signaling involved in the
acute modulation of Pomc cellular activity. ER stress inhibits
leptin signaling via Ptp1b signaling in a dispersed cell culture
system and in the liver (Delibegovic et al., 2009; Hosoi et al.,
2008). In support of this, we found that pretreatment of hypotha-
lamic slices with activators of ER stress resulted in increased
mRNA for both Ptp1b and Socs3 (p < 0.05; Figure 7E). Constitu-
tive expression of Xbp1s in Pomc neurons suppressed the
expression of both Ptp1b and Socs3 in the arcuate nucleus (for
Ptp1b: t(9)= 2.371, p < 0.05; for Socs3: t(9)= 3.179, p < 0.05; Fig-
ure 7F). Thus, we hypothesized that Ptp1b and/or Socs3 may be
required in Pomc neurons to regulate acute ER stress-induced
Figure 4. Improved Glucoregulation in Mice
that Constitutively Express Xbp1s in Pomc
(B) Exogenous glucose infusion rate (GIR) needed
to clamp blood glucose.
(C and D) Endogenous rates of glucose appear-
ance (endo Ra) and disappearance (Rd) were
determined using a constant infusion of [3-3H]
glucose and Steele’s steady-state calculations.
(E) Body weight of PIXs mice on day of experiment
(n = 7).
leptin and insulin resistance. Also, Xbp1s
alone in Pomc neurons may improve
acute leptin and insulin signaling subse-
quent to ER stress.
In order to first assess the requirement
of Ptp1b or Socs3 in the acute ER
stress-induced leptin and insulin resis-
tance in Pomc neurons, we generated
Pomc reporter mice by mating Pomc-cre
mice with the tdtomato reporter mouse
(Jackson Laboratory, #007908). Similar
to previous reports (Al-Qassab et al., 2009; Hill et al., 2008; Wil-
liams et al., 2010), Pomc neurons from Pomc-cre::tdtomato
mice were activated in response to leptin and inhibited in
response to insulin (Figures 7B and 7D). Pomc-cre::tdtomato
reporter mice were subsequently mated to either Ptp1b-lox
tdtomato::Socs3-lox) mice. Tunicamycin failed to blunt the lep-
tin-induced activation of Pomc neurons selectively deficient for
either Ptp1b or Socs3 (for Pomc-cre::tdtomato::Ptp1b-lox:
t(18) = 1.918, p > 0.05; for Pomc-cre::tdtomato::Socs3-lox:
t(18)= 1.167, p > 0.05; Figure 7B). Similarly, tunicamycin failed
to blunt the insulin-induced inhibition of Pomc neurons selec-
tively deficient for either Ptp1b (for Pomc-cre::tdtomato::Ptp1b-
lox: t(14)= 0.9115, p > 0.05; Figure 7D). Notably, Pomc neurons
deficient for Socs3 were significantly more responsive to insulin
compared to Pomc neurons deficient for Ptp1b alone or control
mice (for insulin in Pomc-cre::tdtomato::Socs3-lox cells com-
pared to Pomc-cre::tdtomato::Ptp1b-lox mice: t(12) = 4.457,
p < 0.05; for insulin in Pomc-cre::tdtomato::Socs3-lox cells
compared to control PLT mice: t(14)= 3.849, p < 0.05 Figure 7D).
these data suggest that Soc3 may have a more potent ER
stress-induced activity related to the suppression of acute leptin
and insulin signaling in arcuate Pomc neurons.
In order to assess the role of Xbp1s in the acute ER stress-
induced leptin and insulin resistance, Pomc reporter PIXs mice
were generated by mating PIXs mice with tdtomato reporter
mouse (Jackson Laboratory, #007908). Tunicamycin failed to
blunt the leptin-induced activation of Pomc neurons from PIXs
mice fed a Dox-enriched diet (t(19)= 1.320, p > 0.05; Figure 7B).
Similarly, tunicamycin failed to blunt the insulin-induced inhi-
bition of Pomc neurons from PIXs mice fed a Dox-enriched
diet (t(17)= 0.3917, p > 0.05; Figure 7D).
ER Stress in Obesity and Diabetes
Cell Metabolism 20, 471–482, September 2, 2014 ª2014 Elsevier Inc. 475
Constitutive expression of Xbp1s in Pomc neurons (PIXs mice)
obesity independent of changes in ad libitum food intake, and
directly (i.e., independently of changes in body weight) improves
glucose homeostasis with decreases in circulating insulin and
blood glucose. In addition to these physiological aberrations,
PIXs mice were sensitized to the acute effects of leptin pharma-
cologically to suppress food intake. On a cellular level, PIXs mice
exhibited decreased Ptp1b and Socs3 expression concomitant
with improved leptin and insulin signaling in Pomc neurons sub-
sequent to ER stress. Increased XBP1s expression only in Pomc
neurons was sufficient to upregulate of the XBP1s axis in the
liver. Together, these data support a model in which a ‘‘fed’’
signal in Pomc neurons abrogates HFD-induced obesity while
at the same time inducing a ‘‘fed’’ transcriptional program in
the liver, improving glucose homeostasis.
production. It is of particular interest that Xbp1s expression in
Pomcneurons has profound effects on bodyweight and glucose
homeostasis. Cellular stress and inflammatory pathways, in-
cluding ER stress and the UPR, have been linked to leptin and
insulin resistance as well as obesity and diabetes (Kaneto
et al., 2005; Tsiotra and Tsigos, 2006; Wellen and Hotamisligil,
2005). Notably, Xbp-1+/?heterozygous mice are more sensitive
to diabetes caused by obesity and HFD (Ozcan et al., 2004).
Similar results were obtained in mice deficient for Xbp1s selec-
observed in mice that constitutively express Xbp1s in Pomc
neurons was dependent upon a hypermetabolic phenotype
(increased energy expenditure and heat production) inde-
pendent of altered food intake. Notably, these responses are
phenotypic signatures of improved leptin action within Pomc
neurons (Balthasar et al., 2004; Berglund et al., 2012; Williams
et al., 2011). The hypermetabolic phenotype was also supported
by increased markers for thermogenesis in BAT as well as
iWAT (Cypess et al., 2009; van Marken Lichtenbelt et al., 2009;
Virtanen et al., 2009; Wu et al., 2012). The increased thermogen-
esis of iWAT suggests that Xbp1s expression in Pomc neurons
Figure 5. Regulation of Xbp1s and GalE in the Arcuate Nucleus and the Liver
(A) Relative mRNA expression of Xbp1s and GalE in the liver and the arcuate nucleus of mice fasted mice (18 hr) and mice refed (2 hr) after fasting (18 hr).
(B) Relative mRNA expression of Xbp1s as well as GalE, Edem1, Erdj4, and Bip, which are known Xbp1s target genes, in the arcuate nucleus from PIXs and WT
mice fed either non-Dox- or Dox-containing diet.
(C and D) Relative mRNA expression of Xbp1s, GalE, Edem1, Erdj4, Bip, Atf4, and Atf6 in FACS-Pomc neurons. Data in (C) represent WT mice fasted (18 hr)
and WT mice refed (2 hr) after fasting (18 hr). Data in (D) represent PIXs and WT mice chronically fed Dox-containing diet.
are known Xbp1s target genes in liver. *p < 0.05 compared with control. In (A)–(E), fold change is relative to 18S mRNA. Error bars indicate SEM.
ER Stress in Obesity and Diabetes
476 Cell Metabolism 20, 471–482, September 2, 2014 ª2014 Elsevier Inc.
promotes browning/‘‘beige-ing’’ of white adipose tissues,
contributing to improvements in body weight. Similar to Xbp1s
activity in the liver (Deng et al., 2013), we found that constitutive
expression of Xbp1s in Pomc neurons alone was also sufficient
to improve blood glucose and insulin levels, supportive of
improved insulin signaling and reminiscent of a fed state even
in the absence of caloric intake.
A neuronal cell-nonautonomous regulation of Xbp1 tran-
scription in the intestinal cell of C. elegans has recently been
described (Taylor and Dillin, 2013). Notably, the improved glu-
coregulation in the current study may be due to elevated
Xbp1s levels in the liver via a similar cell-nonautonomous mech-
anism. Hypothalamic neuronal regulation of peripheral tissues
such as liver and pancreas is likely due to hypothalamic regu-
lation of the activity of key autonomic control neurons in the
brainstem and spinal cord. Although this occurs via multisynap-
tic connections that may involve melanocortin 4 receptor-ex-
pressing neurons in key autonomic circuits (Rossi et al., 2011;
agate the Xbp1s transcriptional signal to the liver.
Another salient finding is the relationship between Xbp1s
and both Ptp1b and Socs3 in the ER stress-induced acute leptin
and insulin resistance of arcuate Pomc neurons. Recently, ER
Figure 6. ER Stress Blunts the Leptin and
Insulin Activity in the Arcuate Nucleus
(A) Blots represent changes in the protein levels
for leptin-induced phospho-STAT3 and phospho-
eif2a in response to ER stress.
(B) Quantitative densitometry for protein expres-
sion of leptin-induced pSTAT3 in control and ER
(C) ER stress blunts leptin-induced pSTAT3
immunoreactivity in the arcuate nucleus of the hy-
pothalamus. *p < 0.05; values are means ± SEM
from 3–6 independent experiments; error bars
(D) Blots represent changes in the protein levels for
insulin-induced phospho-AKT and phospho-eif2a
in response to ER stress.
(E) Quantitative densitometry for the protein ex-
pression of insulin-induced pAKT.
stress has been shown to induce leptin
resistance within the hypothalamus as
well as peripheral insulin resistance in
the liver and muscle (Ozcan et al., 2006,
2009; Thaler and Schwartz, 2010; Thaler
et al., 2012). In the current study, our
findings point to a fundamental role of
ER stress in regulating leptin and insulin
signaling in hypothalamic Pomc neurons.
Classical suppressors of cytokine sig-
naling have been likely candidates in ER
stress-induced leptin and insulin resis-
tance (Howard and Flier, 2006; Myers
et al., 2008; White et al., 2009; Zabolotny
et al., 2008). In particular, both Socs3
and Ptp1b are increased within the hypo-
thalamus in a state of excess nutrition or
obesity (Bjørbaek et al., 1998; Enriori et al., 2007; Mu ¨nzberg
et al.,2004; White etal., 2009; Zabolotny etal., 2008). Brain-spe-
cific Socs3 knockout mice or haploinsufficient mice were signif-
icantly protected against the development of DIO associated
with leptin resistance (Howard et al., 2004; Mori et al., 2004).
Ptp1b knockout mice were similarly resistant to DIO (Bence
et al., 2006; Cook and Unger, 2002; Zabolotny et al., 2002).
Notably, both Ptp1b and Socs3 can be induced by noncytokine
Yoshimura et al., 2007; Zhang et al., 2008). Ptp1b also mediates
ER stress-induced hypothalamic leptin resistance independent
of Socs3 activity (Hosoi et al., 2008). Recently, liver-specific
deficiency of Ptp1b attenuated the induction of ER stress in
response to HFD (Delibegovic et al., 2009). These data are
supported in the current study with the demonstration that
ER stress stimulates Ptp1b and Socs3 in the arcuate nucleus
of the hypothalamus. Pomc neurons selectively deficient for
either Ptp1b or Socs3 demonstrated improved acute leptin
and insulin signaling subsequent to ER stress activation. The
mRNA levels of both Ptp1b and Socs3 were lowered in PIXs
mice. Constitutive expression of Xbp1s in Pomc neurons also
blunted the ability of strong inducers of ER stress to induce
cellular leptin and insulin resistance in Pomc neurons. It should
ER Stress in Obesity and Diabetes
Cell Metabolism 20, 471–482, September 2, 2014 ª2014 Elsevier Inc. 477
be noted that several groups have reported that ER stress
stimulates the production and phosphatase activity of PTP1B
and Socs3 in multiple tissues (Agouni et al., 2011; Bettaieb
et al., 2011; Hosoi et al., 2008; Panzhinskiy et al., 2013). How-
ever, it has been unclear how ER stress-induced PTP1B or
SOCS3 activity may affect acute hypothalamic leptin and insulin
Figure 7. ER Stress Blunts the Leptin-Induced Activation and the Insulin-Induced Inhibition of Pomc Neurons
(A) Brightfield illumination (1) of Pomc-hrGFP::Lepr-cre::tdtomato neuron from PLT mice. Panels 2 and 3 show the same neuron under FITC (hrGFP) and Alexa
Fluor 594 (tdtomato) illumination. Complete dialysis of Alexa Fluor 350 from the intracellular pipette is shown in panel 4. Panel 5 is a merge image illustrating
colocalization of hrGFP, tdtomato, and Alexa Fluor 350, indicative of a Pomc neuron that expresses Leprs. Bottom panel labeled ‘‘Control’’: Electrophysiological
study demonstrates a Pomc-hrGFP::Lepr-cre::tdtomato (green/red) neuron that is depolarized in response to leptin. The panel below demonstrates a current
clamp recording of a separate Pomc-hrGFP::Lepr-cre::tdtomato (green/red) neuron in which ER stress blunted the leptin-induced depolarization.
(B) Histogram demonstrating that multiple activators of ER stress blunted the leptin-induced activation of Pomc neurons (n = 8–15 per group). Deletion of either
Ptp1b or Socs3 restores the leptin-induced excitation of arcuate Pomc neurons after ER stress induction. Similarly, constitutive expression of Xbp1s in Pomc
neurons restores the leptin-induced excitation of arcuate Pomc neurons after ER stress induction. *p < 0.05. Error bars indicate SEM.
(C) Panel 1 shows a brightfield illumination of Pomc-hrGFP neuron from PLT mice. Panels 2 and 3 show the same neuron under FITC (hrGFP) and Alexa Fluor
594 (tdtomato) illumination. Panel 4 shows complete dialysis of Alexa Fluor 350 from the intracellular pipette. Panel 5 is a merge illustrating colocalization of
hrGFP and Alexa Fluor 350, indicative of a Pomc neuron that does not expresses Leprs. Bottom panel labeled ‘‘Control’’: Electrophysiological study demon-
strates that a Pomc-hrGFP (green) neuron is hyperpolarized in response to insulin. The panel below shows a separate Pomc-hrGFP (green) neuron in which
ER stress blunts the insulin-induced hyperpolarization.
(D) Histogram illustrating that chemical activation of ER stress blunts the insulin-induced inhibition of arcuate Pomc neurons (n = 8–18 per group). Deletion of
either Ptp1b or Socs3 restores the insulin-induced inhibition of arcuate Pomc neurons after ER stress induction. *p < 0.05, error bars indicate SEM.
(E) Relative mRNA expression of Socs3 and Ptp1b in organotypic slices following pretreatment with ER stress activators.
(F) Relative mRNA of Ptp1b and Socs3 in the arcuate nucleus from PIXs and WT mice fed HFD-Dox. *p < 0.05; values are means ± SEM from 3–6 independent
experiments; error bars indicate SEM.
ER Stress in Obesity and Diabetes
478 Cell Metabolism 20, 471–482, September 2, 2014 ª2014 Elsevier Inc.
PTP1b-dependent, ER stress-induced acute leptin and insulin
resistance in the current study may be explained by a lack of
IRE1-XBP1 activation. In contrast, ER stress-induced sulfhydra-
tion of PTP1B inhibits its native activity and thereby promotes
PERK activity during the response to ER stress (Krishnan et al.,
2011). Thus, although it is apparent that PTP1B activity is inter-
twined with ER stress and the UPR, the current study extends
previous observations and supports a requirement of PTP1B
and SOCS3 in ER stress-induced acute leptin and insulin resis-
tance of POMC neurons.
Overall, these observations underscore a physiologically
important role for ER stress and the UPR to alter the ability
of arcuate Pomc neurons to properly respond to humoral
signals, ultimately abrogating diet-induced obesity and dia-
betes. Notably, the improvements in leptin and insulin signaling
observed in the current study during times of ER stress link
Xbp1s with both Ptp1b and Socs3 in Pomc neurons. These
data also demonstrate that Pomc neurons induce changes in
the metabolic flux in the liver in a cell-nonautonomous mecha-
nism that contributes to improved glucose homeostasis inde-
pendent of altered body weight. In addition, these data clarify
the roles of these molecules in the regulation of metabolism
and may highlight useful targets for regulating obesity and
related metabolic disorders.
Male (4- to 16-week-old) pathogen-free POMC-hrGFP mice (Parton et al.,
2007; Ramadori et al., 2010) were used for all experiments. To identify
POMC neurons with or without leptin receptors, we generated PLT mice as
previously described (Sohn et al., 2011). Briefly, LepR reporter mice were
made by mating LepR-cre mice (Scott et al., 2009) with the tdtomato reporter
mouse (Jackson Laboratory, #007908). LepR-cre::tdtomato reporter mice
were subsequently mated with POMC-hrGFP mice to produce POMC::
LepR-cre::tdtomato (PLT) mice. To identify Pomc neurons with or without
Ptp1b or Socs3, we generated Pomc-cre::tdtomato::Ptp1b-lox or Pomc-
cre::tdtomato::Socs3-lox mice, respectively. Briefly, Pomc-cre reporter mice
were made by mating Pomc-cre mice with the tdtomato reporter mouse.
Pomc-cre::tdtomato mice were subsequently mated with either Ptp1b-lox or
Socs3-lox mice. Subsequent matings generated mice that were deficient for
either Ptp1b or Socs3 in Pomc neurons.
All mice were housed under standard laboratory conditions (12 hr on/off;
lights on at 7:00 a.m.) and temperature-controlled environment with food
and water available ad libitum. All experiments were performed in accordance
with the guidelines established by the National Institute of Health Guide for
the Care and Use of Laboratory Animals and approved by the University of
Texas Institutional Animal Care and Use Committee.
Western Blot Analysis
The arcuate nucleus from age-matched male mice was microdissected with a
scalpel under a microscope. Whole-cell proteins were extracted by homo-
genizing the hypothalamic blocks in IP lysis buffer (25mM Tris-HCl [pH 7.4],
150 mM NaCl, 1 mM EDTA, 1% NP-40, and 5% glycerol [87787 Pierce])
with protease and phosphatase inhibitor cocktails (1:100, 78440, Pierce).
Equal amounts of the samples (10 mg) were separated by SDS-PAGE and
transferred to a nitrocellulose membrane by electroblotting. Antibodies used
here are the following: a phospho-STAT3 antibody (1:1,000, Cell Signaling
Technology, 9131), a phospho-ERK antibody (1:1,000, Cell Signaling Technol-
ogy, 4370), an antibody against STAT3 (1:1,000, Cell Signaling Technology,
9139), pAKT (1:1,000, Cell Signaling Technology, 4060), AKT (1:1,000, Cell
Signaling Technology, 4691), an antibody against SOCS3 (1:600, Abcam,
ab16030), a PTP1B antibody (1:333, Abcam, ab52650), Bip (1:1,000, Cell
Signaling Technology, 3177), eif2a (1:1,000, Cell Signaling Technology,
3398), XBP1 (1:200, Santa Cruz Biotechnology, SC-7160), and anti-b-actin
antibodies (1:10,000, beta Actin antibody, mAbcam 8226). After incubation
in primary antibodies for 72 hr, the membranes were incubated for 1 hr in
HRP-conjugated secondary antibodies (1:7,000, Southern Biotech, 1010-05
and 4050-05), followed by chemiluminescent detection using West Pico
Chemiluminescent Substrate (Thermo Fisher Scientific). To measure the fluo-
rescent intensity, the Odyssey IR imaging system (LI-COR Biosciences) was
used. After incubation for primary antibodies, the membrane was incubated
in the secondary antibody conjugated to a fluorescent entity: IRDye 800-
conjugated goat anti-rabbit IgG and/or Alexa Fluor 680-conjugated goat
ature. At the end of the incubation period, membranes were washed twice
with phosphate -buffered saline (PBS) with 0.05% Tween (PBS-T). The
membrane was visualized and analyzed on the Odyssey IR imaging system
(LI-COR Biosciences). Phospho-proteins were normalized to the levels of
the corresponding total protein.
Whole-cell patch-clamp recordings from POMC-hrGFP neurons maintained
in hypothalamic slice preparations and data analysis were performed as pre-
viously described (Hill et al., 2008). Briefly, 4- to 16-week-old male mice
were anesthetized and transcardially perfused with a modified ice-cold artifi-
cial cerebrospinal fluid (ACSF) (described below), in which an equiosmolar
amount of sucrose was substituted for NaCl. The mice were then decapitated,
and the entire brain was removed and immediately submerged in ice-cold,
carbogen-saturated (95% O2and 5% CO2) ACSF (126 mM NaCl, 2.8 mM
KCl, 1.2 mM MgCl2, 2.5 mM CaCl2, 1.25 mM NaH2PO4, 26 mM NaHCO3,
and 5 mM glucose). Coronal sections (250 mm) were cut with a Leica
VT1000S Vibratome and then incubated in oxygenated ACSF at room temper-
ature for at least 1 hr before recording. Slices were transferred to the recording
chamber and allowed to equilibrate for 10–20 min before recording. The slices
were bathed in oxygenated ACSF (32?C–34?C) at a flow rate of ?2 ml/min.
The pipette solution for whole-cell recording was modified to include an
intracellular dye (Alexa Fluor 594 or Alexa Fluor 350) for whole-cell recording:
120 mM K-gluconate, 10 mM KCl, 10 mM HEPES, 5 mM EGTA, 1 mM CaCl2,
1 mM MgCl2, and 2 mM MgATP and either 0.03 mM Alexa Fluor 594 or Alexa
Fluor 350 hydrazide dye (pH 7.3). Epifluorescence was briefly used to target
fluorescent cells, at which time the light source was switched to infrared
differential interference contrast imaging to obtain the whole-cell recording
(Zeiss Axioskop FS2 Plus equipped with a fixed stage and a QuantEM:512SC
electron-multiplying charge-coupled device camera). Electrophysiological
signals were recorded using an Axopatch 700B amplifier (Molecular Devices),
low-pass filtered at 2–5 kHz, and analyzed offline on a PC with pCLAMP pro-
grams(MolecularDevices).Recording electrodes hadresistancesof2.5–5MU
when filled with the K-gluconate internal solution. Input resistance was as-
sessed by measuring voltage deflection at the end of the response to a hyper-
polarizing rectangular current pulse steps (500 ms of ?10 to ?50 pA).
Leptin (100 nM, provided by A.F. Parlow through the National Hormone and
Peptide Program) or insulin (50 nM, Humulin-R 100 U/ml, Lilly) was added
to the ACSF for specific experiments. Solutions containing leptin or insulin
were typically perfused for 2–4 min. A drug effect was required to be associ-
ated temporally with peptide application, and the response had to be stable
within a few minutes. A neuron was considered depolarized or hyperpolarized
if a change in membrane potential was at least 2 mV in amplitude.
Energy Expenditure and Locomotor Activity
Weight- and body composition-matched 8-week-old WT and PIXs mice
were used for metabolic assessment. Three separate cohorts of animals
were used to produce the metabolic data, with measurements sorted into
12 hrlight/dark periods. To exclude the possibility that changes inbody weight
or composition would contribute to energy expenditure measurements, meta-
bolic assessment was done using weight- and body composition-matched
mice (Butler and Kozak, 2010; Tscho ¨p et al., 2012). Data, where applicable,
ER Stress in Obesity and Diabetes
Cell Metabolism 20, 471–482, September 2, 2014 ª2014 Elsevier Inc. 479
of30.75(orbyexpressingdataonaperanimalbasis) (ButlerandKozak, 2010;
Tscho ¨p et al., 2012).
Mice were first acclimatized to the metabolic cages and housed individually
for 4 days before measurements were taken. Mice were analyzed in the meta-
bolic chambers for 4 days and were provided with food ad libitum. Energy
expenditure was measured by indirect calorimetry, while locomotor activity
was assessed using an infrared light beam detection system (Labmaster,
TSE Systems GmbH). Data were collected using a TSE Labmaster monitoring
24 hr period. Data were averaged over the 4 day period of measurement.
Body Weight and Composition and Fat Distribution
at 4–5 weeks of age. Body weight was measured weekly up to 25 weeks.
Body fat composition of 25-week-old ad libitum fed mice was assessed using
nuclear magnetic resonance spectroscopy using an NMR spectrometer
(EchoMRI). For fat distribution, mice were anesthetized with 1% isoflurane
inhalation and then the trunk (from base of the skull as the spinal canal begins
to widen and the distal end of the tibia) of each mouse was scanned at an
isotropic voxel size of93 mm(80kV, 450mA, and 100msintegrationtime) using
the eXplore Locus micro-CT scanner (GE Health Care). Three-dimensional
images were reconstructed from two-dimensional grayscale image slices
and visualized using Microview Software (GE Medical System). Density values
for soft tissue and bone were calibrated from a phantom (GE Health Care)
containing air bubble, water, and hydroxyl apatite rod. The separation of
fat regions was obtained from the appropriate grayscale value (upper
threshold, ?165; lower threshold, ?360). The abdominal muscular wall was
used as the differentiation line to separate visceral adipose tissue from subcu-
taneous adipose tissue. The contour lines were drawn around the viscera and
three-dimensional ROI was generated. The visceral fat was determined from
the histogram of these segmented viscera using the same thresholds. Subcu-
taneous fat was obtained by subtracting visceral fat from the total body fat.
Statistical analysis was carried out using GraphPad 5. All data were evaluated
using a two-tailed Student’s t test with a p value of less than 0.05 being con-
sidered significant. In all instances, data are presented as mean ± SEM. Body
weight curves were compared using a linear regression analysis. Degrees of
freedom (DF) for t statistics are marked as t(DF).
Supplemental Information includes Supplemental Experimental Procedures,
three figures, and one table and can be found with this article online at
periments and performed all experiments except gene expression and immu-
noblotting, analyzed the data, and wrote the manuscript. Tiemin Liu designed
and performed experiments except gene expression and immunoblotting,
analyzed the data, and wrote the manuscript. X.K. analyzed gene expression
in adipose depots, performed immunohistochemistry in BAT and iWAT,
organotypic slice experiments including immunoblotting and gene expression,
analyzed data, and reviewed the manuscript. E.D.B. designed and performed
hyperinsulinemic-euglycemic clamp experiments, analyzed the data, and re-
viewed the manuscript. Y.D., J.-W.S., Z.D., Y.G., Tianya Liu, L.J., T.F., D.K.,
M.M.S., S.L., C.E.L., K.S., and Y.C. assisted in performing experiments.
P.E.S. and J.K.E. are co-senior authors: they supervised development of the
mouse models, designed experiments, and edited the manuscript.
We thank Dr. Jeffrey Friedman (Rockefeller University) for kindly providing us
with the Lepr-cre mice. We also thank Dr. Bradford Lowell (Beth Israel
Deaconess Medical Center) for kindly providing us with the Pomc-hrGFP
mice. This work was supported by grants to K.W.W. (K01DK087780),
Tiemin Liu (American Diabetes Association 7-11-MN-16), M.F. (American
Heart Association 9SDG2080223), J.-W.S. (American Heart Association
E.D.B. (NIH F32 DK092083 and K01 DK098317), X.K. (American Heart
Association 13POST16710016), T.F. (Juvenile Diabetes Research Foundation
3-2011-405), and J.K.E. (R01DK53301, R01DK088423, and RL1DK081185).
This work was also supported by PL1 DK081182 and UL1RR024923, as well
as P01DK088761 (P.E.S. and J.K.E.) and R01DK55758 (P.E.S.).
Received: March 6, 2014
Revised: May 20, 2014
Accepted: May 29, 2014
Published: July 10, 2014
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