Page 1 of 60 Diabetes · 2018. 1. 8. · SGK1 allele (SGK1loxp/loxp) mice (18) were kindly provided by Dr Geza Fejes-Toth and Dr Aniko Naray-Fejes-Toth (Dartmouth Medical School,

1

SGK1/FOXO3 signaling in hypothalamic POMC neurons

mediates glucocorticoid-increased adiposity

Yalan Deng1, Yuzhong Xiao

1, Feixiang Yuan

1, Yaping Liu

2, Xiaoxue Jiang

1,

Jiali Deng1, Geza Fejes-Toth

3, Aniko Naray-Fejes-Toth

3, Shanghai Chen

1, Yan Chen

1,

Hao Ying1, Qiwei Zhai

1, Yousheng Shu

#2 and Feifan Guo

#1

1 Key Laboratory of Nutrition and Metabolism, Institute for Nutritional Sciences,

Shanghai Institute for Biological Sciences, Chinese Academy of Sciences, University

of Chinese Academy of Sciences, 320 Yueyang Road, Shanghai, China 200031

2 State Key Laboratory of Cognitive Neuroscience and Learning and IDG/McGovern

Institute for Brain Research, School of Brain and Cognitive Sciences, Beijing Normal

University, Beijing, China 100875

3Department of Physiology, Dartmouth Medical School, Lebanon, New Hampshire

03756-0001, USA

Contact Information

# Correspondence should be addressed to Feifan Guo; E-mail: [email protected];

Address: 320 Yueyang Road, Shanghai, China 200031 and Yousheng Shu; E-mail:

[email protected]; Address: Beijing Normal University, Beijing, China 100875.

E-mail: [email protected] Tel: 86 21 54920945; Fax: 86 21 54920291.

[email protected] Tel: 86 10 58804976; Fax: 86 10 58804976.

Page 1 of 60 Diabetes

Diabetes Publish Ahead of Print, published online January 10, 2018

2

Running title: POMC SGK1/FOXO3 signaling regulates adiposity.

The word count: 4161 The number of tables and figures: 0 and 6

ABSTRACT

Although central nervous system has been implicated in glucocorticoid-induced

fat mass gain, the underlying mechanisms are poorly understood. The aim of our

current study was to investigate the possible involvement of hypothalamic serum- and

glucocorticoid-regulated kinase 1 (SGK1) in glucocorticoid-increased adiposity. It is

well-known that SGK1 expression is induced by acute glucocorticoid treatment,

interestingly, we found its expression was decreased in the arcuate nucleus of the

hypothalamus, including POMC neurons, following chronic dexamethasone (Dex)

treatment. To study a role of SGK1 in POMC neurons, mice with development or

adult-onset SGK1 deletion in POMC neurons (PSKO) were then produced. As

observed in Dex-treated mice, PSKO mice exhibited increased adiposity and

decreased energy expenditure. Consistently, mice overexpressing constitutively active

SGK1 in POMC neurons (PSOE) had the opposite phenotype and prevented from

Dex-increased adiposity. Finally, Dex decreased hypothalamic α-melanocyte

stimulating hormone (α-MSH) content and its precursor Pomc expression via

SGK1/Forkhead box O3 (FOXO3) signaling and intracerebroventricular injection of

α-MSH or adenovirus-mediated FOXO3 knockdown in ARC largely reversed the

metabolic alterations in PSKO mice. These results demonstrate that POMC

SGK1/FOXO3 signaling mediates glucocorticoid-increased adiposity, providing new

Page 2 of 60Diabetes

3

insights into mechanistic link between glucocorticoid and fat accumulation and

important hints for possible treatment targets for obesity.

INTRODUCTION

In addition to the overwhelming beneficial effects of glucocorticoid for

anti-inflammatory purposes, chronic glucocorticoid treatment is shown to cause

numerous adverse metabolic outcomes, including fat mass gain (1). Recent studies

have elucidated several peripheral mechanisms underlying glucocorticoid-induced fat

mass increase. For example, glucocorticoid induces adipocyte differentiation (1-3),

alters lipid metabolism in adipose tissue (1-3) and inhibits browning of white adipose

tissue (WAT) or thermogenesis of brown adipose tissue (BAT) (4; 5). In fact, body fat

mass is also largely controlled by the central nervous system (CNS) (6-8). Specific

populations of neurons in the arcuate nucleus (ARC) of hypothalamus also play

fundamental roles in the regulation of energy balance and lipid metabolism (6-8). In

particular, neurons coexpressing orexigenic neuropeptides agouti-related protein

(AgRP) and neuropeptide Y (NPY) along with neurons coexpressing anorexigenic

pro-opiomelanocortin (POMC) precursor and cocaine and amphetamine-related

transcript (CART) are extensively involved in the regulation of appetite, body weight

and metabolism (6-8). POMC is a protein expressed and secreted from POMC

neurons and cleaved by prohormone convertases to produce α-melanocyte stimulating

hormone (α-MSH) (8). α-MSH binds to the melanocortin 4 receptor (MC4R) and

functions as a key hub linking the CNS to peripheral organs through the sympathetic


4

nervous system (SNS), whereas dysfunction of this signaling axis leads to obesity in

mice and humans (9; 10). Activation of SNS promotes the release of norepinephrine

(NE) that binds to β-adrenergic receptor 3 (ADRB3) and stimulates WAT lipolysis

and BAT thermogenesis (11-14). Although previous studies have shown

glucocorticoid regulates food intake and energy expenditure (15; 16), the central

signals mediating glucocorticoid’s effect are poorly understood.

Serum- and Glucocorticoid-regulated Kinase 1 (SGK1) belongs to the family of

serine/threonine kinases and its coding region was originally isolated from rat

mammary tumour cells (17). SGK1 is ubiquitously expressed in various tissues,

including hypothalamus (17) and functions via activation of glucocorticoid receptor

(GR), retinoid X receptor (RXR), peroxisomeproliferator-activated receptor γ (PPARγ)

and nuclear factor κB (NF-κB) (17). It has been shown that SGK1 is involved in the

regulation of many processes, including hypertension, epithelial sodium channel

activity and insulin sensitivity (17-19). SGK1 also mediates many important functions

of glucocorticoids, including insulin secretion and hippocampal neurogenesis (20; 21).

Although extensive studies have been carried out, a role of hypothalamic SGK1 in the

regulation of energy homeostasis is unknown. Furthermore, it is well-known that

SGK1 is an early response gene that can be induced by acute glucocorticoid treatment

in various cells and animal models (20-22), however, the effect of chronic

glucocorticoid treatment on SGK1 expression remains largely unknown. In fact, the

expression of SGK1 in the context of glucocorticoid-induced metabolic effects could

be very important, as studies show that sometimes SGK1 may have opposing effects


5

of glucocorticoid (23).

Despite the above unknown facts, as a downstream target of glucocorticoid (17)

expressed in the hypothalamus (17), it is conceivable to speculate that SGK1 may

contribute to the central action of glucocorticoid. Therefore, the aim of our current

study was to test this hypothesis first by determining the expression of SGK1 in the

hypothalamus and followed by investigating its possible contribution to

glucocorticoid-increased adiposity.

By constructing mice with development or adult-onset knockout of SGK1, or

over-expression of SGK1 in POMC neurons, we demonstrate a crucial role for SGK1

expressed in POMC neurons in glucocorticoid-increased adiposity and provide a

novel mechanistic link between glucocorticoid treatment and body fat mass gain.

RESEARCH DESIGN AND METHODS

Mice and diets

The POMC-Cre mice (24) and POMC-cre:ERT2

(24) were kindly provided by

Prof. Joel K. Elmquist and Tiemin Liu from Southwestern Medical Center, and floxed

SGK1 allele (SGK1loxp/loxp

) mice (18) were kindly provided by Dr Geza Fejes-Toth

and Dr Aniko Naray-Fejes-Toth (Dartmouth Medical School, Hanover, NH, USA).

To generate POMC neuron-specific SGK1 knockout mice, POMC-Cre mice were

crossed with SGK1loxp/loxp

mice. To generate an inducible POMC specific SGK1

knockout mice, POMC-cre:ERT2

mice were crossed with SGK1loxp/loxp

mice.

Tamoxifen (0.15 g/kg; Sigma, MO, USA) suspended in corn oil (Sigma, MO, USA)


6

was intraperitoneally (i.p.) injected to 8-week-old male SGK1loxp/loxp

or SGK1loxp/loxp

×

POMC-cre:ERT2

littermate mice for 5 consecutive days to generate mice with adult

onset of SGK1 deletion in POMC neurons (PSKO-ER). Dexamethasone (Dex)

treatment were conducted with male WT, control or PSKO mice, or male AAV-CA

SGK1/AAV-null ARC injection POMC-cre mice by i.p. injection with PBS or 5

mg/kg Dex every other day for 6 weeks or 2 hours (1; 25). All the mice were in

C57BL/6J background. Mice were maintained on a 12:12 hr light-dark cycle (lights

on 7:00/off 19:00) at 25 ℃ with free access to water and standard chow diet. In vivo

studies were conducted in accordance with the guidelines of the Institutional Animal

Care and Use Committee of Shanghai Institute for Nutritional Sciences, Chinese

Academy of Sciences.

Intracerebroventricular (i.c.v.) cannulation and ARC administration

experiments

I.c.v. cannulation experiments were conducted as previously described (26). Five

days after recovery, mice were infused with 2 ul of α-MSH peptide (Abcam,

Cambridge, UK) at a concentration of 1 nmol/uL or 2ul artificial cerebrospinal fluid

(ACSF) (Tocris, Bristol, UK) and experiments were conducted 24 h later. ARC

administration experiments were conducted as previously described (6). Mice were

anesthetized and received bilateral stereotaxic injections of adenovirus expressing

Forkhead box O3 (FOXO3)-specific short hairpin RNA against FOXO3

(Ad-shFOXO3) or scrambled control adenovirus (Ad-scrambled), adeno-associated


7

virus expressing constitutively active mutant rat SGK1 (S422D) (AAV-CA SGK1) or

control AAV-null into ARC (1.5 mm posterior to the bregma, ± 0.2 mm lateral to

midline and 6 mm below the surface of the skull). AAV-CA SGK1 expression

plasmid was constructed in pAAV-Ef1a-DIO-mCherry-2A plasmid (Addgene, MA,

USA), and SGK1 started to express only in the presence of CRE recombinase.

Metabolic parameter measurements

The mice body composition was measured by a nuclear magnetic resonance

system (Bruker, Rheinstetten, GER). Indirect calorimetry was performed in a

comprehensive laboratory animal-monitoring system (Columbus Instruments, OH,

USA), as previously described (27). Rectal temperature of mice was measured at

14:00 and 17:00 by a rectal probe attached to a digital thermometer (Physitemp,

Clifton, NJ). The measurement of food intake was conducted as reported previously

(6).

POMC neuron identification, count and area

AI9 (tdTomato) reporter mice (Jackson Laboratory) were mated with or

AAV-CA SGK1 and AAV-null expressed mCherry red fluorescent-protein were ARC

injected to POMC-Cre mice to reflect POMC neurons, demonstrated by the

colocolization with POMC antibodies. The distribution and number of POMC

neurons were determined as described previously (6). Average somatic area was

analyzed in > 500 POMC neurons (n = 4 mice/genotype). The area occupied by


8

POMC neurons was manually scored using Image J software.

Hypothalamic α-MSH protein content

Hypothalamus was prepared as previously described (6) and α-MSH were

quantified by ELISA kit (Phoenix Pharmaceuticals, CA, USA), according to

manufacture’s instructions.

Hypothalamic nuclear and cytoplasmic fractions

Hypothalamic nuclear and cytoplasmic fractions were isolated as previously

described (28).

Immunofluorescence staining

Immunofluorescence stainings were performed as previously described (29) with

anti-SGK1 and anti-p-N-myc downstream-regulated gene 1 (p-NDRG1) (Abcam,

Cambridge, UK), anti-POMC (Phoenix pharmaceuticals, inc, CA, USA), anti-FOXO3

(Cell Signaling Technology, MA, USA), anti-p-SGK1 and anti-GR (Santa Cruz

Biotechnology, CA, USA) and anti-α-MSH (Merck Millipore, Frankfurter, GER).

Immunofluorescence staining of p-FOXO3 was performed using the Tyramide Signal

Amplifcation (TSA) Cyanine 3 system (Perkin-Elmer, Boston, MA), and

anti-p-FOXO3 primary antibody (Cell Signaling Technology, MA, USA) was

co-incubated with anti-mCherry (Abbkine, California, USA).


9

RNA isolation and relative quantitative RT-PCR

RNA isolation and RT-PCR were performed as previously described (27). The

sequences of primers used in this study are available upon request.

Western blot analysis

Western blot analysis was performed as previously described (27) with the

following primary antibodies, anti-p-FOXO3, anti-FOXO3, anti-lamin B1 and

anti-p-GR (Cell Signaling Technology, MA, USA), anti-SGK1 and anti-GR (Abcam,

Cambridge, UK), anti-uncoupling protein 1 (UCP1) and anti-p-SGK1 (Santa Cruz

Biotechnology, CA, USA), anti-α-tublin and anti-β-actin (Sigma, MO, USA).

Primary hypothalamic neurons isolation and treatments

Primary cultures of hypothalamic neurons were prepared as previously

described (27). On day 7, primary cultured hypothalamic neurons were infected with

adenovirus expressing SGK1-specific short hairpin RNA (Ad-shSGK1; 108 pfu/60

cm2 cells) or scrambled control adenovirus, constructed as described previously (19).

Primary hypothalamic neurons were transfected with siRNA for FOXO3 by

X-tremeGene siRNA Transfection Reagent (Roche Diagnostics, Mannheim,

Germany). Constitutively active mutant rat SGK1 (S422D) was subcloned into

PCMV-MYC plasmid and transfected into primary cultured hypothalamic neurons

using Lipofectamine 2000 (Life Technologies).


10

Statistical analysis

All values are presented as means ± SEM. Differences between groups were

analyzed by either Student t test or one-way ANOVA followed by the

Student-Newman-Keuls (SNK) test. Differences in which P was < 0.05 were

considered statistically significant.

RESULTS

Chronic Dex treatment decreases SGK1 expression in hypothalamic POMC

neurons

To investigate the metabolic effects of Dex, C57B6J wild-type (WT) mice were

i.p. injected with Dex for 6 weeks, the way of which has been commonly used to

study the role of Dex (1; 30). Dex treatment did not change body weight, though the

total body fat and abdominal fat mass was increased compared with control treatment,

possibly due to the decreased lean mass (Fig. S1A-D). Body fat mass is maintained by

a balance between energy intake and energy expenditure (7). Dex treatment did not

change food intake, but decreased energy expenditure as measured by 24-h-indirect

calorimetry (Fig. S1E and S1F). No difference was observed in locomotor activity,

but the body temperature, levels of BAT thermogenic marker UCP1 (11) and serum

NE levels were significantly lower in Dex-treated mice (Fig. S1G-J).

To investigate the possible involvement of hypothalamic SGK1 in Dex-increased

adiposity, we examined hypothalamic SGK1 expression under this condition and

interestingly found that hypothalamic SGK1 and p-SGK1 were decreased in


11

Dex-treated mice (Fig. 1A and 1B). Furthermore, immunofluorescence staining

showed that SGK1 and p-SGK1 was decreased in ARC of the hypothalamus of

Dex-treated mice (Fig. 1C-F). Immunofluorescence staining of tdTomato and SGK1

showed that SGK1 was colocalized with POMC neurons in PBS-treated mice, but

decreased significantly in POMC neurons of Dex-treated mice (Fig. 1G and 1H). In

contrast, SGK1 expression was increased in ARC of the hypothalamus by acute

treatment (Fig. 1I-L).

Deletion of SGK1 in POMC neurons causes obesity and decreases energy

expenditure

Based on the above results, we speculated that knockout of SGK1 expression in

POMC neurons might mimic Dex-induced metabolic alterations. To test this

hypothesis, we generated POMC neuron-specific SGK1 knockout (PSKO) mice.

Immunofluorescence staining of tdTomato and SGK1 showed that SGK1 was

colocalized with POMC neurons (more than 90 % overlapping with tdTomato) in

control mice, but almost absent in POMC neurons of PSKO mice (Fig. 2A and

S2A-C), with no difference in SGK1 staining in PVN and VMH between PSKO and

control mice (Fig. S2D-G). Consistently, Sgk1 mRNA levels were decreased about

50 % in ARC, as there are other neurons or neurogliocytes (31; 32), but not other

brain areas and tissues, of PSKO mice (Fig. 2B). POMC neuron differentiation and

survival, however, as anatomical assessment of POMC neurons throughout the ARC

area revealed no significant alterations in neuronal population size, distribution and


12

somatic area in PSKO mice (Fig. S3A and S3B). Because POMC promoter also

drives CRE recombinase expression in the pituitary (33), we examined serum contents

of hormones secreted from pituitary, including corticosterone and growth hormone

(34), and found that the levels of these two hormones were not altered in PSKO mice

(Fig. S3C and S3D).

Male PSKO mice exhibited a significant increased body weight from the age of

9-weeks old compared with control mice (Fig. 2C), accompanied by a significant

increase in total body fat and abdominal fat mass (Fig. 2D and 2E), with unchanged

lean mass (Fig. S2H). Food intake was not altered, but the energy expenditure was

markedly decreased and respiratory exchange ratio (RER; VCO2/VO2) was higher in

PSKO mice (Fig. 2F-H). Again, locomotor activity was not changed, but body

temperature, BAT UCP1 and serum NE levels were significantly lower in PSKO mice

(Fig. 2I-L). As observed for male mice, female PSKO mice also displayed similar

phenotypes (Fig. S4), so we undertook all of the subsequent studies in male mice.

Inducible loss of SGK1 in POMC neurons in adult mice recapitulates aberrant

energy homeostasis

We next asked whether adult-onset loss of SGK1 in POMC neurons had similar

effects to those of ablation during development. We employed a tamoxifen-inducible

POMC-cre mouse model (POMC-cre:ERT2

) (24) that allows temporal control of CRE

recombinase activity and can be combined with SGK1flox/flox

mice producing mice with

adult-onset deletion of SGK1 (PSKO-ER). Similar phenotypes were observed as


13

using constitutive POMC-cre mice (Fig. S5).

Mice with over-expression of SGK1 in POMC neurons are lean and resistant to

Dex-induced fat accumulation

We then asked whether over-expression of SGK1 in POMC neurons in mice

would have the opposite phenotype as observed in PSKO mice and prevented from

Dex-increased adiposity. For this purpose, we generated POMC neuron-specific

SGK1 over-expression (PSOE) mice by ARC bilateral stereotaxic injection of

adeno-associated virus expressing constitutively active mutant rat SGK1 (S422D)

(AAV-CA SGK1) or control AAV-null to male POMC-Cre mice. The effect of SGK1

over-expression was validated by immunofluorescence staining of the phosphorylated

levels of NDRG1 that reflects the activation status of SGK1 (35) (Fig. 3A) and

increased signals of SGK1 in POMC neurons (more than 90 % overlapping with

mCherry), but not in PVN and VMH, of PSOE mice (Fig. S6A-H). As predicted, the

body weight was decreased (starting from 6 weeks after AAV injection), accompanied

by a decrease in total body fat and abdominal fat mass in PSOE mice (Fig. 3B-D).

Food intake was not affected, but the energy expenditure was increased and RER was

decreased in PSOE mice (Fig. 3E-G). No difference was observed in locomotor

activity, but body temperature, BAT UCP1 and serum NE levels were increased in

PSOE mice (Fig. 3H-K). Furthermore, PSOE mice were resistant to Dex-induced fat

accumulation and other metabolic alterations (Fig. 4), with Dex injected 5 weeks after

AAV injection under no difference in lean mass and fat mass between control and


14

PSOE mice (Fig. S6I and S6J). In contrast, Dex had very mild effect on PSKO mice,

as demonstrated by the slightly decreased body weight and lean mass, increased fat

mass, with no significant effect on food intake and energy expenditure (Fig. S7).

Dex decreases hypothalamic αααα-MSH content via SGK1 and administration of

α-MSH reverse obese phenotype in PSKO mice

Because previous studies have shown that α-MSH plays a critical role in the

regulation of energy homeostasis (33), we asked whether it might be involved in

Dex-induced metabolic alterations. As predicted, a dramatic reduction of α-MSH

staining was observed in PVN of Dex-treated mice (Fig. 5A and 5B). Similar results

were obtained in PSKO mice (Fig. 5C and 5D). Consistently, the content of α-MSH

was significantly decreased in the hypothalamus of PSKO mice when analyzed by

ELISA (Fig. 5E). Notably, Dex-reduced α-MSH staining was reversed in PSOE mice

(Fig. 5F and 5G).

To investigate whether α-MSH could mediate SGK1 regulation of energy

homeostasis, we i.c.v. administered α-MSH peptide to PSKO or control mice. I.c.v.

injection of α-MSH to PSKO mice markedly reduced body weight and abdominal fat

mass and increased rectal temperature compared with mice injected with control

vehicle (Fig. 5H-J). I.c.v. injection of α-MSH in PSKO mice also blocked UCP1

protein decrease (Fig. 5K). Similar effects were observed in control mice following

i.c.v. injection of α-MSH (Fig. 5H-K).


15

Dex reduces α-MSH precursor POMC expression via SGK1/FOXO3 dependent

pathway and down-regulation of FOXO3 largely reversed the obesity phenotype

in PSKO mice

α-MSH levels are determined by the levels of its precursor POMC, and the

expression of prohormone convertases that are responsible for the cleavage of POMC

to α-MSH (8). The reduced α-MSH concentration in Dex-treated mice did not seem to

be the consequence of decreased expression of processing enzymes, including

prohormone convertase 1 (Pc1/3), prohormone convertase 2 (Pc2), carboxypeptidase

E (Cpe), α-amidating monooxygenase (Pam) and prolylcarboxypeptidase (Prcp) (8),

as gene expression of these enzymes was unchanged (Fig. 6A). On the other hand,

POMC expression was decreased in Dex-treated mice (Fig. 6A-C). Similar results

were obtained in PSKO mice (Fig. S8A-C). The effect of Dex on reducing POMC

expression, however, was reversed by over-expression of SGK1 (Fig 6D and 6E).

Similarly, SGK1 knockdown decreased Pomc expression and SGK1 over-expression

increased Pomc expression in primary cultured hypothalamic neurons (Fig. S8D and

S8E).

We then investigated the downstream signaling of SGK1 in mediating

Dex-decreased POMC expression. Previous study shows that SGK1 phosphorylates

FOXO3 (36), and another member from the same FOXO family FOXO1 inhibits

Pomc expression (37), suggesting that FOXO3 might have similar function to FOXO1

as downstream of SGK1 in Dex-induced metabolic alterations. Consistent with this

possibility, hypothalamic FOXO3 phosphorylation was decreased in Dex-treated mice


16

(Fig. 6F). Similar reduction was observed in hypothalamic ARC of PSKO mice (Fig.

S9A). Furthermore, Dex-decreased hypothalamic FOXO3 phosphorylation was

reversed in PSOE mice (Fig. 6G and 6H). Similar regulatory effects of SGK1 on

p-FOXO3 were observed in primary cultures of hypothalamic neurons (Fig. S9B and

S9C).

Because the inhibitory effect of SGK1 knockdown on Pomc expression was

reversed by siRNA-mediated FOXO3 inhibition (Fig. S9D), promoting us to

investigate the in vivo function of FOXO3 as downstream of SGK1. For this purpose,

we knocked down FOXO3 expression in ARC of PSKO and control mice by ARC

administration (6) of adenovirus expressing shRNA directed against the coding region

of FOXO3 (Ad-shFOXO3) (38) or adenoviruses expressing scrambled sequences

(Ad-scrambled). The effect of Ad-shFOXO3 was demonstrated by the decreased

expression of Foxo3 and the corresponding change in Pomc expression in ARC of

PSKO mice (Fig. 6I). Consistently, immunofluorescence showed that FOXO3 was

decreased in ARC, but not PVN and VMH, in these mice (Fig. S10A and S10B).

Ad-shFOXO3 decreased the body weight, total body fat and abdominal fat mass in

PSKO mice (Fig. 6J-L). Although food intake was not affected (Fig. S10C), the

decreased energy expenditure and increased RER in PSKO mice were largely

reversed by Ad-shFOXO3 (Fig. 6M and 6N). No significant difference in locomotor

activity was detected (Fig. S10D), however, the decreased body temperature, BAT

UCP1 and serum NE in PSKO mice were upregulated by Ad-shFOXO3 (Fig. 6O-Q).

Moreover, the reduced α-MSH staining in PSKO mice was also blocked by


17

Ad-shFOXO3 (Fig. S10E and S10F). Except for the unaltered body weight, similar

effects were observed in control mice following administration of Ad-shFOXO3 (Fig.

6I-Q and Fig. S10).

As glucocortitoid functions via GR (28), we investigated the spatial regulation of

GR and SGK1/FOXO3, with GR antibodies validated previously (39). Though

hypothalamic Gr mRNA was unchanged, total GR and phosphorylated GR expression

were significantly decreased, in Dex-treated mice (Fig. S11A and S11B). Furthermore,

these three proteins were all expressed in POMC neurons, and hypothalamic nuclear

p-GR was decreased and FOXO3 was increased, whereas cytoplasmic total and

phorphorylated proteins examined were all decreased, in Dex-treated mice (Fig. S11C

and S11D).

DISCUSSION

Fat mass accumulation is a serious side effect of glucocorticoid therapy (1).

Recent studies have elucidated several peripheral mechanisms underlying

glucocorticoid-induced fat mass gain (1-5). In this study, we demonstrated a novel

central mechanism mediated by SGK1 underlying glucocorticoid-increased adiposity.

SGK1 is a well-known downstream target of Dex (20-22). It has been widely

demonstrated that acute Dex treatment induces SGK1 (20-22). Interestingly, we found

that SGK1 expression was decreased in ARC POMC neurons of Dex-treated mice.

The importance of POMC SGK1 in mediating Dex-induced adiposity was

demonstrated by the observation that knockout of SGK1 expression in POMC


18

neurons increased adiposity, while overexpression of SGK1 in POMC neurons

resulted in lean phenotype and prevented Dex-induced fat mass gain in mice.

Furthermore, the Dex-induced fat mass gain was much less in PSKO compared with

control mice. The fat mass, however, could still be increased by Dex treatment in

PSKO mice, suggesting the existence of other central or peripheral signals involved in

Dex-increased adiposity (40). Our study provides a novel mechanistic link between

glucocorticoid treatment and fat mass gain. This is important for understanding the

mechanisms of glucocorticoid-induced metabolic phenotypes, and also providing

important hint for the possible treatment target for glucocorticoid-induced side effects.

In addition, our study reports an unrecognized novel function of SGK1 in POMC

neurons of the hypothalamus in the regulation of energy homeostasis. These results

are important for understanding the signals in specific neurons that are critical for

metabolic control.

Body fat mass is maintained by a balance between energy intake and energy

expenditure (7). BAT oxidizes fat to produce heat via increased expression of UCPs,

which is stimulated by activation of SNS. Deletion of UCP1 induces obesity and

upregulation of UCP1 increases thermogenesis and energy expenditure in mice (11).

Consistently, other studies also showed that disruption of SNS activity has significant

negative impact on energy expenditure (6; 41; 42). Our study showed that Dex

increased adiposity mainly by decreasing energy expenditure, as food intake was not

changed in Dex-treated mice. Furthermore, the decreased energy expenditure by Dex

treatment was most likely due to decreased thermogenesis in BAT as demonstrated by


19

the decreased body temperature, BAT UCP1 expression and serum NE in these mice.

Lipolysis in WAT is also regulated by SNS activity (41; 42), which might also affect

Dex-induced adiposity, and should be studied in the future.

Extensive evidence indicates that the melanocortin signaling in hypothalamus

plays an important role in regulating energy homeostasis and lipid metabolism

through affecting SNS activity in BAT (11-14). In this study, we demonstrated a

possible role of α-MSH in mediating Dex regulation of adiposity, as α-MSH levels

were decreased in Dex-treated mice via SGK1 and restoration of hypothalamic

α-MSH levels by i.c.v. administration of this peptide normalized the inadequate

energy homeostasis in PSKO mice. Although the beneficial effects of the

pharmacological treatment are most likely mediated through direct actions on POMC

neurons, we can not exclude its potential effects on other hypothalamic areas due to

the delivery route used.

Our results suggest that the reduced α-MSH content in Dex-treated mice was not

caused by an altered proteolysis process, but the decreased Pomc expression possibly

due to glucocorticoid resistance, as Dex is shown to induce Pomc expression (43).

Furthermore, we found Dex-decreased Pomc expression via SGK1/FOXO3 dependent

pathway, as the inhibitory effect of Dex on Pomc expression was blocked in mice

with SGK1 over-expression or FOXO3 inhibition. Many studies, including those

conducted on FOXO3 knockout mice, have demonstrated that FOXO3 is vital for

many functions in CNS, including neural stem cell homeostasis, stress and

Huntington's Disease (44; 45). We showed that it functions as downstream signal of


20

SGK1 in the regulation of energy homeostasis. We also demonstrated the spatial

relationships among GR, SGK1 and FOXO3, providing the basis for the interaction

and regulation among these proteins.

In this study, we also demonstrated that adult-onset loss of SGK1 in POMC

neurons results in a phenotype similar to that of ablation during development. This is

a key issue, because some works report that multiple hypothalamic neurons express

POMC in adult mice (24) and pre- and post-natal ablation of certain neurons results in

disparate feeding behavior, suggesting that phenotypes caused by prenatal ablation

may be influenced by developmental compensation (24). POMC promoter also drives

CRE recombinase expression in corticotrophs and melanotrophs (46). The

contribution from pituitary might not be that significant in the current study, as no

changes were observed in serum corticosterone and growth hormone content, which

reflect the function of pituitary (34), between PSKO and control mice.

Previous studies have shown that POMC neurons are involved in the regulation

of food intake (29; 47). For reasons unknown, however, we found food intake was not

significantly affected by Dex treatment, or in PSKO or PSOE mice. Consistent with

our study, however, previous works also indicate that genetic blockade of

CNS-MC3R promotes fat accumulation in the absence of hyperphagia (48).

In contrast to the stimulatory effect of glucocorticoid on SGK1 expression

(20-22), we observed decreased hypothalamic SGK1 expression following chronic

Dex treatment, which is to our knowledge a novel observation. We speculate that this

inhibition is not a direct effect of Dex on SGK1 expression, but rather a consequence


21

of attenuated Dex-mediated signaling, as it has been previously shown that prolonged

Dex treatment causes glucocorticoid resistance (49). Because glucocorticoid normally

functions via GR (28), the difference in SGK1 expression under acute or chronic Dex

treatment may be caused by differences in GR activity under different conditions, as

shown by our work and those of others (28; 39). In addition, because chronic Dex

treatment affects the activity of several regulatory molecules that influence SGK1

transcription and/or mRNA decay (28; 50) and hypothalamic signals might also be

affected by peripheral events (6; 16; 26), the possible contribution from these factors

to hypothalamic SGK1 expression in Dex-treated mice cannot be excluded. These

possibilities will be explored in future studies.

In summary, our results demonstrate that SGK1/FOXO3 signaling in POMC

neurons is crucial for Dex-induced adiposity, which provide novel insights into the

central mechanisms underlying Dex-induced obesity. In this study, we also

established that SGK1 in POMC neurons as an essential regulator of systemic energy

balance. This previously unrecognized role for hypothalamic SGK1 also indicates a

potential novel drug target in treating obesity and its related metabolic disorders.

ACKNOWLEDGMENTS

This work was supported by grants from the National Natural Science

Foundation (81325005, 81390350, 81471076, 81570777, 81130076, 31271269,

81400792, 81500622 and 81600623), Basic Research Project of Shanghai Science and

Technology Commission (16JC1404900 and 17XD1404200) and CAS/SAFEA


22

international partnership program for creative research teams. Feifan Guo was also

supported by the One Hundred Talents Program of CAS.

AUTHOR CONTRIBUTIONS

Y.D. researched data, wrote, reviewed and edited the manuscript. Y.X., F.Y.,

Y.L., X.J., J.D. researched data. S.C. provided research material. A.N-F-T and G.F-T

generated and provided the floxed SGK1 mice. Q.Z, H.Y., Y.C. directed the project

and contributed to discussion. F.G. and Y.S. directed the project, contributed to

discussion and wrote, reviewed, and edited the manuscript. F.G. and Y.S. is the

guarantor of this work and, as such, had full access to all the data in the study and

takes responsibility for the integrity of the data and the accuracy of the data analysis.

COMPETING FINANCIAL INTERESTS

The authors declare that they have no competing interests.

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FIGURE LEGENDS

Figure 1. SGK1 expression in hypothalamic POMC neurons under chronic or

acute Dex treatment.

(A) Sgk1 expression in hypothalamus;

(B) SGK1 and p-SGK1 western blot and densitometric quantification in

hypothalamus;

(C and D) Immunofluorescence for SGK1 in ARC (arcuate nucleus) sections (C) and

integrated density quantification (D);

(E and F) Immunofluorescence for p-SGK1 in ARC sections (E) and integrated

density quantification (F);

(G and H) Immunofluorescence for POMC neurons (red), SGK1 (green) and merge

(yellow) in ARC sections (G) and integrated density quantification in POMC neurons

and colocalization (H);

(I) Sgk1 expression in hypothalamus;

(J) SGK1 western blot and densitometric quantification in hypothalamus;

(K and L) Immunofluorescence for POMC neurons (red), SGK1 (green) and merge

(yellow) in ARC sections (K) and integrated density quantification in POMC neurons


27

and colocalization (L).

Studies were conducted in 14 to 15-weeks old male wild-type mice (for A-F) or

POMC-tdTomato indicator mice (for G and H) treated without (- Dex) or with Dex (+

Dex) for 6 weeks. Studies were conducted in 9-weeks old male wild-type mice (for I

and J) or POMC-tdTomato indicator mice (for K and L) treated without (- Dex) or

with Dex (+ Dex) for 2 hours. Data are expressed as mean ± SEM (n = 6-11/group). *:

p < 0.05 for the effect of with versus without Dex treatment group.

Figure 2. PSKO mice exhibit obese phenotype and decreased energy expenditure

as Dex-treated mice.

(A) Immunofluorescence for POMC neurons (red), SGK1 (green) and merge (yellow)

in ARC (arcuate nucleus) sections from male POMC-tdTomato indicator mice;

(B) Sgk1 expression in different tissues (ARC; COR: cortex; WAT: white adipose

tissue; BAT: brown adipose tissue; LV: liver);

(C) Body weight curve;

(D) Total body fat mass;

(E) Abdominal fat mass;

(F) Daily food intake;

(G) Daily energy expenditure;

(H) Daily RER (respiratory exchange ratio, VCO2/VO2);

(I) Daily locomotor activity;

(J) Basal rectal temperature;


28

(K) UCP1 western blot and densitometric quantification in BAT;

(L) Serum NE (norepinephrine).

All studies were conducted in 12 to 14-weeks old male control (- PSKO) and PSKO

(+ PSKO) mice. Data are expressed as mean ± SEM (n = 6-16/group). *: p < 0.05 for

the effect of PSKO group versus control group.

Figure 3. PSOE mice show lean phenotype and increased energy expenditure.

(A) Immunofluorescence for POMC neurons (red), p-NDRG1 (green) and merge

(yellow) in ARC (arcuate nucleus) sections;

(B) Body weight curve;

(C) Total body fat mass;

(D) Abdominal fat mass;

(E) Daily food intake;

(F) Daily energy expenditure;

(G) Daily RER (respiratory exchange ratio, VCO2/VO2);

(H) Daily locomotor activity;

(I) Basal rectal temperature;

(J) UCP1 western blot and densitometric quantification in BAT (brown adipose

tissue);

(K) Serum NE (norepinephrine).

All studies were conducted in 19 to 20-weeks old male control (- PSOE) and PSOE (+

PSOE) mice. Data are expressed as mean ± SEM (n = 6-9/group). *: p < 0.05 for the


29

effect of PSOE group versus control group.

Figure 4. PSOE mice are resistant to Dex-induced fat accumulation and

decreased energy expenditure.

(A) Body weight curve;

(B) Total body fat mass;

(C) Abdominal fat mass;

(D) Daily food intake;

(E) Daily energy expenditure;

(F) Daily RER (respiratory exchange ratio, VCO2/VO2);

(G) Daily locomotor activity;

(H) Basal rectal temperature;

(I) UCP1 western blot and densitometric quantification in BAT (brown adipose

tissue);

(J) Serum NE (norepinephrine).

All studies were conducted in 19 to 20-weeks old male control (- PSOE) and PSOE (+

PSOE) mice treated without (- Dex) or with Dex (+ Dex). Data are expressed as mean

± SEM (n = 6-12/group). *: p < 0.05 for the effect of PSOE group versus control

group.

Figure 5. Dex decreases hypothalamic α-MSH content via SGK1 and i.c.v

administration of α-MSH reverses obese phenotype in PSKO mice.


30

(A and B) Immunofluorescence for α-MSH in PVN (paraventricular nucleus) sections

(A) and integrated density quantification (B) in 14 to 15-weeks old male wild-type

mice treated without (- Dex) or with Dex (+ Dex);

(C-E) Immunofluorescence for α-MSH in PVN sections (C), integrated density

quantification (D) and relative hypothalamic α-MSH content by ELISA (E) in 12 to

14-weeks old male control and PSKO mice;

(F-G) Immunofluorescence for α-MSH in PVN sections (F) and integrated density

quantification (G) in 19 to 20-weeks old male control and PSOE mice treated without

(- Dex) or with Dex (+ Dex);

(H-K) Body weight (H), abdominal fat mass (I), basal rectal temperature (J) and

UCP1 western blot and densitometric quantification in BAT (brown adipose tissue)

(K) in 10 to 12-weeks old male control (- PSKO) and PSKO (+ PSKO) mice treated

without (- α-MSH) or with α-MSH (+ α-MSH).

Data are expressed as mean ± SEM (n = 6-8/group). *: p < 0.05 for any treatment

compared with control group for A-E. *: p < 0.05 for the effect of any group versus

control mice treated without Dex; #: p < 0.05 for the effect of PSOE mice versus

control mice following Dex treatment for G. *: p < 0.05 for the effect of any group

versus control mice treated without α-MSH; #: p < 0.05 for the effect of with versus

without α-MSH in PSKO mice for H-K.

Figure 6. Dex reduces α-MSH precursor POMC expression via SGK1/FOXO3

dependent pathway and down-regulation of FOXO3 in ARC largely reverses the


31

obesity phenotype in PSKO mice.

(A-C) Neuropeptides expression in hypothalamus (A), immunofluorescence for

POMC in ARC (arcuate nucleus) sections (B) and integrated density quantification (C)

in 14 to 15-weeks old male wild-type (WT) mice treated without (- Dex) or with Dex

(+ Dex);

(D and E) Immunofluorescence for POMC in ARC sections (D) and integrated

density quantification (E) in 19 to 20-weeks old male control and PSOE mice treated

without (- Dex) or with Dex (+ Dex);

(F) P-FOXO3 and FOXO3 western blot and densitometric quantification in

hypothalamus of 14 to 15-weeks old male WT mice treated without (- Dex) or with

Dex (+ Dex);

(G and H) Immunofluorescence for POMC neurons (red), p-FOXO3 (green) and

merge (yellow) in ARC sections (G) and integrated density quantification in POMC

neurons and colocalization (H) in 19 to 20-weeks old male control and PSOE mice

treated without (- Dex) or with Dex (+ Dex);

(I-Q) The expression of Sgk1, Foxo3 and Pomc in ARC (arcuate nucleus) (I), body

weight (J), total body fat mass (K), abdominal fat mass (L), daily energy expenditure

(M), daily RER (respiratory exchange ratio, VCO2/VO2) (N), basal rectal temperature

(O), UCP1 western blot and densitometric quantification in BAT (brown adipose

tissue) (P) and serum NE (norepinephrine) (Q) in 16 to 18-weeks old male control (-

PSKO) and PSKO (+ PSKO) mice injected with Ad-scrambled (- Ad-shFOXO3) or

Ad-shFOXO3 (+ Ad-shFOXO3).


32

Data are expressed as mean ± SEM (n = 6-11/group). *: p < 0.05 for the effect of with

versus without Dex treatment group for A, C and F. *: p < 0.05 for the effect of any

group versus control mice treated without Dex; #: p < 0.05 for the effect of PSOE

mice versus control mice following Dex treatment for E and H. *: p < 0.05 for the

effect of any group versus control mice injected without Ad-shFOXO3; #: p < 0.05

for the effect of with versus without Ad-shFOXO3 injection in PSKO mice for I-Q.


E F

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RE

R

* * *

G

0 0.1

0.2

0.3

0.4

0.5

EE

(k

ca

l/h

r)

* * *

Light Dark Total

tdTomato SGK1 Merge

100 um

Co

ntr

ol

PS

KO


Control PSOE

0

0.2

0.4

0.6

0.8

Light Dark Total E

E (

kc

al/h

r)

* * *

0

0.5

1.0

Light Dark Total

RE

R

* * * F E G D

0

1

2

3

4

5

Fo

od

in

tak

e

(g/d

ay) *

0

0.2

0.4

0.6

Ab

do

min

al

fat

ma

ss

(g

)

Fig. 3

B A C

0

1

2

Fa

t m

as

s (

g)

* 100 um

mCherry

mCherry

p-NDRG1 Merge

Merge p-NDRG1

Co

ntr

ol

PS

OE

Control

PSOE

* 20

25

30

35

0 2 4 6 8 10

Time (weeks)

Bo

dy w

eig

ht

(g)

0

500

1000

Light Dark Total Lo

co

mo

tor

ac

tivit

y

(co

un

ts)

H

35

36

37

14:00 17:00

Re

cta

l

tem

pe

ratu

re (℃

)

* *

I J K

*

0

200

400

Se

rum

NE

(n

g/l

)

UCP1

Actin

PSOE: - + - + - +

0

100

200

300

Arb

itra

ry

un

its

*


Control + Dex PSOE + Dex

0

2

4

6

Fo

od

in

tak

e

(g/d

ay)

14:00 17:00

Re

cta

l

tem

pe

ratu

re (℃

)

35

36

37

* *

0

0.4

0.8

1.2

Light Dark Total

RE

R

* * *

0

0.2

0.4

0.6

0.8

Light Dark Total

EE

(k

ca

l/h

r)

* * *

0

500

1000

1500

Light Dark Total Lo

co

mo

tor

ac

tivit

y

(co

un

ts)

0

0.5

1.0

Ab

do

min

al

fat

ma

ss

(g

)

*

Fig. 4

Dex

* 20

25

30

35

0 1 2 3 4 5 6 7 8 9 10 11

Time (weeks)

Bo

dy w

eig

ht

(g)

Control

PSOE

A B C D

E F G

H

0

200

400

Se

rum

NE

(n

g/l

)

* J I

0

100

200

300

Arb

itra

ry u

nit

s

*

0

1

2

3

4

5

Fa

t m

as

s (

g)

*

UCP1

Actin

PSOE: - + - + - +

Dex: + + + + + +


Fig. 5

E

100 um

a- MSH

Control PSKO

100 um

a- MSH

- Dex + Dex

100 um

a- MSH

Control PSOE

G F

D C B A

0

50

100

150

*

a-

MS

H (

arb

itra

ry

flu

ore

sc

en

ce

un

its)

Dex: - + 0

50

100

150

*

a-

MS

H (

arb

itra

ry

flu

ore

sc

en

ce

un

its)

H I J

15

20

25

Bo

dy w

eig

ht

(g)

*

* #

0

0.1

0.2

Ab

do

min

al

fat

ma

ss

(g

)

*

*

#

K

34

35

36

37

Rec

tal

tem

pe

ratu

re (℃

) #

*

*

PSKO a- MSH Control a- MSH

Control + a- MSH PSKO + a- MSH

- -

0

50

100

150

Rela

tive

a-

MS

H

Co

nte

nt

(%)

*

0

100

200

300

*

*

#

a-

MS

H (

arb

itra

ry

flu

ore

sc

en

ce

un

its)

PSOE

Control

Dex: - - + +

Control PSOE

- Dex + Dex

0

100

200

Arb

itra

ry

un

its

* *

*

#

a- MSH: - +

UCP1

Actin

PSKO: - - + +

+ -


E D

100 um

POMC

Control PSOE

F Dex: - + - + - +

p-FOXO3

FOXO3

Actin

Arb

itra

ry

un

its

0

50

100

150

* *

+ Dex Dex -

Control PSOE

- Dex + Dex

0

100

200 *

*

PO

MC

(a

rbit

rary

flu

ore

sc

en

ce

un

its

)

#

Dex：- - + +

PSOE Control

0

50

100

150

Pc1/3 Pc2 Cpe Pam Prcp Re

lati

ve

mR

NA

(%

)

Pomc

*

Fig. 6 + Dex Dex -

0

50

100

150

*

- Dex + Dex B C

Dex：- +

A

PO

MC

(a

rbit

rary

flu

ore

sc

en

ce

un

its

)

100 um

POMC

H G

100 um

mCherry

p-FOXO3

Control PSOE

- D

ex

+ D

ex

0

100

200

P-F

OX

O3 (

arb

itra

ry

flu

ore

scen

ce u

nit

s)

PSOE Control

*

*

#

Dex：- - + +

0

50

100

150

p-F

OX

O3

/mC

he

rry

co

localizati

on

(%

)

Dex：- - + +

*

*

#

Merge


Fig. 6

35

36

37

14:00 17:00

Re

cta

l

tem

pa

ratu

re (℃

)

*

*

*

* # #

*

#

0

100

200

NE

(n

g/l

)

*

Q P O Ad-shFOXO3: - + -

UCP1

Actin

PSKO: - - + + +

0

100

200

Arb

itra

ry

un

its

*

* #

Control Ad-shFOXO3

Control + Ad-shFOXO3

PSKO Ad-shFOXO3

PSKO + Ad-shFOXO3

- -

0

0.5

1.0

Ab

do

min

al

fat

ma

ss

(g

)

*

*

#

0

0.2

0.4

0.6

0.8

Light Dark Total

EE

(k

ca

l/h

r)

* * #

# #

* * * * *

*

0

0.4

0.8

1.2

Light Dark Total

RE

R

* * * * * * * #

#

#

L M N

0

100

200

Sgk1 Foxo3 Pomc Re

lati

ve

mR

NA

(%

)

* * * *

*

*

* #

#

0

2

4

6

8

Fa

t m

as

s (

g)

*

*

#

0

10

20

30

40

Bo

dy w

eig

ht

(g)

* # I J K


SUPPLEMENTAL MATERIAL

FIGURE LEGENDS

Figure S1. Chronic Dex treatment increases adiposity and decreases energy

expenditure.



(C) Lean mass;

(D) Abdominal fat mass;


(F) Daily energy expenditure;


(H) Basal rectal temperature;

(I) UCP1 western blot and densitometric quantification in BAT (brown adipose

tissue);

(J) Serum NE (norepinephrine).

All studies were conducted in 14 to 15-weeks old male wild-type mice treated without

(- Dex) or with Dex (+ Dex) for 6 weeks. Data are expressed as mean ± SEM (n =

6-16/group). *: p < 0.05 for the effect of with versus without Dex treatment group.

Figure S2. SGK1 in POMC neurons and metabolic parameters in PSKO mice.

(A and B) Immunofluorescence for tdTomato (red), POMC (green) and merge

(yellow) in ARC (arcuate nucleus) sections (A) and colocalization (B);


2

(A) Integrated density quantification in POMC neurons and colocalization for SGK1

in ARC sections;

(D and E) Immunofluorescence for SGK1 in PVN (paraventricular nucleus) sections

(D) and integrated density quantification (E);

(F and G) Immunofluorescence for SGK1 in VMH (ventromedial nucleus) sections (F)

and integrated density quantification (G);

(H) Lean mass.

All studies were conducted in 12 to 14-weeks old male control and PSKO mice. Data

are expressed as mean ± SEM (n = 4-6/group). *: p < 0.05 for the effect of PSKO

group versus control group.

Figure S3. POMC neuron anatomy and pituitary-adrenal axis function in PSKO

mice.

(A) Relative POMC neuron area;

(B) POMC neuron number and distribution throughout ARC (arcuate nucleus);

(C) Serum corticosterone content;

(D) Serum growth hormone content.

All studies were conducted in 12 to 14-weeks old male control and PSKO mice. Data

are expressed as mean ± SEM (n = 6-8/group). *: p < 0.05 for the effect of PSKO

group versus control group.

Figure S4. Metabolic phenotypes in female PSKO mice.


3



(C) Daily food intake;

(D) Daily energy expenditure;

(E) Daily RER (respiratory exchange ratio, VCO2/VO2);

(F) Daily locomotor activity;

(G) Basal rectal temperature.

All studies were conducted in 15 to 16-weeks old female control and PSKO mice.

Data are expressed as mean ± SEM (n = 6-13/group). *: p < 0.05 for the effect of

PSKO group versus control group.

Figure S5. Metabolic phenotypes in PSKO-ER mice.




(D) Daily food intake;

(E) Daily energy expenditure;

(F) Daily RER (respiratory exchange ratio, VCO2/VO2);


(H) Basal rectal temperature.

All studies were conducted in 12 to 13-weeks old male control and PSKO-ER mice

treated with tamoxifen (T) at 8 weeks of age. Data are expressed as mean ± SEM


4

(n=6-8/group). *: p < 0.05 for the effect of PSKO-ER group versus control group.

Figure S6. Identification of POMC neurons and SGK1 expression in PSOE mice.

(A and B) Immunofluorescence for mCherry (red), POMC (green) and merge (yellow)

in ARC (arcuate nucleus) sections (A) and colocalization (B);

(C and D) Immunofluorescence for POMC neurons (red), SGK1 (green) and merge

(yellow) in ARC sections (C) and integrated density quantification in POMC neurons

(D);

(E and F) Immunofluorescence for SGK1 in PVN (paraventricular nucleus) sections

(E) and integrated density quantification (F);

(G and H) Immunofluorescence for SGK1 in VMH (ventromedial nucleus) sections

(G) and integrated density quantification (H);

(I) Lean mass;

(J) Total body fat mass.

Studies were conducted in 19 to 20-weeks old male POMC-Cre mice injected with

AAV-null (for A and B). Studies were conducted in 19 to 20-weeks old male control

and PSOE mice (for C-H). Studies were conducted in 13 to 14-weeks old male control

and PSOE mice 5 weeks after ARC injection of AAV-null or AAV-CA SGK1 prior to

Dex treatment (for I and J). Data are expressed as mean ± SEM (n = 4-12/group). *: p

< 0.05 for the effect of PSOE group versus control group.

Figure S7. The metabolic phenotypes of PSKO mice under Dex treatment.


5

(A) Body weight;



(D) Lean mass;


(F) Daily energy expenditure.

All studies were conducted in 14 to 16-weeks old male control and PSKO mice

treated without (- Dex) or with Dex (+ Dex) for 6 weeks. Data are expressed as mean

± SEM (n =10-13/group). *: p < 0.05 for the effect of any group versus control mice

treated without Dex; #: p < 0.05 for the effect of with versus without Dex treatment in

PSKO mice.

Figure S8. SGK1 regulates POMC expression.

(A-C) Neuropeptide expression in hypothalamus (A), immunofluorescence for POMC

in ARC (arcuate nucleus) sections (B) and integrated density quantification (C) in 12

to 14-weeks old male control and PSKO mice;

(D) Sgk1 and Pomc expression in primary hypothalamic neurons treated without (-

Ad-shSGK1) or with Ad-shSGK1 (+ Ad-shSGK1);

(E) Sgk1 and Pomc expression in primary hypothalamic neurons treated without (- CA

SGK1) or with CA SGK1 plasmid (+ CA SGK1).

Data are expressed as mean ± SEM (n = 6-12/group). *: p < 0.05 for the effect of CA

SGK1 group versus control group.


6

Figure S9. SGK1 regulates POMC expression by phosphorylating FOXO3.

(A) P-FOXO3 and FOXO3 western blot and densitometric quantification in ARC

(arcuate nucleus) of 12 to 14-weeks old male control (- PSKO) and PSKO (+ PSKO)

mice;

(B) P-FOXO3, FOXO3 and SGK1 western blot and densitometric quantification in

primary hypothalamic neurons treated without (- Ad-shSGK1) or with Ad-shSGK1 (+

Ad-shSGK1);

(C) P-FOXO3, FOXO3 and SGK1 western blot and densitometric quantification in

primary hypothalamic neurons treated without (- CA SGK1) or with CA SGK1

plasmid (+ CA SGK1);

(D) Sgk1, Foxo3 and Pomc expression in primary hypothalamic neurons treated with

Ad-scrambled (- Ad-shSGK1) or Ad-shSGK1 (+ Ad-shSGK1) in the presence of

control reagent (- siFOXO3) or siFOXO3 (+ siFOXO3).

Data are expressed as mean ± SEM (n = 6-8/group). *: p < 0.05 for any treatment

compared with control group for A-C. *: p < 0.05 for the effect of any group versus

group without Ad-shSGK1 and siFOXO3; #: p < 0.05 for the effect of with versus

without siFOXO3 in the presence of Ad-shSGK1 for D.

Figure S10. Down-regulation of FOXO3 largely reverses hypothalamic α-MSH

content and in PSKO mice.

(A and B) Immunofluorescence for FOXO3 in ARC (arcuate nucleus), PVN


7

(paraventricular nucleus) and VMH (ventromedial nucleus) sections (A) and

integrated density quantification (B) in PSKO mice injected with Ad-scrambled (-

Ad-shFOXO3) or Ad-shFOXO3 (+ Ad-shFOXO3);

(C) Daily food intake;

(D) Daily locomotor activity;

(E and F) Immunofluorescence for α-MSH in PVN sections (E) and integrated density

quantification (F).

All studies were conducted in 16 to 18-weeks old male control and PSKO mice

injected with Ad-scrambled (- Ad-shFOXO3) or Ad-shFOXO3 (+ Ad-shFOXO3).

Data are expressed as mean ± SEM (n = 6-11/group). *: p < 0.05 for the effect of any

group versus control mice injected without Ad-shFOXO3; #: p < 0.05 for the effect of

with versus without Ad-shFOXO3 injection in PSKO mice for B and E.

Figure S11. Levels of hypothalamic nuclear and cytoplasma GR, SGK1 and

FOXO3 in mice treated with Dex.

(A) Gr expression in hypothalamus;

(B) GR and p-GR western blot and densitometric quantification in hypothalamus;

(C) Immunofluorescence for POMC neurons (red), GR/SGK1/FOXO3 (green) and

merge (yellow) in ARC (arcuate nucleus) sections from male POMC-tdTomato

indicator mice;

(D) Total and phosphorylated GR, SGK1 andFOXO3 western blot in nuclear and

cytoplasm in hypothalamus.


8

Studies were conducted in 14 to 15-weeks old male wild-type mice treated without (-

Dex) or with Dex (+ Dex) for 6 weeks (for A, B and D) or 9-weeks old male

POMC-tdTomato indicator mice (for C). Data are expressed as ± SEM (n =

6-11/group). *: p < 0.05 for the effect of with versus without Dex treatment group.


20

25

30

0 1 2 3 4 5 6

Time (weeks)

Bo

dy w

eig

ht

(g)

0

2

4

6

Fo

od

in

tak

e

(g/d

ay)

0

0.2

0.4

0.6

0.8

Light Dark Total

EE

(k

ca

l/h

r)

* *

0

500

1000

1500

Light Dark Total Lo

co

mo

tor

acti

vit

y

(co

un

ts)

A B C

E F G

35

36

37

38

14:00 17:00

Re

cta

l

Te

mp

era

ture

(℃

)

* *

H

Arb

itra

ry

un

its

0 50

100 150

*

UCP1

Actin

Dex： - + - + - + I

0

100

200

Se

rum

NE

(n

g/l

)

*

J

Fig. S1

0

0.2

0.4

0.6

Ab

od

om

ina

l

fat

ma

ss

(g

) *

*

0

1

2

3

0 1 2 3 4 5 6

Time (weeks) F

at

ma

ss

(g

)

- Dex + Dex + Dex Dex -

D

15

20

25

Le

an

ma

ss

(g

)

Time (weeks)

*

0 1 2 3 4 5 6


A

Fig. S2

B

0

50

100

PO

MC

/ t

dTo

ma

to

co

-lo

ca

lisa

tio

n (

%)

tdTomato

POMC

100 um 50 um

Merge

C

0

50

100

150

*

SG

K1

/td

To

ma

to

co

localizati

on

(%

)

*

SG

K1

(a

rbit

rary

flu

ore

sc

en

ce

un

its

)

0

50

100

150 D E

F G

100 um

SGK1

Control PSKO

SGK1

Control PSKO

100 um

SG

K1

(a

rbit

rary

flu

ore

sc

en

ce

un

its

)

0

50

100

150

SG

K1 (

arb

itra

ry

flu

ore

sc

en

ce

un

its

)

0

50

100

150

PV

N

VM

H

0

10

20

30

Le

an

ma

ss

(g

) H

Control PSKO


Fig. S3

Control PSKO

A B

0

100

200

300

400

1 2 3 4 5 6 7 8 9 10 11 12

ARC section

PO

MC

ne

uro

n

co

un

ts

0

50

100

Co

rtic

os

tero

ne

(ng

/ml)

0

1

2

3

4

Gro

wth

ho

rmo

ne

(ng

/ml)

C D

0

50

100

150

Re

lati

ve

PO

MC

ne

uro

n a

rea

(%

)


A B C D

G

0

1000

2000

Light Dark Total Lo

co

mo

tor

ac

tivit

y

(co

un

ts)

E F

Control PSKO

Fig. S4

Re

cta

l

tem

pe

ratu

re (℃

)

35

36

37

*

*

0

1

2

3

Fa

t m

as

s (

g)

0

2

4

6

Fo

od

In

tak

e

(g/d

ay)

0

0.4

0.8

1.2

Light Dark Total

RE

R

* * *

0

0.2

0.4

0.6

Light Dark Total

EE

(k

ca

l/h

r)

* * *

Age (week)

15

20

25

30

6 8 10 12 14 16

Bo

dy w

eig

ht

(g)

*

Control

PSKO


Fig. S5

Control + T PSKO-ER + T

A B

0

0.4

0.8

1.2

Light Dark Total

RE

R

*

0

500

1000

1500

Lo

co

mo

tor

ac

tivit

y

(co

un

ts)

Light Dark Total 35

36

37

Re

cta

l

tem

pe

ratu

re (℃

)

*

C D

H F G

0

0.2

0.4

0.6

Light Dark Total

EE

(k

ca

l/h

r)

* * *

E

0

2

4

6

Fo

od

in

tak

e

(g/d

ay)

15

20

25

30

5 7 9 11 13

Bo

dy w

eig

ht

(g) * * *

Age (weeks)

Tamoxifen

PSKO-ER Control

Age (weeks)

* *

0

1

2

3

7 9 10 11

Fat

mass (

g)

0

0.1

0.2

0.3

Ab

do

min

al

fat

ma

ss

(g

)

*


0

50

100

PO

MC

/ m

Ch

err

y

co

-lo

ca

lisa

tio

n (

%)

200 um 50 um

mCherry

POMC

Fig. S6

A B

Merge

Co

ntr

ol

PS

OE

C D E F

G H

Co

ntr

ol

PS

OE

100 um

SGK1

100 um

SGK1

Control PSOE

SG

K1

(a

rbit

rary

flu

ore

sc

en

ce

un

its

)

0

50

100

150

SG

K1

(a

rbit

rary

flu

ore

sc

en

ce

un

its

)

0

50

100

150

mCherry SGK1

100 um

*

SG

K1

(a

rbit

rary

flu

ore

sc

en

ce

un

its

) 0

100

200

300

Control PSOE

Merge

PVN

VM

H

0

1

2

3

Fa

t m

as

s (

g)

J I

0

10

20

30

Le

an

ma

ss

(g

)


Control Dex

Control + Dex

PSKO Dex

PSKO + Dex

- -

0

10

20

30

40

0 6

Time (weeks)

Bo

dy w

eig

ht

(g) * * * *

#

0

1

2

3

4

5

0 6

Time (weeks) F

at

ma

ss

(g

)

* * * * * #

0

10

20

30

Le

an

ma

ss

(g

)

0 6

Time (weeks)

* #

0

0.5

1.0

Ab

do

min

al

fat

ma

ss

(g

)

* * *

0

2

4

6

Fo

od

in

tak

e

(g/d

ay)

0

0.2

0.4

0.6

0.8

Light Dark Total

EE

(k

ca

l/h

r)

* * * * * * * * * #

A B C

D E F

Fig. S7 Page 55 of 60 Diabetes

Fig. S8

B

C

100 um

POMC

Control PSKO

*

Pc1/3 Pc2 Cpe Pam Prcp 0

50

100

150

Pomc Re

lati

ve

mR

NA

(%

) PSKO Control

A

0

50

100

150

*

PO

MC

(a

rbit

rary

flu

ore

sc

en

ce

un

its

)

0

50

100

150 R

ela

tive

mR

NA

(%

)

Sgk1 Pomc

* *

D

*

0

200

400

600

800

Sgk1 Pomc Re

lati

ve

mR

NA

(%

)

*

E Ad-shSGK1 + Ad-shSGK1

- CA SGK1

+ CA SGK1

-


Fig. S9

Actin

FOXO3

P-FOXO3

Ad-shSGK1: - + - + - +

SGK1

B

D

Ad-shSGK1 siFOXO3

+ Ad-shSGK1 siFOXO3

Ad-shSGK1 + siFOXO3

+ Ad-shSGK1 + siFOXO3

0

100

200

Sgk1 Foxo3 Pomc Re

lati

ve

mR

NA

(%

)

* * * * *

* # #

0

50

100

150

Arb

itra

ry u

nit

s

* * *

p-FOXO3

FOXO3

SGK1

Actin

CASGK1: - + - + - +

0

100

200

Arb

itra

ry u

nit

s

* *

C

*

Ad-shSGK1

+ Ad-shSGK1

-

CA SGK1

+ CA SGK1

-

- -

-

-

A

FOXO3

p-FOXO3

PSKO: - + + - +

Actin

0

50

100

150

Arb

itra

ry

un

its

* *

PSKO Control

-


*

FO

XO

3 (

arb

itra

ry

flu

ore

sc

en

ce

un

its

)

* #

B

Fig. S10

A

FOXO3

- Ad-shFOXO3 + Ad-shFOXO3 - Ad-shFOXO3 + Ad-shFOXO3

Control PSKO Control PSKO

Documents

Page 1 of 60 Diabetes · 2018. 1. 8. · SGK1 allele (SGK1loxp/loxp) mice (18) were kindly provided by Dr Geza Fejes-Toth and Dr Aniko Naray-Fejes-Toth (Dartmouth Medical School,