Báo cáo khoa học: Ablation of Sax2 gene expression prevents diet-induced obesity docx

To further define the role of Sax2 in energy homeo-stasis, age-matched adult wild-type, Sax2 heterozygous and null mutant animals were exposed to a high-fat diet.. Unlike their counterpar

Ruth Simon1,2, Stefan Britsch2* and Andrew Bergemann1*

1 Department of Pathology, Mount Sinai School of Medicine, New York, NY, USA

2 Institute for Molecular and Cellular Anatomy, University of Ulm, Germany

Introduction

Obesity is increasingly becoming a major health hazard

throughout all industrialized societies Easy access to

high caloric food and a sedentary life style are the

main causes for the increase in obesity and related

health risks, including diabetes mellitus and

cardiovas-cular diseases Great efforts are being made to

under-stand the regulation of energy homeostasis and to find

ways of reducing the obesity epidemic and related

health risks The regulation of energy homeostasis

occurs by a complex circuitry in the brain, particularly

in the hypothalamus and brainstem These brain circuitries integrate and coordinate several types of sig-nals from the periphery, as well as from other parts of the brain, including neurotransmitters, hormones and nutrients, and translate them into feeding behavior, thereby controlling energy uptake and expenditure Peripheral signals, including adiposity signals arising from adipose tissue and the pancreas, as well as signals from the gastrointestinal tract, interact with specific neurons of the hypothalamus and the brainstem,

Keywords

brainstem; diet-induced obesity; energy

homeostasis; food uptake; neural circuitry

Correspondence

R Simon, Institute for Molecular and

Cellular Anatomy, University of Ulm,

Albert-Einstein-Allee 11, D-89081 Ulm, Germany

Fax: +49 731 500 23102

Tel: +49 731 500 23225

E-mail: ruth.simon@uni-ulm.de

*These authors contributed equally to this

work

(Received 19 July 2010, revised 5

November 2010, accepted 11 November

2010)

doi:10.1111/j.1742-4658.2010.07960.x

Regulation of energy homeostasis is mainly mediated by factors in the hypothalamus and the brainstem Understanding these regulatory mecha-nisms is of great clinical relevance in the treatment of obesity and related diseases The homeobox gene Sax2 is expressed predominantly in the brainstem, in the vicinity of serotonergic neurons, and in the ventral neural tube starting during early development Previously, we have shown that the loss of function of the Sax2 gene in mouse causes growth retardation start-ing at birth and a high rate of postnatal lethality, as well as a dramatic metabolic phenotype To further define the role of Sax2 in energy homeo-stasis, age-matched adult wild-type, Sax2 heterozygous and null mutant animals were exposed to a high-fat diet Although food uptake among the different groups was comparable, Sax2 null mutants fed a high-fat diet exhibited a significantly lower weight gain compared to control animals Unlike their counterparts, Sax2 null mutants did not develop insulin resis-tance and exhibited significantly lower leptin levels under both standard chow and high-fat diet conditions Furthermore, neuropeptide Y, an important regulator of energy homeostasis, was significantly decreased in the forebrain of Sax2 null mutants on a high-fat diet These data strongly suggest a critical role for Sax2 gene expression in diet-induced obesity Sax2 gene expression may be required to allow the coordinated crosstalk

of factors involved in the maintenance of energy homeostasis, possibly regu-lating the transcription of specific factors involved in energy balance

Abbreviations

BAT, brown adipose tissue; H&E, hematoxylin and eosin; 5-HT, serotonin; NPY, neuropeptide Y; PAS, periodic acid-Schiff;

POMC, pro-opiomelanocortin; WAT, white adipose tissue.

respectively [1] Adiposity signals, such as leptin and

insulin, interact specifically in a reciprocal way with

two neuron groups located in the arcuate nucleus of

the hypothalamus: the orexigenic neurons expressing

neuropeptide Y (NPY) and the anorectic neurons that

express pro-opiomelanocortin (POMC) High levels of

leptin and insulin prevent food intake by suppressing

the expression of NPY mRNA and by activating

POMC mRNA expression, whereas low levels activate

NPY mRNA expression, which in turn inhibits the

expression of POMC mRNA, leading to an increase in

appetite and potentially to obesity [2–6] In addition,

there are NPY and POMC expressing neurons located

in nuclei of the brainstem involved in the regulation of

energy homeostasis, as well as receptors for leptin and

insulin allowing the crosstalk of the hypothalamus with

the brainstem and vice versa [7,8] The brainstem nuclei

in turn receive information from the gastrointestinal

tract, through signals such as ghrelin and peptide YY,

relaying them to nuclei in the hypothalamus [9–14]

Possible candidates for the crosstalk between brainstem

and hypothalamus are serotonin (5-HT) and the

mela-nocortin pathway Heisler et al [15] reported specific

serotonin receptors, 5-HT2CR and 5-HT1BR, located on

POMC and NPY neurons, respectively Antagonists,

particularly to 5-HT2CR, regulate energy balance by

activating the melanocortin pathway [16,17] In turn,

melanocortin 4 receptors and NPY receptors are

located in the midbrain, the pons and the ventral

medulla, further suggesting an interaction between

sero-tonergic neurons and NPY and POMC neurons in the

hypothalamus [16,18,19] The homeobox gene Sax2,

which is expressed predominantly in the brainstem,

plays a critical role in the regulation of serotonin, NPY

and POMC activities during early postnatal

develop-ment Taken together with the dramatic metabolic

phe-notype exhibited by Sax2 null mutants, these data

strongly suggest an important function for Sax2 in the

regulation of energy homeostasis [20]

In the present study, we report that adult Sax2 null

mutants (Sax2) ⁄ )) are resistant to diet-induced obesity,

although their food uptake is comparable to wild-type

(Sax2+⁄ +) as well as Sax2 heterozygous (Sax2) ⁄ +)

animals Sax2) ⁄ ) animals on a high-fat diet exhibit

normal glucose metabolism and do not develop insulin

resistance In addition, NPY mRNA levels in the

fore-brain of Sax2) ⁄ ) on a high-fat diet are

down-regu-lated, whereas leptin levels are decreased independent

of the diet The data obtained in the present study

sug-gest that glucose metabolism and energy storage

path-ways are indirectly affected by a lack of Sax2 gene

expression, most likely through an impairment of food

absorption

Results

Comparison of body weight and food uptake of adult wild-type, Sax2 heterozygous and null mutants

During early postnatal development all Sax2) ⁄ ) ani-mals show significant growth retardation independent

of their gender [21] Examination of both male and female adult Sax2) ⁄ ) animals revealed a significantly smaller size compared to age-matched wild-type or Sax2 heterozygous counterparts Although there was

no difference in body weight between male Sax2) ⁄ + and Sax2+⁄ + animals (n = 8; P > 0.05), the female counterparts exhibited a significant weight difference,

as analyzed by a two-tailed Student’s t-test (n = 14;

P < 0.05; Fig 1A), implying a heterozygous pheno-type for females as a result of a dosage effect Com-paring Sax2+ ⁄ + and Sax2) ⁄ + animals to Sax2) ⁄ ) mice of the same gender revealed a more dramatic dif-ference in body weight (females: Sax2+⁄ + and Sax2) ⁄ +, n= 14; Sax2) ⁄ ), n= 16; Sax2+⁄ +⁄ Sax2) ⁄ +, P < 0.05; Sax2+⁄ +⁄ Sax2) ⁄ ), P < 0.0005 and Sax2) ⁄ +⁄ Sax2) ⁄ ), P < 0.005; males: Sax2+⁄ + and Sax2) ⁄ +, n = 8; Sax2) ⁄ ), n = 7; Sax2+⁄ +⁄ Sax2) ⁄ )and Sax2) ⁄ +⁄ Sax2) ⁄ ), P < 0.0001) In addi-tion, the difference in average weight between control and mutant animals was 2.5-fold greater for males than females (Fig 1A) To determine the cause of these weight differences, we determined the average daily food uptake of male and female animals of all three genotypes over a period of 10 days As shown in Fig 1B, the amount of food uptake of male Sax2) ⁄ ) did not differ significantly from their counterparts (Sax2+ ⁄ + and Sax2) ⁄ +, n = 10; Sax2) ⁄ ), n = 6;

P > 0.05; Fig 1B) This is different for female ani-mals Although there was no significant difference when comparing Sax2) ⁄ + animals with Sax2+⁄ + or Sax2) ⁄ ), there was a small, but significant difference between Sax2+⁄ +and Sax2) ⁄ )females (Sax2+⁄ +and Sax2) ⁄ +, n= 10; Sax2) ⁄ ), n= 11; Sax2+⁄ +⁄ Sax2) ⁄ ) P< 0.05; Fig 1B) Comparing the food uptake between male and female animals of the corre-sponding genotype revealed a significantly higher amount (P < 0.05) for male Sax2+⁄ + and Sax2) ⁄ ) compared to the female counterparts, whereas there was no difference between male and female Sax2) ⁄ + animals Furthermore comparing the ratio of food uptake to body weight of the different genotypes and genders revealed no significant differences for female animals but a hyperphagic behavior for Sax2) ⁄ )male animals (P < 0.0005; Fig 1C) These data imply a gender-specific role for Sax2 in relation to food uptake

and⁄ or metabolism Although, for female animals, the

slightly reduced food intake might account for the

weight difference, this is not the case for the male

ani-mals The discrepancy between food uptake and body

weight suggests that Sax2) ⁄ )mice either utilize energy

sources less efficiently for storage at least in the case of

male mutants or undergo higher energy expenditure

To address the latter possibility, we measured the body

temperature of adult animals for 5 days in succession

at the same time of day We found that Sax2 mutant

animals exhibited a significant lower body temperature

compared to control animals (Fig 1D) The low body

temperature could be an indicator for fasting

condi-tions of the mutant mice, which does not correlate

with the food uptake data In addition, we examined

the relative mRNA expression levels of specific

mark-ers involved in thermogenesis and metabolism, such as

uncoupling protein 1, peroxisome proliferator-activated

receptor c and peroxisome proliferator-activated

recep-tor coactivarecep-tor 1a, amongst others, in white (WAT)

and brown (BAT) adipose tissue by quantitative

real-time RT-PCR The mRNA expression level of these

markers did not exhibit a difference between control and mutant tissues, suggesting that energy expenditure

as well as energy storage are not affected by a loss of Sax2expression (data not shown)

Sax2 null mutants are resistant to diet-induced obesity

To further identify a role for the Sax2 gene in the reg-ulation of energy homeostasis Sax2+⁄ +, Sax2) ⁄ +, as well as Sax2) ⁄ ) animals, were exposed to a high-fat diet Age-matched male and female animals of all three genotypes were single housed and exposed either to standard chow or a high-fat diet for 6–11 weeks The weight gain of all animals was determined weekly at the same time of day and day of the week Animals fed a standard diet showed a small increase in body weight, with no significant differences between Sax2) ⁄ ) animals and their Sax2+⁄ + counterparts (females: Sax2+⁄ +, n = 6, Sax2) ⁄ ) and Sax2) ⁄ +, n = 5; males: Sax2+⁄ + and Sax2) ⁄ +, n= 5; Sax2) ⁄ ),

n= 2; Fig 2A,B, left panel) By contrast Sax2+⁄ +

Fig 1 Determination of body weight, food uptake and body temperature of Sax2 + ⁄ +

, Sax2) ⁄ +and Sax2) ⁄ )mice Body weight (A), average daily food uptake (B) and food uptake per gram of body weight (C) of single housed male and female Sax2+⁄ +, Sax2) ⁄ +and Sax2) ⁄ )mice

at the age of 4 months (D) Determination of body temperature of adult Sax2 + ⁄ + and Sax2) ⁄ )animals All symbols with error bars are the mean ± SEM and asterisks indicate the statistical significance: *P < 0.05; **P < 0.005; ***P < 0.0005; ****P < 0.0001.

and Sax2) ⁄ + animals on the high-fat diet exhibited a

dramatic weight gain compared to animals on standard

chow, as well as Sax2) ⁄ ) animals on a high-fat diet

(females: Sax2+⁄ +, n = 11; Sax2) ⁄ +, n = 12; Sax2) ⁄ ),

n= 7; males: Sax2+⁄ +and Sax2) ⁄ +, n = 5; Sax2) ⁄ ),

n= 3; Fig 2A, B, right panel) Both male and female

Sax2) ⁄ )animals gained weight during the first week on

the high-fat diet (females 6.4% and males 15.1% of the

initial body weight) but substantially less than their

Sax2+⁄ +and Sax2) ⁄ + counterparts Although female

Sax2) ⁄ ) animals gained weight over the subsequent

6 weeks (12.6% of the initial body weight compared to

25.5% of the Sax2+⁄ +animals) (Fig 2C), male Sax2) ⁄ )

animals unexpectedly lost weight after 3 weeks on a

high-fat diet (Fig 2D) These data strongly suggest

that Sax2 gene expression is required for diet-induced

obesity and that its role is gender specific

Histological analysis of adult WAT, BAT and liver

tissue

Previously, we have shown that the postnatal Sax2) ⁄ )

phenotype exhibits a lack of fat incorporation in WAT

and BAT, as well as low glycogen storage in the liver [20] The lack of energy storage most likely contributes

to the premature death of the majority of Sax2) ⁄ ) mice during early postnatal development [21] To determine whether the Sax2) ⁄ ) animals surviving to adulthood exhibit a similar phenotype, we examined WAT, BAT and liver tissues of Sax2+⁄ +and Sax2) ⁄ ) mice at the age of 6 months (Fig 3A–H) In addition,

we performed histological staining assays on tissues obtained from female animals fed a high-fat diet for

11 weeks to determine whether a special diet could res-cue the phenotype (Fig 3I–P) Hematoxylin and eosin (H&E) staining analysis of animals fed standard chow revealed very little fat incorporation into epididymal WAT of Sax2) ⁄ ) mice compared to Sax2+⁄ + WAT (Fig 3A,B) Although there was an increase of fat incorporation in WAT of Sax2) ⁄ ) mice on a high-fat diet, the incorporation was far less than that into WAT of Sax2+⁄ + animals Indeed, the high-fat diet did not even rescue incorporation into mutants to the levels seen in wild-type animals on a standard diet, as indicated by the smaller size of the adipose cells (Fig 3I,J,B) Analysis of BAT revealed a similar

Fig 2 Sax2 expression is required for diet-induced obesity (A–D) Determination of weight gain of single housed female (A, C) and male (B, D) Sax2 + ⁄ + , Sax2) ⁄ +and Sax2) ⁄ )mice at 6 weeks (A, B, D) or 11 weeks (C) on a high-fat diet HFD, high-fat diet; STD, standard chow; All symbols with error bars are the mean ± SEM and asterisks indicate the statistical significance: *P < 0.01 (A, B); P < 0.05 and 0.01 (C);

P < 0.05 to 0.0005 (D); **P < 0.005; ***P < 0.0005.

pattern Although BAT of Sax2+⁄ + animals on a

high-fat diet for 11 weeks demonstrated drastically

increased fat incorporation compared to Sax2+⁄ + fed

a standard chow (Fig 3C,K), fat incorporation in

BAT of Sax) ⁄ )remained unchanged (Fig 3D,L)

In addition to adipose tissue, we also examined liver

tissue by H&E as well as periodic acid-Schiff (PAS)

staining for morphological and glycogen storage

differ-ences, respectively Liver tissue obtained from

Sax2+⁄ +and Sax2) ⁄ )animals fed standard chow did

not show any structural differences with little or no fat

incorporation (Fig 3E,F) In addition PAS staining

revealed less glycogen storage in the mutant animal

(Fig 3G,H), which corresponds to the results obtained

in postnatal animals [20] Examining the tissues of

ani-mals fed a high-fat diet, again we found a dramatic

increase of fat incorporation in the wild-type tissue

and to a very small extent in the mutant (Fig 3M,N)

Furthermore H&E and PAS staining revealed high

gly-cogen and fat storage in the tissues of Sax2+⁄ +

animals, whereas Sax2) ⁄ ) liver tissues showed only

slightly elevated glycogen storage levels comparable to the levels of Sax2+⁄ + animals fed standard chow (Fig 3O,P) Taken together, these data confirm that the postnatal phenotype maintains through adulthood Furthermore, these data strongly suggest that the high-fat diet cannot rescue the phenotype (e.g increasing incorporation of lipid and glycogen into respective tis-sues) In addition, it is demonstrated that the necessary specialized cells and molecular pathways required for lipid and glycogen storage are present and functional

in Sax2) ⁄ )mice

Determination of blood glucose levels and serum hormone assays

Deregulation of glucose metabolism could be one explanation for resistance to diet-induced obesity To explore this possibility, we examined the glucose metabolism of Sax2+⁄ + and Sax2) ⁄ ) animals by determining fasting blood glucose levels, as well as by performing glucose tolerance tests Unlike their male

WAT

BAT

Liver

H&E

Liver

PAS

Fig 3 Histological analysis of WAT, BAT and liver tissues of female Sax2 + ⁄ + and Sax2) ⁄ )animals WAT, BAT and liver tissues of Sax2 + ⁄ + (A, C, E, G, I, K, M, O) and Sax2) ⁄ )animals (B, D, F, H, J, L, N, P) fed standard chow (A–H) or a high-fat diet (I–P), respectively The tissues were stained with H&E (A–F, I–N) Liver tissue was also stained with PAS reagent for glycogen incorporation (G, H, O, P) HFD, high-fat diet; STD, standard diet Size bar = 100 lm, WAT (A, B, I, J); 50 lm, BAT and liver (C–H, K to P).

counterparts, female Sax2) ⁄ ) animals fed a standard

chow showed small but significant higher fasting blood

glucose levels compared to Sax+⁄ + (n = 4 for all

groups; females, P < 0.05; Fig 4A; data not shown)

However, neither female, nor male Sax2) ⁄ ) exhibited any significant differences in the glucose tolerance tests, suggesting that glucose metabolism per se is not directly affected by lack of Sax2 gene expression

Fig 4 Analysis of blood glucose, insulin and leptin levels of female Sax2 + ⁄ +

and Sax2) ⁄ )animals (A) Blood glucose levels of Sax2 + ⁄ +

and Sax2) ⁄ )animals fed standard (STD) chow or a high-fat diet (HFD) after a 16 h fast (B–D) Glucose tolerance tests of Sax2+⁄ +and Sax2) ⁄ ) animals fed standard chow (B) or a high-fat diet for 7 weeks (C) and 11 weeks (D) (E, F) Determination of blood insulin (E) and leptin levels (F) of Sax2 + ⁄ +

and Sax2) ⁄ )animals fed standard chow and high-fat diet, respectively The inset in (F) represents an enlargement of the STD data to better demonstrate the ratio between Sax2+⁄ +and Sax2) ⁄ )leptin levels HFD, high-fat diet; STD, standard diet All symbols with error bars are the mean ± SEM and asterisks indicate the statistical significance: *P < 0.05; **P < 0.01; ***P < 0.005.

(n = 4 for all groups; Fig 4B; data not shown) This

is in contrast to the data obtained from animals fed a

high-fat diet Because of the smaller number of male

Sax2) ⁄ ) animals and the more severe reaction to the

high-fat diet, this analysis involved only female

ani-mals Mutant animals fed a high-fat diet exhibited

lower blood glucose levels, although there was only a

significant difference at 7 weeks on the diet (n = 5 for

both groups) and not at 11 weeks (Sax2+⁄ +, n = 4;

Sax2) ⁄ ), n = 5; Fig 4A) Furthermore, glucose

toler-ance tests performed on animals fed a high-fat diet for

7 weeks did not show a difference between control and

mutant animals (n = 5 for both groups; Fig 4C) This

changed when glucose tolerance tests were performed

on animals fed a high-fat diet for 11 weeks Although

the data obtained from Sax2) ⁄ )animals on a high-fat

diet are comparable to animals on standard chow,

Sax2+⁄ + animals developed insulin resistance

(Sax2+⁄ +, n = 4; Sax2) ⁄ ), n = 5; Fig 4D)

Exami-nation of serum insulin levels in Sax2+⁄ + and

Sax2) ⁄ )animals, both on a standard as well as a

high-fat diet, further confirmed these data, as indicated by

significantly elevated insulin levels only in the

Sax2+⁄ + animals fed a high-fat diet (standard chow:

Sax2+⁄ +, n = 4; Sax2) ⁄ ), n = 3; P > 0.05; high-fat

diet: Sax2+⁄ +, n = 3; Sax2) ⁄ ), n = 4; P < 0.05;

Fig 4E) Serum insulin levels of Sax2) ⁄ ) animals

remained the same on both diets

To further establish the cause for resistance to

diet-induced obesity of Sax2) ⁄ )mice, we determined serum

leptin levels, an additional major player in the

regula-tion of energy homeostasis Leptin, an adipokine factor,

is expressed predominantly in WAT, and the secretion

of leptin occurs proportionally to the size of adipose

tis-sue [22] As shown in Fig 4F, serum leptin levels in

Sax2) ⁄ ) animals were significantly lower for both

die-tary groups compared to Sax2+ ⁄ + animals (standard

chow: Sax2+⁄ +, n = 3; Sax2) ⁄ ), n = 4; P < 0.01;

high-fat diet: Sax2+⁄ + and Sax2) ⁄ ), n= 4;

P< 0.005; Fig 4F) Although serum leptin levels in

animals fed a high-fat diet increased 16-fold compared

to animals fed standard chow, the ratio of serum leptin

levels between Sax2+⁄ +and Sax2) ⁄ )animals remained

the same under the different diets (Fig 4F)

Analysis of Sax2 expression in the adult brain

To ensure that Sax2 expression of adult animals

occurs in the same pattern as during postnatal

devel-opment, we examined the brains of Sax2 heterozygous

and mutant animals by b-galactosidase staining As

shown in Fig 5A,B,D,E, Sax2 expression in the adult

brain was comparable to the expression pattern during

postnatal development [20,21] Sax2 expression occured in the hindbrain in the vicinity of the parame-dian raphe (Fig 5D) and the B3 raphe (Fig 5E) nuclei In addition, we also found b-galactosidase staining in the midbrain in Sax2 mutants, which is absent from Sax2 heterozygous brains (Fig 5B) Dur-ing postnatal development, NPY and POMC expres-sion, two critical factors in energy homeostasis, are affected by the loss of Sax2 expression To further determine how Sax2 is involved in the regulation of energy homeostasis, we performed co-localization assays using an antibody recognizing b-galactosidase

as a marker for Sax2 expression and antibodies for POMC, NPY and serotonin The immunofluorescence assays were performed on cryostat sections corre-sponding to the hindbrain region, indicated by (e) in Fig 5C, representing the area of the nucleus of the sol-itary tract (NTS), as well as the B3 raphe and Raphe oralis regions In the area of the NTS, POMC showed co-localization with b-galactosidase (Fig 5F–H), whereas NPY was present in the vicinity of Sax2 expressing cells but did not co-localize (Fig 5I–K) Serotonin positive cells also were present in the vicinity

of Sax2 expressing cells but did not overlap, as shown for the B3 raphe region (Fig 5L–N) These data sug-gest that Sax2 might be involved in the regulation of energy homeostasis via the melanocortin pathway During postnatal development, we found an increase

of serotonin levels in the hindbrain of Sax2 mutants, which most likely contributes to the phenotype These elevated serotonin levels were not found in the adult hindbrain, as shown for the raphe oralis (Fig 5O–P)

Determination of NPY and POMC mRNA expression by real-time RT-PCR

It is well established that leptin is regulating energy homeostasis in the brain by interacting with the leptin receptor (ObRb), particularly receptors located on NPY and POMC neurons in the arcuate nucleus of the hypothalamus, as well as on nuclei of the brainstem such as the NTS [12,14,23] Under obese conditions, humans and mice develop leptin resistance, resulting in the loss of the inhibitory effect of leptin on NPY expression [24] Previously, we reported the deregula-tion of NPY and POMC mRNA expression in Sax2) ⁄ ) mice during postnatal development [20] The expression levels of NPY and POMC mRNA in the mutant hindbrain imply a fasting status compared to wild-type, whereas forebrain NPY mRNA levels sug-gest satiation [20] (data not shown), indicating that Sax2expression might be required for the coordinated crosstalk between factors involved in energy homeostasis

To determine whether NPY and POMC mRNA

expression might be involved in the resistance of

Sax2) ⁄ ) mice to diet-induced obesity, we performed

real-time RT-PCR assays RNA was isolated from the

hind- and forebrain of Sax2+⁄ + and Sax2) ⁄ )animals

either fed standard chow or a high-fat diet RT-PCR

was performed employing specific primers for NPY

and POMC mRNAs, as well as primers for GAPDH

mRNA as an internal standard As shown in Fig 6,

there was no significant difference in both NPY and

POMC mRNA levels in the fore- and hindbrain for

Sax2+⁄ + and Sax2) ⁄ ) animals on a standard chow

diet Although POMC levels in the mutant were

reduced compared to the wild-type, this was not

statistically significant Unlike during postnatal devel-opment, NPY and POMC expression levels no longer indicated fasting conditions for Sax2) ⁄ ) animals In addition, there was also no significant difference of NPY and POMC expression in the hindbrain of ani-mals on a high-fat diet, with the exception of signifi-cantly lower POMC levels in the Sax) ⁄ )compared to Sax2+⁄ + on standard chow This differs from the forebrain where the expression of NPY mRNA was significantly lower in the Sax) ⁄ ) brain on a high-fat diet compared to those from Sax+ ⁄ + on a high-fat diet In addition, there was a significant difference of the NPY expression levels of Sax2 mutants on standard and high-fat diet The decrease of NPY

D

N

E

Fig 5 Analysis of Sax2 expression pattern

in adult animals (A) Lateral view of b-galac-tosidase stained adult brains Top: Sax2 heterozygous brain Bottom: Sax2 mutant brain (B, D, E) Coronal sections of b-galac-tosidase stained brains from the regions indicated in (C) (b, d, e) (F–N) Co-expression analysis by immunofluorescence of cryostat sections of the hindbrain region indicated in (C) (e) using an antibody recognizing b-galac-tosidase as marker for Sax2 expression,

as well as antibodies for POMC, NPY and serotonin; ·63 magnification; scale bar = 7.5 lm (O, P) Comparison of serotonin concentration in control and Sax2 mutant animals; ·63 magnification; scale bar = 25 lm lacZ, green; POMC, NPY and serotonin, red; *b-galactosidase staining; B3, B3 raphe nuclei; NTS, nucleus of the solitary tract; RPM, raphe paramedian;

RO, raphe oralis.

mRNA levels further indicates the requirement of

Sax2 expression for diet-induced obesity Obese

ani-mals develop leptin resistance, which is manifested in

the loss of the inhibitory effect of leptin on NPY

mRNA expression [24] We further conclude from

these data, unlike during postnatal development, NPY

and POMC mRNA expression in the adult hindbrain

is no longer as strongly affected by the loss of Sax2

expression

Discussion

Sax2, also called Nkx1.1, is a homeobox gene of the

Nkx1 gene family located on chromosome 5 of the

mouse genome The human homolog is located on

chromosome 4 in the vicinity of the Wolf–Hirschhorn syndrome To date, no involvement in this disorder has been determined for Sax2 [25] In the mouse, loss of Sax2 gene expression causes postnatal lethality and a dramatic metabolic phenotype [20,21] Few of the Sax2 mutants survive to adulthood further exhibiting a lean phenotype We have shown previously that serotonin levels in the mutant are increased in the postnatal hind-brain [20] The data obtained in the present study sug-gest that, during postnatal development, Sax2 might be involved in the regulation of serotonin synthesis but loses this function later in development (Fig 5O–P) Serotonin plays an important role during pre- and post-natal development of the brain It is possible that seroto-nin levels of the surviving mutants are more moderately increased, thereby allowing a closer to normal develop-ment of the brain and survival to adulthood

In the present study, we demonstrate that the abla-tion of Sax2 gene expression prevents diet-induced obesity in adult mice There are several possibilities that might cause the metabolic phenotype of Sax2 (i.e either deregulation of food uptake, food absorption and⁄ or a defect in the metabolic pathways to store energy) Glucose tolerance tests, as well as serum insu-lin levels, suggest that the glucose metabolism per se is not affected by Sax2 deficiency Furthermore, our his-tological analysis of adipose and liver tissues demon-strates that Sax2) ⁄ )mice are able to incorporate fat as well as glycogen also to a lesser extent compared to Sax2+⁄ + mice These data strongly suggest that the pathways required for energy storage (e.g storage of lipids and glycogens) are not directly affected by lack

of Sax2 gene expression In addition, food uptake by Sax2) ⁄ ) animals is comparable to Sax2+⁄ + and Sax2) ⁄ + animals, although female mutants take up slightly less than their counterparts Overall, the differ-ence in food uptake does not account for the size dif-ferences, particularly not in the case of male animals Loss of Sax2 expression could also affect energy expenditure Although we did not observe hyperactive behavior of the mutant animals, it is possible that increased energy expenditure is responsible for their lean phenotype Both male and female mutants on a high-fat diet exhibit wet fur starting in the neck, which

is an area close to BAT It is possible that this specific diet causes a rise in surface temperature, as was shown for the DGAT1 mice However, unlike the DGAT1 mutants, Sax2 mutants do not exhibit increased UCP1 mRNA expression, which is an important factor in the regulation of body temperature and energy expenditure [26] (data not shown) Indeed, we have demonstrated that Sax2 mutants under normal feeding condi-tions exhibit a lower body temperature compared to

A

B

Fig 6 Determination of mRNA expression levels of NPY and

POMC by real-time RT-PCR Determination of relative NPY (A) and

POMC (B) mRNA levels in the fore- and hindbrain of female

Sax2 + ⁄ + and Sax2) ⁄ )animals fed standard chow (STD) or a

high-fat diet (HFD), respectively, by real-time RT-PCR Statistical analysis

by the 2)DDCTmethod with Sax2 +⁄ + on STD as reference; FB,

fore-brain; HB, hindfore-brain; *P < 0.05.

wild-type animals, which suggests that the lean

pheno-type is not the result of energy expenditure and rather

indicates a fasting status of the animals

It is possible that the loss of Sax2 expression

impairs the absorption of nutrients Several studies

link leptin to the regulation of intestinal absorption of

nutrients in addition to its regulatory role in the brain

[27–30] These reports demonstrate that leptin levels

correlate with absorption efficiency [27,30] We have

shown that leptin levels are considerably lower in

Sax2) ⁄ )mice, potentially accounting for the reduction

in weight through low absorption efficiencies

How-ever, the question remains as to whether low leptin

lev-els are the cause or effect of the lean phenotype

The data of the present study comparing male and

female Sax2) ⁄ ) animals suggest a gender-specific role

for Sax2 in energy homeostasis The difference in body

weight is much more severe in male than in female

Sax2) ⁄ ) mice, particularly in animals fed a high-fat

diet Unexpectedly, male Sax2) ⁄ ) mice on a high-fat

diet lose weight, falling below the starting weight after

an initial weight gain It is well known that fat storage

occurs differently in males and females [31,32] While

males accumulate fat preferentially in abdominal and

visceral tissues, females store fat subcutaneously [31]

One major factor in the gender-specific distribution of

fat accumulation is estrogen, as shown in rats

under-going an ovariectomy, which led to an increase in

visceral fat and loss of subcutaneous fat; it was

fur-ther demonstrated that estrogen treatment was able to

restore fat distribution [33]

Sax2is a transcription factor and, although we have

not determined a function for its role during early

development and⁄ or in energy homeostasis, it is

possi-ble that Sax2 deficiency prevents the crosstalk of

fac-tors involved in the maintenance of energy

homeostasis During postnatal development, the loss

of Sax2 expression causes an increase in serotonin

lev-els in the brainstem and a deregulation of NPY and

POMC expression in the hind- and forebrain [20],

sug-gesting that the crosstalk between the different regions

of the brain involved in energy balance is affected

This could occur either through an involvement of

Sax2 in the development of morphological structures

(e.g brain circuits) or the regulation of the expression

of factors required for the regulation of energy

homeo-stasis Further studies are required to determine the

pathway(s) through which Sax2 is regulating energy

homeostasis In particular, the identification of target

genes will be an important step forward in defining a

role for Sax2 in energy homeostasis Altogether, Sax2

provides an excellent model for studying the regulation

of energy homeostasis by neurons of the brainstem

Materials and methods

Animals The generation of Sax2) ⁄ ) has been described elsewhere [21] All experiments were performed on animals with a mixed genetic background of S129⁄ C57BL ⁄ 6J Mutant, wild-type and heterozygous animals were all taken from the same litter Food uptake, high-fat diet and glucose toler-ance tests were performed on age-matched adult male and female animals starting at 4 months of age Body tempera-ture was measured on 6 days in succession at the same time

of day using a veterinary thermometer for rodents (Micro-life, Widnau, Switzerland) Experiments were carried out in accordance with the guidelines of the Mount Sinai School

of Medicine Institutional Animal Care and Use Committee (USA)

Analysis of food uptake and high-fat diet Four-month-old male and female Sax2+ ⁄ +, Sax2) ⁄ + and Sax2) ⁄ )animals were single housed 1 week before the start

of the experiment To determine the daily food uptake, the animals were fed a Nutra-Gel diet (BioServ, Frenchtown,

NJ, USA; catalog number S4798) for 10 days in succession and the amount of food consumed was measured daily at the same time of day Daily food intake was determined by averaging the amount of food consumed for the last 5 days

in succession of the experiment After 1 week on standard chow, half the animals were exposed to a high-fat diet (Bio-Serv; catalog number F2685; 35.5% fat, 35% carbohydrate, 20% protein, 0.1% fiber and 3.7% ash; caloric intake amounts to 5.4 kcalÆg)1) for 6–11 weeks, whereas the con-trol group was fed a standard chow (Purina Mills, LLC, Gray Summit, MO, USA; catalog number 5053) The body weight of all animals was determined weekly

Glucose tolerance test Female Sax2+⁄ + and Sax2) ⁄ ) animals fed a standard chow or a high-fat diet were starved for 12–16 h before the experiment Fasting blood glucose levels were determined using a One Touch glucose meter (Lifescan, Johnson and Johnson, Milpitas, CA, USA), followed by an intraperito-neal injection of a 10% glucose solution (2 mg glucoseÆg body weight)1) Blood glucose levels were determined at 5,

15, 30, 60 and 120 min after injection

Blood serum analysis Blood was collected from Sax2+ ⁄ + and Sax2) ⁄ ) animals fed a standard chow as well as a high-fat diet Blood insulin and leptin levels were determined using ELISA kits (Crystal Chem Inc., Downers Grove, IL, USA) in accordance with the manufacturer’s instructions