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

Ablation of Sax2 gene expression prevents diet-induced obesity Ruth Simon1,2, Stefan Britsch2* and Andrew Bergemann1* Department of Pathology, Mount Sinai School of Medicine, New York, NY, USA Institute for Molecular and Cellular Anatomy, University of Ulm, Germany Keywords brainstem; diet-induced obesity; energy homeostasis; food uptake; neural circuitry Correspondence R Simon, Institute for Molecular and Cellular Anatomy, University of Ulm, AlbertEinstein-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 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 mechanisms 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 starting 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 homeostasis, 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 resistance 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 regulating the transcription of specific factors involved in energy balance 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 cardiovascular diseases Great efforts are being made to understand 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 signals 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, 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 FEBS Journal 278 (2011) 371–382 ª 2010 The Authors Journal compilation ª 2010 FEBS 371 Sax2 expression required for diet-induced obesity R Simon et al 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 melanocortin 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 receptors and NPY receptors are located in the midbrain, the pons and the ventral medulla, further suggesting an interaction between serotonergic 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 development Taken together with the dramatic metabolic phenotype 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 not develop insulin resistance In addition, NPY mRNA levels in the forebrain of Sax2) ⁄ ) on a high-fat diet are down-regulated, whereas leptin levels are decreased independent of the diet The data obtained in the present study suggest that glucose metabolism and energy storage pathways are indirectly affected by a lack of Sax2 gene expression, most likely through an impairment of food absorption 372 Results Comparison of body weight and food uptake of adult wild-type, Sax2 heterozygous and null mutants During early postnatal development all Sax2) ⁄ ) animals 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 phenotype for females as a result of a dosage effect Comparing Sax2+ ⁄ + and Sax2) ⁄ + animals to Sax2) ⁄ ) mice of the same gender revealed a more dramatic difference 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 addition, 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 animals 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 corresponding 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 FEBS Journal 278 (2011) 371–382 ª 2010 The Authors Journal compilation ª 2010 FEBS R Simon et al Sax2 expression required for diet-induced obesity A B C D Fig 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 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 ⁄ 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 animals 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 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 conditions of the mutant mice, which does not correlate with the food uptake data In addition, we examined the relative mRNA expression levels of specific markers involved in thermogenesis and metabolism, such as uncoupling protein 1, peroxisome proliferator-activated receptor c and peroxisome proliferator-activated receptor coactivator 1a, amongst others, in white (WAT) and brown (BAT) adipose tissue by quantitative realtime 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 Sax2 expression (data not shown) Sax2 null mutants are resistant to diet-induced obesity To further identify a role for the Sax2 gene in the regulation 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+ ⁄ + FEBS Journal 278 (2011) 371–382 ª 2010 The Authors Journal compilation ª 2010 FEBS 373 Sax2 expression required for diet-induced obesity R Simon et al A B C D Fig 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 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 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 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 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 374 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 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 rescue 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 FEBS Journal 278 (2011) 371–382 ª 2010 The Authors Journal compilation ª 2010 FEBS R Simon et al Sax2 expression required for diet-induced obesity Standard diet +/+ High fat diet –/– +/+ –/– A B I J C D K L E F M N G H O P WAT BAT Liver H&E Liver PAS Fig 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) 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 differences, 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 animals 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 glycogen 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 highfat diet cannot rescue the phenotype (e.g increasing incorporation of lipid and glycogen into respective tissues) 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 FEBS Journal 278 (2011) 371–382 ª 2010 The Authors Journal compilation ª 2010 FEBS 375 Sax2 expression required for diet-induced obesity R Simon et al counterparts, female Sax2) ⁄ ) animals fed a standard chow showed small but significant higher fasting blood glucose levels compared to Sax+ ⁄ + (n = 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 A B C D E F Fig 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 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 < 0.005 376 FEBS Journal 278 (2011) 371–382 ª 2010 The Authors Journal compilation ª 2010 FEBS R Simon et al (n = 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 animals Mutant animals fed a high-fat diet exhibited lower blood glucose levels, although there was only a significant difference at weeks on the diet (n = for both groups) and not at 11 weeks (Sax2+ ⁄ +, n = 4; Sax2) ⁄ ), n = 5; Fig 4A) Furthermore, glucose tolerance tests performed on animals fed a high-fat diet for weeks did not show a difference between control and mutant animals (n = 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) Examination of serum insulin levels in Sax2+ ⁄ + and Sax2) ⁄ ) animals, both on a standard as well as a highfat 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 dietinduced obesity of Sax2) ⁄ ) mice, we determined serum leptin levels, an additional major player in the regulation of energy homeostasis Leptin, an adipokine factor, is expressed predominantly in WAT, and the secretion of leptin occurs proportionally to the size of adipose tissue [22] As shown in Fig 4F, serum leptin levels in Sax2) ⁄ ) animals were significantly lower for both dietary 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 development, 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 Sax2 expression required for diet-induced obesity postnatal development [20,21] Sax2 expression occured in the hindbrain in the vicinity of the paramedian 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) During postnatal development, NPY and POMC expression, 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 corresponding to the hindbrain region, indicated by (e) in Fig 5C, representing the area of the nucleus of the solitary 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 suggest 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 deregulation 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 suggest satiation [20] (data not shown), indicating that Sax2 expression might be required for the coordinated crosstalk between factors involved in energy homeostasis FEBS Journal 278 (2011) 371–382 ª 2010 The Authors Journal compilation ª 2010 FEBS 377 Sax2 expression required for diet-induced obesity A R Simon et al B C D E F G H I J K L M O P N 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 378 Fig Analysis of Sax2 expression pattern in adult animals (A) Lateral view of b-galactosidase stained adult brains Top: Sax2 heterozygous brain Bottom: Sax2 mutant brain (B, D, E) Coronal sections of b-galactosidase 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-galactosidase 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 statistically significant Unlike during postnatal development, 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 animals on a high-fat diet, with the exception of significantly 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 FEBS Journal 278 (2011) 371–382 ª 2010 The Authors Journal compilation ª 2010 FEBS R Simon et al A B Fig 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 highfat diet (HFD), respectively, by real-time RT-PCR Statistical analysis by the 2)DDCT method with Sax2+ ⁄ + on STD as reference; FB, forebrain; HB, hindbrain; *P < 0.05 mRNA levels further indicates the requirement of Sax2 expression for diet-induced obesity Obese animals 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 of the mouse genome The human homolog is located on Sax2 expression required for diet-induced obesity chromosome 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 hindbrain [20] The data obtained in the present study suggest 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 postnatal development of the brain It is possible that serotonin levels of the surviving mutants are more moderately increased, thereby allowing a closer to normal development of the brain and survival to adulthood In the present study, we demonstrate that the ablation 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 insulin levels, suggest that the glucose metabolism per se is not affected by Sax2 deficiency Furthermore, our histological analysis of adipose and liver tissues demonstrates 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 difference in food uptake does not account for the size differences, 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 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 conditions exhibit a lower body temperature compared to FEBS Journal 278 (2011) 371–382 ª 2010 The Authors Journal compilation ª 2010 FEBS 379 Sax2 expression required for diet-induced obesity R Simon et al wild-type animals, which suggests that the lean phenotype 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 However, the question remains as to whether low leptin levels 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 undergoing an ovariectomy, which led to an increase in visceral fat and loss of subcutaneous fat; it was further demonstrated that estrogen treatment was able to restore fat distribution [33] Sax2 is a transcription factor and, although we have not determined a function for its role during early development and ⁄ or in energy homeostasis, it is possible that Sax2 deficiency prevents the crosstalk of factors involved in the maintenance of energy homeostasis During postnatal development, the loss of Sax2 expression causes an increase in serotonin levels in the brainstem and a deregulation of NPY and POMC expression in the hind- and forebrain [20], suggesting 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 homeostasis 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 380 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 tolerance tests were performed on age-matched adult male and female animals starting at months of age Body temperature was measured on days in succession at the same time of day using a veterinary thermometer for rodents (Microlife, 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 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 days in succession of the experiment After week on standard chow, half the animals were exposed to a high-fat diet (BioServ; catalog number F2685; 35.5% fat, 35% carbohydrate, 20% protein, 0.1% fiber and 3.7% ash; caloric intake amounts to 5.4 kcalỈg)1) for 6–11 weeks, whereas the control 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 intraperitoneal injection of a 10% glucose solution (2 mg glucosg body weight)1) Blood glucose levels were determined at 5, 15, 30, 60 and 120 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 FEBS Journal 278 (2011) 371–382 ª 2010 The Authors Journal compilation ª 2010 FEBS R Simon et al Histological analysis Brains, WAT, BAT and liver tissues were collected and fixed in 4% paraformaldehyde overnight, washed in NaCl ⁄ Pi, dehydrated through graded ethanol, followed by two changes in Americlear (Fisher Scientific Co., Pittsburgh, PA, USA) and embedded in Paraplast (Fisher Scientific Co.) Both H&E and PAS staining methods were performed as described previously [20] Sax2 expression studies b-Galactosidase (Carl Roth GmbH, Karlsruhe, Germany) stained adult Sax2 heterozygous and homozygous brains were embedded in 1% sucrose and 50 lm vibratom sections were prepared Co-expression studies were performed on 14 lm cryostat sections of adult Sax2 heterozygous and homozygous brains employing monoclonal anti-b-galactosidase serum (Sigma-Aldrich, St Louis, MO, USA) (dilution : 2000) as marker for Sax2 expression, as well as anti-serotonin (Immunostar, Inc., Hudson, WI, USA) (dilution : 3000), anti-NPY (dilution : 5000) and anti-POMC (dilution : 1000) (both Sigma-Aldrich) polyclonal sera Secondary antibodies, CyTM2-conjugated donkey anti-mouse (b-galactosidase), CyTM3-conjugated donkey anti-rabbit (serotonin, NPY) and CyTM3-conjugated donkey ant-chicken (POMC) (Jackson ImmunoResearch, Newmarket, UK) were used at a dilution of : 500 Images were obtained at a Leica TCS SP5-II (Leica Microsystems, Wetzlar, Germany) confocal laser scanning microscope Quantitative real-time RT-PCR Total RNA was prepared from fore- and hindbrains of Sax2+ ⁄ + and Sax2) ⁄ ) fed a standard chow or a high-fat diet, respectively using RNeasy Minikit (Qiagen, Hilden, Germany) Quantitative real-time RT-PCR was performed as described previously [20] using the oligonucleotides: NPY, 5¢-GCTTGAAGACCCTTCCATTGG-3¢ and 5¢-GG CGGAGTCCAGCCTAGTGG-3¢; POMC, 5¢-CATTAGG CTTGGAGCAGGTC-3¢ and 5¢-GAATGAGAAGACCCC TGCAC-3¢; and GAPDH, 5¢-CCAGAGCTGAACGGGAA G-3¢ and 5¢-TGCTGTTGAAGTCGCAGG-3¢ Statistical analysis Results are expressed as the mean ± SEM Comparisons between groups were made by an unpaired two-tailed Student’s t-test or analyzed using the 2)DDCT method, as described previously [34] (quantitative real-time RT-PCR) P < 0.05 was considered statistically significant Acknowledgements We would like to thank Ms Jacqueline Andratschke for her excellent technical assistance This work was Sax2 expression 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S237–S246 Clegg DJ, Brown LM, Woods SC & Benoit SC (2006) Gonadal hormones determine sensitivity to central leptin and insulin Diabetes 55, 978–987 Livak KJ & Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-delta delta C(T)) method Methods 25, 402– 408 FEBS Journal 278 (2011) 371–382 ª 2010 The Authors Journal compilation ª 2010 FEBS ... = 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+ ... the ablation 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... 2010 FEBS 373 Sax2 expression required for diet-induced obesity R Simon et al A B C D Fig Sax2 expression is required for diet-induced obesity (A–D) Determination of weight gain of single housed