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Endocrinology Vol. 147, No. 10 4542-4549
Copyright © 2006 by The Endocrine Society

SIM1 Overexpression Partially Rescues Agouti Yellow and Diet-Induced Obesity by Normalizing Food Intake

Bassil M. Kublaoui, J. Lloyd Holder, Jr., Kristen P. Tolson, Terry Gemelli and Andrew R. Zinn

Departments of Pediatrics (B.M.K.) and Internal Medicine (B.M.K., A.R.Z.) and McDermott Center for Human Growth and Development (B.M.K., J.L.H., K.P.T., T.G., A.R.Z.), The University of Texas Southwestern Medical School, Dallas, Texas 75390-8591

Address all correspondence and requests for reprints to: Bassil Kublaoui, Departments of Pediatrics and Internal Medicine and McDermott Center for Human Growth and Development, The University of Texas Southwestern Medical School, 5323 Harry Hines Boulevard, Dallas, Texas 75390-8591. E-mail: bassil.kublaoui{at}utsouthwestern.edu; or andrew.zinn{at}utsouthwestern.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Single-minded 1 (SIM1) mutations are associated with obesity in mice and humans. Haploinsufficiency of mouse Sim1 causes hyperphagic obesity with increased linear growth and enhanced sensitivity to a high-fat diet, a phenotype similar to that of agouti yellow and melanocortin 4 receptor knockout mice. To investigate the effects of increased Sim1 dosage, we generated transgenic mice that overexpress human SIM1 and examined their phenotype. Compared with wild-type mice, SIM1 transgenic mice had no obvious phenotype on a low-fat chow diet but were resistant to diet-induced obesity on a high-fat diet due to reduced food intake with no change in energy expenditure. The SIM1 transgene also completely rescued the hyperphagia and partially rescued the obesity of agouti yellow mice, in which melanocortin signaling is abrogated. Our results indicate that the melanocortin 4 receptor signals through Sim1 or its transcriptional targets in controlling food intake but not energy expenditure.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
SINGLE-MINDED 1 (SIM1), which encodes a member of the bHLH-PAS (basic helix-loop-helix Per Arnt Sim) family of nuclear transcription factors (1), is one of six genes associated with human monogenic obesity (2). SIM1 was first implicated in body weight regulation by a balanced translocation that disrupted the gene in a girl with early-onset obesity, hyperphagia, and accelerated linear growth (3). Visible deletions of the region of chromosome 6 containing SIM1 have also been associated with early-onset obesity in boys and girls (4, 5, 6, 7). Heterozygous disruption of the murine Sim1 gene leads to an analogous phenotype of hyperphagic obesity, increased linear growth, and enhanced sensitivity to diet-induced obesity (DIO) (8, 9). Homozygous mutant mice die shortly after birth and exhibit failure of terminal differentiation of the neurons of the paraventricular (PVN) and supraoptic nuclei of the hypothalamus (10). In adult mice the Sim1 gene is expressed in the PVN and supraoptic nuclei as well as the basomedial amygdala and a subset of lateral hypothalamic neurons (8).

The phenotype of Sim1 heterozygous mice is similar to that of melanocortin 4 receptor (Mc4r) knockout mice (11), and the girl with heterozygous SIM1 disruption (3) clinically resembles children with heterozygous MC4R mutations (12, 13). The triad of hyperphagic obesity, increased linear growth, and enhanced DIO sensitivity is also seen in other mouse mutations, including Agouti yellow (Ay) (14, 15), brain-derived neurotrophic factor (Bdnf) conditional knockout (16), and hypomorphic alleles of TrkB, the Bdnf receptor (17). Impaired hypothalamic melanocortin signaling is proposed to underlie this phenotype in all of these models (18), leading us to hypothesize a similar signaling defect in humans or mice with Sim1 haploinsufficiency (3, 8). Although coexpression of Mc4r and Sim1 within hypothalamic neurons has not been directly demonstrated, expression of Mc4r specifically in Sim1 neurons of the PVN and amygdala completely rescued the hyperphagia of Mc4r null mice (19), further supporting a cellular interaction between Sim1 and Mc4r in appetite regulation.

Two mechanisms have been proposed for this interaction. Sim1 heterozygotes may have a developmental defect leading to reduced numbers of PVN neurons, including Mc4r neurons (9). Alternatively, adult Sim1 heterozygotes may have abnormal transcriptional regulation of target genes impinging on the Mc4r signaling pathway (8). The in vivo transcriptional targets of Sim1 are not known, but Mc4r does not appear to be directly regulated by Sim1 because hypothalamic Mc4r expression was not reduced in Sim1 heterozygotes (8). Experiments testing proposed mechanisms of interaction between Sim1 and Mc4r using Sim1 loss-of-function mutations are confounded by the absence of mature PVN neurons in Sim1 homozygous knockout mice. As an alternative means to test whether Sim1 modulates Mc4r signaling in adult mice, we overexpressed Sim1 via BAC transgenesis. We show that the transgene conferred resistance to DIO and also rescued the hyperphagia of Ay mice, in which melanocortin signaling is abrogated. Our findings support a postdevelopmental, physiologic role for Sim1 in feeding regulation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Unless otherwise stated, C57BL/6 mice from the National Cancer Institute, aged 6–9 wk, were used. Ay mice were purchased from Jackson Laboratories (Bar Harbor, ME). Mice were fed ad libitum and kept on a 12-h light,12-h dark cycle (0700–1900 h light). All experimental protocols were approved by the University of Texas Southwestern Institutional Animal Care and Use Committee and are in accord with accepted standards of humane animal care.

Generation of SIM1 transgenic mice
CsCl-purified supercoiled DNA from BAC clone RP11–621C20 was microinjected into C57BL/6 fertilized pronuclei. Three founders showed germline transmission of the transgene, and transgenic lines were established and maintained by backcrossing to C57BL/6 mice. Sim1 transgenic mice were genotyped using PCR primers specific for the human SIM1 gene, 5'-CAA TTG AGA CCT TAA GGG TGC T-3' and 5'-CTC ACA TCG GCC TCC TTC ACA-3'. All experiments used mice from N10 or later generations. RT-PCR with human-specific primers confirmed that the transgene was expressed in kidney and brain, like endogenous mouse Sim1. Two lines showed relatively low expression; one of these was not studied further.

Transgene copy number determination
BamHI-digested mouse liver DNAs were hybridized with a 1:1 mixture of 32P-radiolabeled human SIM1 and mouse Sim1 genomic fragments that detected bands of approximately 5 kb (mouse Sim1) and approximately 10 kb (human SIM1). Hybridization intensities were quantitated using a STORM phosphor imager (Amersham, Piscataway, NJ).

Real-time PCR
Real-time PCR was performed as previously described (8). Briefly, hypothalami from fresh brains were dissected with a block (David Kopf Instruments, Tujunga, CA), using the following landmarks: posteriorly, posterior aspect of median eminence; anteriorly, 5 mm anterior to the median eminence; dorsally, the thalamus; laterally, medial to the dentate gyrus. Total RNA was extracted using Tripure reagent (Roche Applied Science, Indianapolis, IN). Quantitative real-time PCR was performed using an MJ Research Opticon instrument (Bio-Rad, Hercules, CA) and the QuantiTect HotStart SYBR green qPCR kit (QIAGEN, Valencia, CA). Measurements were normalized to ß-actin mRNA levels. Primers sequences were 5'-GCC CTC CTG CTT CAG ACC TC-3' and 5'-CGT TGC CAG GAA ACA CGG-3' (Pomc), 5'-CAG CAG AGG ACA TGG CCA GAT ACT AC-3' and 5'-GGG CGT TTT CTG TGC TTT CCT TCA TT-3' Npy, 5'-TCC CAG AGT TCC CAG GTC TAA GTC-3' and 5'-GCG GTT CTG TGG ATC TAG CAC CTC-3' Agrp, 5'-GAC GAT GCT CCC CGG GCT GTA TTC-3' and 5'-TCT CTT GCT CTG GGC CTC GTC ACC-3' (ß-actin), 5'-GAG GCA GGC AGG TAC TT-3' and 5'-CTG ACC ACA CTA TCT TCA T-3' (human SIM1 and mouse Sim1). All reactions were subjected to 40 cycles of amplification (denaturation at 94 C for 15 sec, annealing at 53 C for 30 sec, and extension at 72 C for 30 sec). Standard curves were generated using reference cDNA prepared from normal mouse hypothalamus and used to normalize measurements from experiment to experiment. All measurements were made in the exponential phase of the real-time PCR, as described by the manufacturer (Bio-Rad). Reactions were performed in triplicate and the results averaged. The coefficient of variation was less than 15% for each set of measurements.

Nissl staining
Mice were deeply anesthetized with pentobarbital (7.5 mg per 0.15 ml, ip) and transcardially perfused with 10 ml heparinized saline (10 U/ml, 2 ml/min) followed by 10 ml phosphate-buffered 4% paraformaldehyde (2 ml/min). Brains were removed, postfixed for 24 h in 4% paraformaldehyde, and then equilibrated in 30% sucrose in PBS for 72 h. Brains were coronally sectioned (8 µm) on a cryostat. Sections containing the PVN were from bregma –0.58 to –1.22 mm according to The Mouse Brain in Stereotaxic Coordinates (20). Sections were delipidated in 1:1 alcohol/chloroform overnight and then rehydrated through 100 and 95% alcohol to distilled water. Sections were stained in 0.1% cresyl violet solution for 3–5 min and rinsed in distilled water. They were then dehydrated through 95 and 100% ethanol for 3 min each and cleared with xylene for 3 min.

Growth and feeding studies
Mice were genotyped and weaned onto their respective diets at 3 wk of age and fed ad libitum with either a low-fat diet (Teklad, Madison, WI; 2.94 kcal/g, with 46.8% available carbohydrate, 4.0% available fat, and 24.0% available protein) or a high-fat diet (Research Diets, New Brunswick, NJ; 5.24 kcal/g, with 26.3% available carbohydrate, 34.9% available fat, and 26.2% available protein). Cohorts used to measure food intake were individually housed. Otherwise mice were group housed. Food consumption and body weight were measured weekly, and data were analyzed and plotted as mean ± SE. Feeding efficiency was calculated by dividing the change in body mass (milligrams) by the food intake (kilocalories) over a 2-wk period.

Metabolic and body composition studies
Indirect calorimetry was performed using a 12-cage equal flow CLAMS calorimeter (Columbus Instruments, Columbus, OH). Mice were habituated to the metabolic cages for 2 d prior to beginning data acquisition. Nuclear magnetic resonance analysis of body composition was performed using a Bruker minispec mq10 nuclear magnetic resonance analyzer. Live mice were individually analyzed by the machine, and then the collected data were further analyzed and plotted as mean ± SE.

Data analysis
Data were analyzed using Microsoft Excel (Redmond, WA) and plotted using Prism software (GraphPad Software, San Diego, CA). Unless otherwise noted, means were compared using unpaired, two-tailed t tests, with Welch’s correction if F test indicated unequal sample variances. Multiple comparisons were performed using one-way ANOVA with Newman-Keuls multiple comparison post hoc test. Differences were considered statistically significant if P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Generation of SIM1 transgenic mice
A human SIM1 BAC transgene was used to distinguish its expression from that of the endogenous mouse Sim1 gene. The amino acid sequences of human SIM1 and mouse Sim1 proteins are 96% identical (21). The BAC clone contains the SIM1 transcription unit and 65 kb of 5'- and 58 kb of 3'-flanking sequences (Fig. 1AGo). The 5' end of the insert contains part of an RNA helicase gene whose pattern of expression is different from SIM1, suggesting that all SIM1 5' regulatory sequences are present within the BAC. Two transgenic lines were obtained, a high copy number line (HSTG18) and a low copy number line (HSTG26) (Fig. 1Go, B and C). Human SIM1 mRNA was quantitated in the hypothalamus by real-time PCR using primers with identical binding sites in human SIM1 and mouse Sim1 transcripts (Fig. 1DGo). The HSTG18 line displayed approximately 35-fold expression and the HSTG26 line displayed 2.5-fold expression of the human SIM1 mRNA relative to the mouse Sim1 mRNA. These measurements were corroborated by direct sequencing of RT-PCR products (data not shown). Despite abundant hypothalamic SIM1 expression, HSTG18 mice had no visible phenotype (Fig. 1EGo) and normal PVN histology (Fig. 1Go, F and G).


Figure 1
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  		FIG. 1. Generation of Sim1 transgenic mice. A, Map of human SIM1 BAC clone. B, Southern blot showing human SIM1 transgene (10 kb band) in transgenic mice and mouse Sim1 gene (5 kb band) in transgenic and wild-type mice. C, Quantitation of Southern blot showing approximate copy number of human SIM1 transgene in both lines. D, Quantitation of total human SIM1 plus mouse Sim1 transcripts in transgenic vs. wild-type mouse hypothalamus by real-time PCR. E, Gross appearance of 20-wk-old male wild-type (left) and transgenic (right) mice on a LF diet. Nissl stain of coronal section of adult brain through the PVN in female wild-type (F) and transgenic (G) HSTG18 mice is shown.

 
Growth and feeding behavior of SIM1 transgenic mice.
Three-week-old mice were weaned onto chow containing 4% [low fat (LF)] or 35% [high fat (HF)] dietary fat, allowed to feed ad libitum, and weighed weekly (Fig. 2Go, A–D). Growth of transgenic and wild-type mice on the LF diet was indistinguishable. By contrast, transgenic mice gained considerably less weight on the HF diet than their wild-type littermates. The high-expressing HSTG18 mice (Fig. 2Go, A and B) were more resistant to DIO than the low-expressing HSTG26 mice (Fig. 2Go, C and D). The transgenes had similar effects on males and females.


Figure 2
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  		FIG. 2. Growth curves of transgenic lines on HF and LF diet. A, Males, HSTG18 vs. wild-type littermates. Curves on LF are superimposed (LF: transgenic, n = 8, wild-type, n = 12; HF: transgenic, n = 5, wild-type, n = 10). *, P < 0.01 from 5 wk onward on HF. B, Females, HSTG18 vs. wild-type littermates (LF: transgenic, n = 5, wild-type, n = 8; HF: transgenic, n = 15, wild-type, n = 7). *, P < 0.05 from 9 wk onward on HF. C, Males, HSTG26 vs. wild-type littermates (LF: transgenic, n = 9, wild-type, n = 9; HF: transgenic, n = 11, wild-type, n = 7. Weights were not significantly different on either diet. D, Females, HSTG26 vs. wild-type littermates (LF: transgenic, n = 9, wild-type, n = 9). Weights were not significantly different on a LF diet (HF: transgenic, n = 9, wild-type, n = 9). *, P < 0.05 at wk 6, 7, and 12. Mice were group housed with the same sex and weighed weekly.

 
Subsequent studies used the high-expressing HSTG18 line. We measured weekly food intake of male HSTG18 mice and wild-type littermates on LF and HF diets (Fig. 3Go, A–D). On LF, weekly food intake differed significantly between transgenic and wild-type mice at only five time points, and cumulative food intake did not differ significantly at any time point, although there was a trend toward decreased food intake in the transgenic mice. On HF, SIM1 transgenic mice ate significantly less than their wild-type littermates at almost every time point after 5–6 wk of age, reflected by both weekly and cumulative food intake measures.


Figure 3
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  		FIG. 3. Feeding studies of male HSTG18 transgenic mice vs. wild-type littermates. Weekly (A) and cumulative (B) food intake on LF diet. *, P < 0.05. Weekly (C) and cumulative (D) food intake on HF diet. *, P < 0.05 from 6 wk onward. Mice were weaned onto their respective diets at 3 wk of age and individually housed. Food intake was determined weekly.

 
Energy expenditure, activity, and body composition of SIM1 transgenic mice
To determine whether resistance to DIO in SIM1 transgenic mice was due solely to decreased food intake, we measured energy expenditure, locomotor activity, and feeding efficiency in transgenic mice and wild-type littermates on LF and HF diets (Fig. 4Go, A–F). Regardless of diet, SIM1 transgenic mice exhibited normal energy expenditure and locomotor activity in both the light and dark cycles. SIM1 transgenic mice also exhibited normal feeding efficiency. Regardless of diet, the oxygen consumption rate, carbon dioxide production rate, and respiratory quotients of transgenic mice were the same as wild-type littermates (data not shown). In both wild-type and transgenic mice, the respiratory quotient was appropriately lower in mice fed a HF diet (data not shown). Body composition of SIM1 transgenic mice on LF was no different from that of wild-type littermates (data not shown). On HF, SIM1 transgenic mice had significantly less fat mass than wild-type littermates (15.3 vs. 18.9%, P < 0.01).


Figure 4
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  		FIG. 4. Energy expenditure, activity, and feeding efficiency in HSTG18 transgenic vs. wild-type mice. A and D, Energy expenditure of males during light and dark cycles. B and E, Total activity of males during light and dark cycles. C and F, Feeding efficiency of males at various ages. A–C, LF diet. D–F, HF diet. Mice were habituated to the CLAMS metabolic cages for 2 d before the data were collected over the following 3 d. For A–F, transgenic, n = 6, wild-type, n = 6.

 
Pomc regulation by HF in SIM1 transgenic mice
Pomc expression in the hypothalamus is induced after switching mice from a LF to a HF diet and has been proposed to be part of the physiologic hypophagic response (22). Because reduced Sim1 dosage impairs this response (8), we asked whether overexpression of SIM1 affects hypothalamic Pomc induction by HF. Figure 5AGo shows that in contrast to wild-type mice, Pomc was not induced by HF in SIM1 transgenic mice. Npy and Agrp mRNA levels did not show any significant changes in response to the HF diet and did not differ significantly between wild-type and transgenic mice (Fig. 5Go, B and C).


Figure 5
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  		FIG. 5. Pomc, Npy, and Agrp expression in HSTG18 transgenic vs. wild-type mice. A, Wild-type mice but not SIM1 transgenic mice induce hypothalamic Pomc when fed a HF diet. B and C, Npy and Agrp expression is not affected by diet or genotype (n = 4 for each condition).

 
Effect of SIM1 transgene on Ay obesity
Given that Sim1 is expressed in Mc4r PVN neurons (19) and Sim1 heterozygous mice are resistant to the anorectic effects of hypothalamic melanocortin signaling (26), we asked whether overexpression of SIM1 would rescue the phenotype of Ay mice. Figure 6Go shows that the SIM1 transgene partially rescued Ay obesity. The effect was more prominent in females than males. To investigate the mechanism of the partial rescue of Ay obesity by the SIM1 transgene, food intake and feeding efficiency were measured in Ay mice and littermates that carry the SIM1 transgene (Fig. 6Go, E–H). The transgene completely normalized food intake of Ay mice but had no effect on feeding efficiency. Body composition analysis revealed that Ay/SIM1 transgenic mice had reduced fat mass, compared with Ay mice. Fat mass was reduced by 17% in females and 19% in males. Lean mass was also slightly reduced in females (4%) but not males (Fig. 7Go).


Figure 6
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  		FIG. 6. Partial rescue of obesity and normalization of food intake of Ay mice by SIM1 transgene. A, Gross appearance of wild-type and HSTG18 transgenic mice. B, Gross appearance of Ay and Ay/HSTG18 transgenic mice. C, Mean weight of females on LF diet (wild-type, n = 5, HSTG18, n = 4, Ay, n = 7, Ay/HSTG18, n = 5). *, P < 0.05 from wk 5 onward for Ay/HSTG18 vs. Ay. D, Mean weight of males on LF diet (wild-type, n = 12, HSTG18, n = 7, Ay, n = 6, Ay/HSTG18, n = 10). *, P < 0.05 from wk 13 onward for Ay/HSTG18 vs. Ay. E, Average daily food intake in females aged 5–7 wk. F, Average daily food intake in males aged 5–7 wk. G, Average feeding efficiency in females aged 6–7 wk. H, Average feeding efficiency in males aged 6–7 wk. Multiple groups were compared using one-way ANOVA with Newman-Keuls Multiple comparison post hoc test. Groups with different letters are significantly different (P < 0.01). Groups with the same letter are not significantly different.

 

Figure 7
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  		FIG. 7. Body composition of wild-type, HSTG18 transgenic, Ay, and Ay/HSTG18 transgenic mice. A and D, Total body mass. B and E, Fat mass. C and F, Lean mass. A–C, Females (wild-type, n = 5, HSTG18, n = 4, Ay, n = 7, Ay/HSTG18, n = 5). D–F, Males (wild-type, n = 12, HSTG18, n = 7, Ay, n = 6, Ay/HSTG18, n = 10).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The phenotype of Sim1 haploinsufficiency is strikingly similar to that of Mc4r knockout mice, and both genes are expressed within the PVN, lateral hypothalamus, and amygdala. Sim1 is necessary during fetal development for normal terminal differentiation of PVN neurons but continues to be expressed in the PVN, amygdala, and lateral hypothalamus in adult mice, suggesting that it may also function physiologically in energy homeostasis. To test this hypothesis, we generated SIM1 BAC transgenic mice and asked whether overexpression of SIM1 leads to either a lean phenotype or resistance to DIO, opposite to the phenotype of reduced Sim1 expression. We used a human SIM1 BAC clone to differentiate it from the mouse gene. Human and mouse Sim1 proteins are 96% identical in amino acid sequence. The BAC we chose includes extensive 5'- and 3'-flanking sequences and showed a copy number-dependent hypothalamic expression level, suggesting that it contains all the necessary regulatory elements to drive appropriate SIM1 expression. Furthermore, the BAC insert spans a genomic region similar to that of a mouse BAC that drives enhanced green fluorescent protein expression (23) with the same pattern as the endogenous Sim1 gene (22, 24). Two transgenic lines were generated, a high expresser and a low expresser. Neither line had a weight phenotype on a LF chow diet. Whereas it might seem surprising for overexpression of a developmental transcription factor to have only subtle phenotypic effects, SIM1 acts as a heterodimer, and its dimerization partner, ARNT2 (aryl hydrocarbon receptor nuclear translocator-2) in the hypothalamus (25), may be limiting in the transgenic mice. Alternatively, there could be translational regulation of the SIM1 transcript. No suitable SIM1 antibody is presently available to test this possibility.

Although neither transgenic line showed any body weight phenotype on a chow diet, we previously showed that Sim1 heterozygous mice have enhanced susceptibility to DIO (8). Consequently we examined the effect of the transgene on weight gain of animals fed a HF diet. The high-expressing line of mice was resistant to DIO, whereas the low-expressing line showed a similar trend that did not reach statistical significance, probably due to interanimal variability. These data suggest a dose-dependent action of SIM1 on feeding behavior. We examined the mechanism of DIO resistance in the high-expressing line. These mice ate significantly less of the HF diet than their wild-type littermates but showed no difference in basal metabolic rate or total energy expenditure. Additionally, Sim1 heterozygous mice are hyperphagic but have normal energy expenditure (9). Our results are consistent with the findings of Balthasar et al. (19), who showed that Mc4r- and Sim1-expressing neurons in the PVN or amygdala function solely in the control of food intake, with energy expenditure being regulated by Mc4r neurons elsewhere in the brain. The fact that SIM1-overexpressing mice on a LF diet are not lean may be due to redundant mechanisms that defend normal body weight.

Overexpression of SIM1 partially rescued the obesity of the Ay mouse, suggesting that Sim1 acts downstream of Mc4r. There is also direct evidence from the Sim1 heterozygous mouse that Sim1 functions downstream of the MC4R (26). The fact that the transgene completely rescued the hyperphagia of Ay mice but had no effect on their feeding efficiency and only partially rescued their body weight suggests that it has little or no effect on their energy expenditure, which is known to be reduced (27, 28, 29). This inference is also consistent with the model that Mc4r PVN neurons act specifically in feeding regulation (19, 26) and with the findings of normal energy expenditure in both Sim1 heterozygous mice (9, 26) and SIM1 transgenic mice.

Short-term exposure to the HF diet-induced hypothalamic Pomc expression in wild-type but not SIM1 transgenic mice. The mechanism of this induction is not clear, but it may be part of the physiologic response to dietary fat (22), whereby the increased caloric density of the HF diet leads to increased Pomc expression causing a compensatory reduction in food intake. The lack of Pomc induction by the HF diet in the transgenic mice may reflect an already maximal activation of Mc4r signaling and negative feedback on Pomc expression. The mechanism of feedback is unclear but could involve projections from the PVN to the arcuate nucleus (30). The lack of change in Npy or Agrp expression argues against a leptin-mediated effect.

The increased weight of Sim1 heterozygotes is due mostly to increased fat mass (8). However, they also exhibit increased lean mass and increased length. The SIM1 transgenic mice on HF had decreased fat as well as lean mass, compared with wild-type littermates. Ay and Mc4r–/– mice are also longer than littermates (11, 31, 32). The mechanism of the changes in lean mass in any of these models is unknown.

Sim1 has been implicated in the regulation of food intake in the homeostatic response to dietary fat (8). Reduction and increase in Sim1 dosage have opposite effects on body weight regulation. Reduced gene dosage leads to increased sensitivity to DIO, and increased gene dosage leads to resistance, with higher SIM1 expression correlated with greater resistance. The molecular mechanisms underlying this Sim1 dosage effect are unclear. The most parsimonious explanation for the phenotype of SIM1 overexpression is that in addition to its developmental role, Sim1 has a physiologic function in energy homeostasis. Taken together with our previous work and the work of Balthasar et al. (19), we propose that Sim1 acts downstream of Mc4r within PVN neurons to regulate food intake. Identification of relevant Sim1 transcriptional targets in hypothalamic melanocortin neurons is needed to elucidate the molecular mechanism of its effect on feeding regulation.


   Acknowledgments
 
We thank Ward Wakeland and Fanglin Wei for assistance generating transgenic mice, Ed Esplin for initially characterizing transgene expression, Yuhe Zhao for genotyping and growth studies, and John Shelton and James Richardson for help with brain sections. We thank Michael Brown for reviewing the manuscript.


   Footnotes
 
This work was supported by grants from the American Heart Association, the American Diabetes Association, the Texas Higher Education Coordinating Board, and National Institutes of Health Grant P20 RR020691.

Disclosure statement: The authors have nothing to disclose.

First Published Online May 18, 2006

Abbreviations: Ay, Agouti yellow; DIO, diet-induced obesity; HF, high fat; LF, low fat; MC4R, melanocortin 4 receptor; PVN, paraventricular nucleus; SIM1, single-minded 1.

Received April 10, 2006.

Accepted for publication May 10, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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