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Endocrinology, doi:10.1210/en.2003-0834
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Endocrinology Vol. 145, No. 4 1503-1513
Copyright © 2004 by The Endocrine Society

Deletion of the Nhlh2 Transcription Factor Decreases the Levels of the Anorexigenic Peptides {alpha} Melanocyte-Stimulating Hormone and Thyrotropin-Releasing Hormone and Implicates Prohormone Convertases I and II in Obesity

Enxuan Jing, Eduardo A. Nillni, Vanesa C. Sanchez, Ronald C. Stuart and Deborah J. Good

Department of Veterinary and Animal Sciences and Center for Neuroendocrine Studies (E.J., D.J.G.), University of Massachusetts, Amherst, Massachusetts 01003; and Division of Endocrinology, Department of Medicine (E.A.N., V.C.S., R.C.S.), Brown Medical School, Rhode Island Hospital, Providence, Rhode Island 02903

Address all correspondence and requests for reprints to: Dr. Deborah J. Good, Department of Veterinary and Animal Sciences, 304 Paige Laboratory, University of Massachusetts, Amherst, Massachusetts 01003. E-mail: goodd{at}vasci.umass.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Body weight is controlled by the activation of signal transduction pathways in both the brain and peripheral tissues. Interestingly, although many hypothalamic neuropeptides and receptors have been implicated in the regulation of body weight, the transcriptional and posttranscriptional mechanisms through which these genes are expressed in response to changes in energy balance remain unclear. Our laboratory studies a mouse in which targeted deletion of the neuronal basic helix-loop-helix (bHLH) transcription factor, nescient helix-loop-helix 2 protein (Nhlh2), results in adult-onset obesity. The aim of this work was to use the phenotype of the Nhlh2 knockout mouse and the expression pattern of Nhlh2 to identify genes that are regulated by this transcription factor. In this article, we show that Nhlh2 is expressed throughout the adult hypothalamus. Using dual-label in situ hybridization, we demonstrate that, in the arcuate nucleus of the adult hypothalamus (ARC), Nhlh2 expression can be found in rostral proopiomelanocortin (POMC) neurons, whereas in the paraventricular nucleus (PVN), Nhlh2 is expressed in TRH neurons. In addition, we find that hypothalamic POMC-derived {alpha}MSH in the ARC and TRH in the PVN are regulated posttranscriptionally via Nhlh2-mediated control of prohormone convertase I and II mRNA levels. This is the first report in which regulation of body weight is linked to the action of a neuronal bHLH transcription factor on prohormone convertase mRNA levels. Furthermore, this work supports a direct role for transcriptional control of neuropeptide processing enzymes in the etiology of adult-onset obesity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IT IS WELL ESTABLISHED that the hypothalamus is a primary site of energy balance and body-weight control in the central nervous system. Many of the proteins that regulate this balance between energy intake and energy expenditure have been identified using over 20 different knockout, transgenic, and spontaneous mutant rodent models. Most of these proteins are hormones, receptors, and intracellular signaling factors, whereas only five neuronal transcription factors have been identified. Consequently, there is not a full understanding of the pathways that act to transcriptionally regulate expression of genes important to the control of energy balance by the central nervous system.

Our laboratory studies a neuronally expressed member of the basic helix-loop-helix (bHLH) transcription factor family, nescient helix-loop-helix 2 protein (Nhlh2). Although most other obese mouse models begin to gain weight around the time of puberty, Nhlh2 knockout mice (N2KO) clearly have adult-onset obesity, characterized by increased weight gain after 12 wk of age (1, 2, 3). We have shown that N2KO mice are not overtly hyperphagic before the onset of obesity and that these animals show a lack of physical activity, which precedes adult-onset obesity. In humans, adult-onset obesity is more common than childhood obesity, yet of more than 25 obese mouse models, only the N2KO mouse, the melanocortin-4 receptor (MC4-R) knockout mouse (4), the serotonin, 5-hydroxy tryptamine (5-HT), subtype 2C receptor knockout mouse (5), the Cpefat/fat mouse (6, 7), and the tubby mouse (6) clearly show adult-onset obesity.

Body weight is regulated by complex feedback mechanisms involving the brain and the peripheral organs. One of the best studied of these pathways, leptin protein and its hypothalamic receptor were identified as key molecules in the regulation of energy stores using rodent models and spontaneous mutations in humans (8, 9). Leptin protein is produced in adipocytes and is transported to the brain capillaries where it eventually binds to the long form of the leptin receptor. In the arcuate nucleus of the hypothalamus (ARC), leptin receptor has been colocalized with neurons that produce proopiomelanocortin (POMC) and neuropeptide Y (10, 11). The POMC-derived neuropeptide {alpha}MSH acts to decrease food intake and increase energy expenditure, whereas neuropeptide Y has the opposite physiological activity (12). In the paraventricular nucleus of the hypothalamus (PVN), the leptin receptor also colocalizes with pro-TRH neurons, and leptin regulates TRH mRNA expression (13, 14). Of note, {alpha}MSH from POMC neurons binds to the MC4-R on PVN TRH neurons (14), and targeted deletion of MC4-R results in adult-onset obesity in mice. The N2KO mice show slightly elevated food intake, reduced physical activity, and adult-onset obesity, which are phenotypes similar to the MC4-R knockout mouse. Thus, in our attempt to identify the gene regulatory pathways altered by deletion of the Nhlh2 transcription factor, we have focused on the POMC and TRH neurons.

In this report, we show that the Nhlh2 transcription factor is expressed in rostral ARC POMC neurons and PVN TRH neurons. In addition, we show that functional Nhlh2 transcription factor is necessary for normal expression of a key neuropeptide processing enzyme, prohormone convertase I (PC1). We propose that decreased expression of posttranscriptional processing enzymes reduces levels of fully processed and bioactive neuropeptides, which in turn diminishes the hypothalamic response to increased energy availability and results in adult-onset obesity in the N2KO mice.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
All work involving animals was done in compliance with the Institutional Animal Care and Use Committee at the University of Massachusetts, Amherst. Heterozygous breeders between 2 months and 1 yr of age were used for maintenance of the Nhlh2 line of mice. For heterozygous crosses, the expected 1:2:1 ratio of normal to heterozygous to knockout animals, with a 1:1 ratio of males to females, was obtained. At 3 wk of age, all pups were weaned into new cages by sex and given a unique four-digit ear tag. At the same time, a 1-cm tail biopsy was taken and immediately placed on dry ice for DNA preparation and genotype analysis. N2KO and normal mice were maintained in 12 h light, 12 h dark conditions with ad libitum food (4.5% crude fat). For all experiments reported herein, only male mice were used to eliminate the need to take estrous cycles into account in the females.

cRNA probes
The cRNA probe for mouse Nhlh2 was made using a full-length cDNA template corresponding to the entire published sequence for the mRNA (15), which was cloned into pBLUESCRIPT SK+ (Stratagene, La Jolla, CA). T7 was used for the antisense probe, and T3 was used for the sense probe. Sense RNA probes showed no specific signal (data not shown). The cRNA probe for murine POMC was made using a 920-bp HindIII/EcoRI fragment corresponding to nucleotides 46–963 (GenBank accession no. NM 008895) of the murine POMC mRNA, which was cloned into pBLUESCRIPT. T7 was used to produce antisense probe, and T3 was used to produce the sense probe, which was negative for signal (data not shown). The murine PC1 and prohormone convertase II (PC2) cDNA were a gift from Dr. Nabil Seidah (Clinical Research Institute of Montreal, Montreal, Quebec, Canada) and contain the entire coding regions of both genes. The murine TRH plasmid was obtained from Dr. Joel Elmquist (Beth Israel Deaconess Medical Center, Boston, MA) and originally constructed by M. Yamada (Gunma University Graduate School of Medicine, Maebashi, Japan), and it contains nucleotides 309–573 of the murine prepro-TRH gene (GenBank accession no. NM 009426).

In situ hybridization
Mice were euthanized by decapitation, brains were isolated by dissection, and a hypothalamic tissue block was made by trimming between the optic chiasm and the mammillary bodies. This block was fresh-frozen on dry ice and sectioned on a cryostat at 12 µm. Sections containing the PVN were determined visually based on the position of densely grouped cells surrounding the third ventricle. Sections containing the ARC were identified based on the position of a dense group of cells in the region of the median eminence of the hypothalamus. The lateral hypothalamus (LH) was identified based on its location relative to the ARC and PVN in the consecutive slide sections. The cRNA probes (riboprobes) were prepared using linearized plasmid according to the manufacturer’s directions (T3/T7 Riboprobe kit; Promega Corporation, Madison, WI). The methods for prehybridization and hybridization of the slides have been published (16). Approximately 1 x 106 cpm of 33P-labeled probe was diluted into 25 µl of hybridization buffer and added to the center of each tissue section on slides. The slides were coverslipped and hybridized for 16–18 h at 52 C in the Boekel Slide Moat (Feasterville, PA). After several washes to remove unbound probes, the slides were exposed to phosphor imager screens overnight. The intensity of the signal was used to determine the length of exposure to emulsion. The slides were then dipped in NTB3 liquid emulsion (Kodak, Rochester, NY), dried overnight, exposed at 4 C for a predetermined amount of time (usually 1–4 wk), and developed using Kodak products.

Dual-label in situ hybridization
The procedure for dual-label in situ hybridization is identical to the procedure for in situ hybridization with the following exceptions. The POMC and TRH probes were prepared according to the manufacturer’s directions (Promega T3/T7 Riboprobe kit) with digoxigenin (DIG)-uridine triphosphate added in place of the 33P-uridine triphosphate described above. The Nhlh2 riboprobe was prepared using 33P-uridine triphosphate as described above. Approximately 1 x 106 cpm of 33P-labeled probe and 10–25 ng of DIG-labeled probe in 25 µl of hybridization buffer was added to the center of each slide and hybridized for 16–18 h at 52 C. The slides were washed as described (16), and the DIG-labeled probe was detected using an anti-DIG-horseradish peroxidase-conjugated antibody and developed with diaminobenzidine according to the directions in the TSA Biotin Amplification System (PerkinElmer Life Sciences, Boston, MA). After detection of the nonradioactive signals, the slides were exposed to phosphor imager screens overnight. The intensity of the 33P signal was used to determine the length of exposure to emulsion. The slides were dipped in Kodak NTB3 liquid emulsion, dried overnight, exposed at 4 C for a predetermined amount of time (usually 1–4 wk), and developed using Kodak products.

Immunohistochemistry
Brains were fixed overnight in Histochoice (Amresco, Solon, OH) and then embedded in paraffin blocks. Fourteen-micron sections were placed on glass slides (VWR Superfrost Plus, West Chester, PA) and stored at 4 C until use. The Universal Vector Elite ABC kit (Vector Laboratories, Burlingame, CA) was used according to the manufacturer’s directions. Anti-{alpha}MSH (made in sheep, AB5087) was obtained from CHEMICON International, Inc. (Temecula, CA) and used at a dilution of 1:1000. This antibody is made to the C-terminal end of mammalian {alpha}MSH, and at the time of this publication, the company has not disclosed a list of species-specific reactivities. Anti-TRH (rat) (17) was obtained from Dr. Theo Visser (Erasmus University, Rotterdam, The Netherlands) and used at a dilution of 1:1000. This antibody recognizes fully processed TRH peptide. Anti-PC1 (mouse) was obtained from Dr. Nabil Seidah (Clinical Research Institute of Montreal) and used at a dilution of 1:500.

Peptide extraction
The hypothalamus was dissected as described (18) and frozen in liquid nitrogen. For electrophoresis, samples were boiled for 10 min in a buffer containing 2 N acetic acid, 2 mM EDTA, 2 mM EGTA, and various enzyme inhibitors (0.1% concentrations each for phenylmethylsulfonylfluoride, aprotinin, bacitracin, bestatin, and pepstatin). Samples were sonicated, further homogenized using a Dounce homogenizer (Dounce Kontes Glass Co., Vineland, NJ), and centrifuged at 15,000 x g for 30 min. The supernatant was removed, and a small aliquot was taken from the pellet for protein analysis.

HPLC fractionation
The HPLC used was a Varian ProStar Gradient System (Varian, Inc., Palo Alto, CA) to fractionate the tissue samples. The column used was a Varian C18 Reverse phase column (Microsorb MV 300–5; Varian), and for the POMC peptides, a linear gradient was used from 20–40% B in 20 min using the following mobile phases: 0% acetonitrile-0.1% trifluoroacetic acid and 90% acetonitrile-0.1% trifluoroacetic acid. The flow rate was 1.0 ml/min, and there were equilibration times used on either side of the gradient. Fractions were collected over the course of the 20 min in 0.5-ml fractions. These fractions were then evaporated using an ultra-cold speed vacuum system and then reconstituted in 0.5 ml buffer used in the RIA. Synthetic ACTH peptide was injected on the HPLC to determine retention times. Predicted retention times allowed for analysis of specific regions along the gradient for RIA analysis. The assays used for POMC-derived peptides were developed in our laboratory using commercially synthesized or standard available peptides and primary antibodies developed in our laboratory. The trace was iodinated using the chloramine T oxidation-reduction method followed by HPLC purification.

Electrophoresis
Supernatants were lyophilized, resuspended in 0.0625 M Tris (pH 6.8), 1% sodium dodecyl sulfate, 15% glycerol, and 15 mM dithiothreitol buffer, boiled for 5 min, and loaded onto a 1.5-mm discontinuous polyacrylamide tricine-sodium dodecyl sulfate gel. After electrophoresis, gels were cut into 2-mm slices with a gel slicer (Hoeffer Scientific Instruments, San Francisco, CA). Each slice was placed into 2 N acetic acid for protein extraction before RIA and incubated for 4 d, and the gel slices were removed. Recovery of proteins/peptides from the gel slices was approximately 90%. The molecular masses of the sample proteins/peptides were determined using the following prestained molecular mass markers: 29, 20.4, 14.4, 6.5, and 2.8 kDa (Diversified Biotech, Newton, MA). Extracted peptides from each gel slice were then subjected to TRH and {alpha}MSH RIAs. Synthetic TRH and MSH (Peninsula Laboratories, Belmont, CA) were iodinated by the chloramine T method, and the RIAs were performed as described previously (19). Three independent experiments were done for each sample.

Peptide RIAs
Custom-made thyrosinated prepro-TRH240–255 was iodinated by the chloramine T method, and the RIAs were performed as described and developed previously in Nillni et al. (19). RIAs were performed in 0.1 M sodium phosphate buffer (pH 7.4) containing 500 mg/liter sodium azide and 2.5 g/liter BSA. We recently developed ACTH and MSH assay protocols. Antibodies were generated in rabbit using custom-made AC-KRRPVKVYPNVAENESAEAFC-AMIDE and AC-SYSMEHFRWGKPVC-amide peptides for ACTH and MSH, respectively. The ACTH antibody does not cross-react with {alpha}MSH and recognizes two prominent peaks from the POMC processing analysis. These peaks represent ACTH and pro-ACTH. Full-length MSH (1–13aa) and ACTH1–38 (one thyrosine removed) were used as iodinated tracers. Anti-{alpha}MSH antibody was diluted 1:12,000, and ACTH was diluted 1:25,000 using about 5,000 cpm of 125I-{alpha}MSH and 125I-ACTH, respectively.

Quantification of in situ hybridization results, immunohistochemistry cell counts, and statistical analysis of data
All gene and protein expression analysis was done using at least four animals per genotype. After development, digitalized images were taken with an Olympus P-10 digital camera using an Olympus BH-2 microscope (Olympus, Melville, NY) with the same brightness and contrast for the same comparison groups. The grayscale-digitized images were quantified using the Bioquant Classic system (BIOQUANT Image Analysis Corporation, Nashville, TN), and the same threshold was used for comparison sets. The signal-to-noise ratio was adjusted, and the total number of positive pixels per unit area (cells) was calculated using at least 50 single neuron measurements for each brain region. Each comparison was done in triplicate, and the sample means were calculated. The F test was used to determine whether variances were equivalent, and significance was tested using the Student’s t test with a one-tailed distribution. Digital photographs of fields at x40 magnification of matched hypothalamic areas from normal and N2KO animals were imported into the Bioquant software program. P <= 0.05 was considered statistically significant. The details on optimization of quantitative analysis using Bioquant software have been published (20).

The immunohistochemistry results for {alpha}MSH and PC1 comparisons were quantified by counting corresponding positive neuron numbers from normal and N2KO mice above the same threshold for each comparison. The computer-assisted positive cell number counting was done using Bioquant Classic system on grayscale-digitized images. The Student’s t test was used to test the significance of differences between multiple comparison groups with a one-tailed distribution and equal variance. P <= 0.05 was considered significant.

The TRH data were analyzed using a Tukey-Kramer post-hoc test on pairwise comparisons assuming equal variances for the two groups. This post-hoc test uses the harmonic mean of the two groups being contrasted and is used for sets with unequal numbers. A critical value of 0.772 was calculated for the analysis of older animals, with the difference between N2KO and WT animals exceeding this with a difference of 2.210. For the young animals, the calculated critical difference of 2.287 was not exceeded, and the difference was not significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Nhlh2 is expressed in hypothalamic POMC and TRH neurons
Previous work has demonstrated expression of the Nhlh2 gene in many areas of the embryonic neuroepithelium (15). After characterization of the N2KO animals (2), we speculated, based on the later onset of obesity in the N2KO animals, that the Nhlh2 transcription factor may be expressed in the adult brain. To test this prediction, we examined Nhlh2 mRNA expression in the specific regions of the adult hypothalamus known to be involved in body-weight maintenance. High levels of Nhlh2 expression were detected in the adult PVN and in neurons scattered throughout the ARC, LH, ventromedial hypothalamus, dorsal medial hypothalamus (Fig. 1Go, A–E), and all regions that contain neurons involved in body-weight control. Nhlh2 is also expressed in the medial habenula (Fig. 1FGo).



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  		FIG. 1. Expression of Nhlh2 in the adult hypothalamus. A–F, In situ hybridization using a 33P-labeled cRNA probe to the mouse Nhlh2 gene on coronal sections of hypothalamus from normal mice. A, Nhlh2 expression in the PVN. Magnification, x10. B, Nhlh2 expression in the ARC. Magnification, x20. C, Individual cells of the ARC express Nhlh2. Magnification, x40. D, Nhlh2 expression in the LH. Magnification, x40. E, Nhlh2 expression in the ventromedial hypothalamus. Magnification, x20. F, Nhlh2 is also expressed in a nonneuroendocrine area of the brain, the habenula. Magnification, x20. III, Third ventricle; ME, median eminence; Hp, hippocampus.

 
Based on the activity of POMC and pro-TRH-derived peptides in reducing food intake and increasing energy expenditure, we asked if Nhlh2 was expressed in ARC POMC neurons and in PVN TRH neurons using dual-label in situ hybridization of normal adult mouse brain. As shown in Fig. 2Go, Nhlh2 is expressed in the POMC neurons of the ARC, as indicated by the increased number of silver grains (Nhlh2 expression) over the brown neurons labeled with DIG-labeled probe against POMC (Fig. 2Go, A–D). Analysis of rostral to caudal portions of the ARC showed that 33% of all rostral POMC-expressing neurons also contained Nhlh2 (Fig. 2Go, A, B, and G), whereas the more caudal POMC neurons were adjacent to Nhlh2-expressing cells, with significantly fewer POMC neurons containing this transcription factor (Fig. 2Go, E–G). Medial regions of the ARC had both dual- and single-labeled POMC neurons (Fig. 2Go, C, D, and G). Using dual-label in situ hybridization, we also found that Nhlh2 is expressed in 41% of PVN TRH neurons in the parvocellular region of the PVN and in 45% of TRH neurons in the LH (Fig. 3Go).



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  		FIG. 2. Colocalization of Nhlh2 and POMC in the ARC. Dual-label in situ hybridization of coronal sections from adult mouse hypothalamus demonstrating colocalization of the probe signals for the 33P-labeled Nhlh2 cRNA probe (black grains, open arrows) and the DIG-labeled POMC cRNA probe (brown cells, filled arrows). Each picture shows different regions (rostral to caudal) of the ARC from normal mice. A and B, POMC-Nhlh2 dual-labeled neurons in the rostral ARC. C and D, POMC-Nhlh2 dual-labeled neurons in the medial ARC. This section is approximately 30–50 µm posterior to the section shown in A. C and D, The medial region of the ARC contains more non-Nhlh2-expressing POMC neurons. E and F, In the caudal part of the ARC, there are fewer POMC-Nhlh2 dual-labeled neurons. Instead, Nhlh2-expressing neurons appear in close proximity to POMC-expressing neurons. This section is approximately 150 µm posterior to the section shown in A. Magnification, x40. G, Histogram showing quantification of percentage of colocalization. Total dual-labeled and single-labeled neurons were counted from at least four sections from each of three animals for each region of the ARC. Data are represented as the percentage of colocalized neurons over POMC only-positive neurons with SEM. Significance is indicated using the following letter series: a, P = 0.025, rostral compared with medial; b, P = 0.0078, rostral compared with caudal.

 


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  		FIG. 3. Colocalization of Nhlh2 and TRH in the PVN. A–D, Dual-label in situ hybridization of coronal sections from adult mouse hypothalamus demonstrating colocalization of the probe signals for the 33P-labeled Nhlh2 cRNA probe (black grains, open arrows) and the DIG-labeled TRH riboprobe (brown cells, filled arrows). E, Quantification of the data indicated that 41 ± 6% of the TRH neurons were colocalized with Nhlh2 in the parvocellular region of the PVN and 45 ± 5% of the TRH neurons were colocalized with Nhlh2 in the LH. Data are represented as percentage colocalized with SEM.

 
Rostral POMC peptide levels are reduced posttranscriptionally in N2KO
Expression of Nhlh2 in POMC neurons is consistent with our hypothesis that one function of the Nhlh2 transcription factor is to control genes involved in body-weight regulation. Because POMC-derived neuropeptides normally act to decrease food intake and increase energy expenditure, we asked whether N2KO animals had decreased levels of POMC neuropeptides. We measured levels of hypothalamic POMC-derived neuropeptides using immunohistochemistry and RIA. Immunohistochemistry revealed that N2KO mice have decreased levels of {alpha}MSH peptide in the cell bodies of rostral ARC POMC neurons (Fig. 4Go, E and F; Fig. 5BGo). {alpha}MSH peptide expression in the caudal ARC appears to be unaffected (Fig. 4Go, G and H; Fig. 5BGo). The regional decrease in {alpha}MSH peptide levels correlates well with the regional coexpression of Nhlh2 in POMC neurons. Furthermore, RIA analysis of {alpha}MSH levels extracted from normal and N2KO animals confirm that there is an overall decrease in the levels of fully processed {alpha}MSH peptide (see Fig. 7AGo).



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  		FIG. 4. POMC mRNA levels are similar, but {alpha}MSH peptide levels are decreased in N2KO animals. In situ hybridization using a cRNA probe to murine POMC in normal (A and C) and N2KO (B and D) mice. There are similar numbers of POMC mRNA-positive neurons for both genotypes in both the rostral (A and B) and caudal (C and D) regions of the ARC. Immunohistochemistry using antibodies to {alpha}MSH (Chemicon) on coronal sections of normal (E and G) and N2KO mice (F and H). Immunohistochemistry for {alpha}MSH in the rostral ARC of normal (E) and N2KO (F) mice. Note the number of darkly stained neurons in the section from normal mice, whereas there are fewer and more weakly stained POMC-positive neurons in the section from N2KO mice. In the caudal ARC, the difference in {alpha}MSH staining intensity between normal (G) and N2KO (H) animals is less dramatic.

 


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  		FIG. 5. Quantification of POMC mRNA and {alpha}MSH peptide levels reveals a rostral to caudal difference in peptide levels without a corresponding change in mRNA levels. A, POMC mRNA signal intensity, measured as pixel counts for N2KO and WT mice in the rostal and caudal ARC. B, Numbers of {alpha}MSH-positive neurons in the ARC of wild-type (WT) and age- and sex-matched N2KO siblings. a, P = 0.0096, Student’s t test. Data are represented as mean ± SEM.

 


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  		FIG. 7. Electrophoretic separation of hypothalamic {alpha}MSH peptide and pro-TRH-derived peptides from wild-type (WT) and knockout mice. A–C, Proteins isolated from nine animals of each genotype were separated by SDS-PAGE and eluted from the gel (A and C) or subjected to HPLC (B) and subjected to RIA using (A) anti-MSH serum, (B) anti-ACTH1–38, or (C) an antiserum that recognizes a C-terminal pro-TRH-derived peptide and its pre-form. D, Detection of mature TRH peptide in cell extracts from WT and N2KO hypothalamus. Animals were divided into young (<11 wk old, three of each genotype) and old (>15 wk old, six of each genotype). Data were analyzed using the Tukey-Kramer test, and the effect of genotype on TRH levels was found to be significant (difference, 2.210; critical difference, 0.772) only in the older age group (a, P = 0.05).

 
Because Nhlh2 is a transcription factor, we hypothesized that decreased hypothalamic POMC peptide levels reflected decreased POMC gene expression in the N2KO animals. bHLH transcription factors regulate POMC gene transcription in the corticotroph cells of the pituitary (21), and the pattern of leptin-mediated up-regulation of POMC expression in the rostral ARC (22) led us to theorize that hypothalamic POMC mRNA expression could be regulated by the bHLH transcription factor Nhlh2. Surprisingly, POMC mRNA levels were identical in both the rostral and caudal ARC of normal and N2KO animals (Fig. 4Go, A–D; Fig. 5AGo). We previously reported that N2KO animals appeared to have fewer POMC-positive neurons in their hypothalami than normal animals (2). Closer examination of these data and inclusion of more animals into our study groups indicated that these preliminary data represented an undercount of very lightly stained POMC neurons after immunohistochemistry. Analysis of our in situ results indicate that the number of POMC-positive neurons throughout the ARC are similar in N2KO and normal animals (Fig. 5AGo). This suggests that deletion of Nhlh2 does not affect the differentiation, development, or total adult number of POMC neurons, but rather, it results in changes in the pattern of peptide expression in rostral POMC ARC neurons.

The POMC-derived product ACTH has been hypothesized to represent the immediate substrate for PC2 proteolytic activity to generate {alpha}MSH (23, 24). Thus, the absence or reduction in PC2 activity would be predicted to result in accumulation of ACTH, and the absence or reduction of PC1 activity would be predicted to result in accumulation of a precursor form of ACTH. In fact, ACTH levels detected by our specific RIA in hypothalamic regions of Nhlh2-deficient mice were increased 29% above controls (see Fig. 7BGo; also see the table published as supplemental data on The Endocrine Society’s Journals Online web site, http://endo.endojournals.org). Because this antibody recognizes ACTH and its larger pro-form, an accumulation of these moieties is also an indication of reduced PC1 and PC2 activity. Thus, our results indicate that the Nhlh2 transcription factor does not regulate POMC mRNA levels and that decreased expression of POMC peptides in the N2KO animals must be due to posttranscriptional control mechanisms.

N2KO animals show reduced levels of prepro-TRH mRNA and pro-TRH peptide in their PVN
We have found that Nhlh2 is expressed in over 40% of the parvocellular PVN and LH TRH neurons, suggesting that this transcription factor acts in these neurons to regulate body weight. We were interested in whether levels of prepro-TRH mRNA and pro-TRH protein were altered in N2KO mice, reflecting an effect of loss of the Nhlh2 transcription factor on TRH expression. In contrast to POMC mRNA levels, N2KO mice show reduced mRNA for prepro-TRH in both the LH (data not shown) and the PVN (Fig. 6Go, A–D). Likewise, there is a visible reduction in mature TRH peptide levels in the PVN (Fig. 6Go, E and F) and LH (data not shown). Because immunohistochemistry can only demonstrate relative levels of peptide, we analyzed one of the C-terminal end products of pro-TRH processing, prepro-TRH208–255 (5.4-kDa peptide) and the TRH peptide quantitatively using electrophoretic separation followed by RIA and TRH from cell extracts. We found that the level of the 5.4-kDa pro-TRH peptide was slightly, but significantly, reduced in N2KO mice compared with normal animals (Fig. 7CGo), and reduced levels of mature TRH peptide were only seen in older N2KO mice (Fig. 7DGo). Interestingly, mRNA and immunohistochemistry assays were able to detect reduced TRH in young preobese animals as well as in older obese N2KO mice. This may reflect the ability of the two latter assays to detect and analyze individual cells rather than analyze contents of an entire area, as done in the RIA assays. Although only a hypothalamic block was taken for extraction for the RIA analysis, it is also possible that TRH stored in nerve terminals away from the site of the LH and PVN nuclei may contribute to the higher levels of TRH detected in the hypothalami of young N2KO mice by RIA analysis.



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  		FIG. 6. Both TRH mRNA and peptide levels are reduced in the PVN of N2KO mice. A–D, In situ hybridization of coronal sections of brain containing PVN from wild-type (WT) (A and C) and N2KO (B and D) mice. A and B, Magnification, x4. C and D, Magnification, x40. E and F, Immunohistochemistry using an antibody to mature TRH in N2KO mice (F) compared with WT mice (E). Magnification, x20.

 
Nhlh2 regulates mRNA levels of the posttranscriptional processing enzymes, PC1 and PC2
POMC, pro-TRH, and other hypothalamic neuropeptide precursors are posttranslationally processed to give rise to bioactive neuropeptides by the action of PCs (25). One of these, PC1 has been shown to mediate the initial processing event for the POMC and pro-TRH precursors (19, 24, 26). Furthermore, PC1 is expressed in ARC POMC neurons and PVN pro-TRH neurons (27, 28). Based on our results, we hypothesized that decreased levels of mature POMC peptides in the rostral ARC in the absence of reduced POMC mRNA levels could be due to a reduction in processing enzyme levels. As shown in Fig. 8Go, mRNA levels for PC1 are decreased in the ARC in N2KO animals compared with age matched normal mice (Fig. 8Go, A and B). The expression of these processing enzymes is decreased in both young preobese and older obese (data not shown) animals, indicating that decreased PC1 expression precedes the onset of obesity in the N2KO animals. PC1 mRNA levels are also reduced in the PVN of N2KO animals (Fig. 8Go, C and D), as well as in the LH of N2KO animals (data not shown). Quantification of these results indicate that there was a 44% reduction in PC1 mRNA levels in the ARC of N2KO mice compared with age- and sex-matched normal mice (Fig. 9AGo). In the PVN, there is a 55% reduction in PC1 mRNA in N2KO animals (Fig. 9AGo). As shown using immunohistochemistry, the net effect of these reduced PC1 mRNA levels is a reduction in the level of PC1 protein (Fig. 8Go, E–H; Fig. 9BGo). We have also found a reduction in ARC levels of PC2 mRNA (see Supplemental Figs. 1Go and 2Go). Thus, these data support our hypothesis that adult-onset obesity in N2KO mice is due to the reduced levels of hypothalamic-processing enzymes, which lead to decreased amounts of mature, fully processed {alpha}MSH and TRH neuropeptides causing increased energy intake and decreased energy expenditure in these animals.



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  		FIG. 8. N2KO animals have reduced expression of hypothalamic PC1 mRNA and protein. A–D, In situ hybridization using a cRNA probe to murine PC1 on coronal sections of hypothalamus from normal (A and C) and N2KO (B and D) mice. Magnification, x40. PC1 mRNA expression was compared for the ARC (A and B) and PVN (C and D). E–H, Immunohistochemistry using an antibody to murine PC1 on coronal sections of hypothalamus from normal (E and G) and N2KO (F and H) mice. PC1 protein expression was compared for the ARC (E and F; magnification, x40) and PVN (G and H; magnification x20). Insets in A–D are at magnification, x10. Arrows in E and F point to neuron cell bodies in the ARC. Note that, in the ARC, there is also background staining of blood vessels. III, Third ventricle adjacent to the PVN.

 


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  		FIG. 9. Quantification of PC1 mRNA and protein levels shows reduced levels of mRNA and protein in N2KO animals. A, Comparison of PC1 mRNA signal intensity, measured as pixel counts between wild-type (WT) and N2KO mice in the rostral ARC (44% reduction) and PVN (55% reduction). a, P = 0.027, Student’s t test; b, P = 0.022, Student’s t test. B, Comparison of PC1-positive neurons in the rostral ARC (71% reduction) and PVN (51% reduction) of N2KO mice compared with age- and sex-matched WT siblings. a, P = 0.009, Student’s t test; b, P = 0.015, Student’s t test. Data are represented as mean ± SEM.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
These data are the first to correlate regulation of PC1 mRNA levels with posttranscriptional control of neuropeptide expression and the regulation of body weight in a transcription factor knockout mouse. We have shown that Nhlh2 is expressed in all regions of the hypothalamus involved in body-weight control. Of note, leptin receptor expression is found in all hypothalamic nuclei where Nhlh2 is expressed (29, 30, 31, 32), and there are putative E-box motifs in the published promoter of the leptin receptor. One of these, at nucleotides 6143–6149, is identical to the consensus-binding site for the related bHLH transcription factor Nhlh1 (33), which opens the possibility that Nhlh2 could influence leptin receptor expression.

Only two other transcription factors with links to body-weight regulation have been colocalized in POMC and TRH neurons—STAT3 (signal transducer and activator of transcription 3) and c-Fos (14, 34, 35, 36, 37). Neither of these knockout mice are useful to the study of adult-onset obesity because STAT3 knockout mice are embryonic lethal (38) and c-Fos knockout mice develop ossification of their bone marrow and show behavioral problems but not obesity (39). Another putative transcription factor, Tub is expressed in the ARC and PVN (Coyle, C. A., and D. J. Good, unpublished data; and Refs. 40 and 41) but has not yet been colocalized in specific neuron subtypes in this area. Thus, N2KO mice are currently the only transcription factor knockout mice in which altered regulation of genes within POMC and TRH neurons leads to adult-onset obesity.

In this article, we show that Nhlh2 is not expressed by all POMC neurons. Rather, 33% of rostral POMC neurons coexpress Nhlh2, whereas the caudal POMC neurons were more likely to be adjacent to Nhlh2-expressing cells, with only about 12% of POMC neurons also showing Nhlh2 expression. Thus, expression of the Nhlh2 transcription factor is more concentrated in the rostral POMC neurons that are also responsive to leptin treatment (22) and express melanocortin-3 receptors (42). We have shown that Nhlh2 is expressed in 41% of PVN TRH neurons, which contain receptors for and are responsive to melanocortins (14). Both POMC and TRH neurons are direct targets of leptin signaling (10, 14). Thus, it is possible that Nhlh2 mediates some of the effects of leptin or other energy balance signals on POMC and TRH synthesis.

Using both immunohistochemistry of specific regions of the ARC and RIA analysis of whole hypothalami, we have shown that levels of fully processed {alpha}MSH are reduced in N2KO mice, whereas unprocessed forms of POMC (pro-ACTH) are elevated. {alpha}MSH binds to melanocortin receptors in the hypothalamus and suppresses food intake, resulting in lower overall body weight. Thus, lower levels of {alpha}MSH would be expected in animals with increased body weight. Surprisingly, Nhlh2 does not directly regulate levels of {alpha}MSH by regulating POMC gene transcription. Rather, levels of this neuropeptide are controlled posttranscriptionally by Nhlh2-mediated regulation of PC1 and PC2 mRNA levels. {alpha}MSH is generated by extensive posttranslational processing of the product of the POMC gene, with PC1 mediating the first and second cleavages of the prohormone (43) and PC2 generating mature {alpha}MSH (23, 24). Thus, reduced levels of PC1 and PC2 enzyme in N2KO animals would be expected to reduce levels of mature {alpha}MSH peptide and increase levels of unprocessed forms of POMC, which is exactly what we have found. One may expect that ACTH levels might also be lower when PC1 is down-regulated. However, because we do not know at this juncture whether the down-regulation of PC1 and PC2 is the same, it is possible that PC2 activity is more inhibited than PC1, resulting in an accumulation of ACTH. Interestingly, unlike POMC mRNA levels, prepro-TRH mRNA levels are reduced in N2KO animals concomitant with reduced levels of mature TRH peptide. One possible explanation is that prepro-TRH mRNA levels could be reduced as a secondary effect of decreased {alpha}MSH in the knockout mice and that levels of mature TRH peptide are even further reduced in the presence of low PC1 enzyme. This hypothesis is supported by the fact that generation of TRH208–255 requires the action of PC1 (26, 44). In addition, it is plausible that lower overall neuropeptide processing in the N2KO mice may result in reduced levels of other neuropeptides that positively or negatively impact food intake or energy expenditure, although the phenotype of the N2KO mice shows that the net effect of Nhlh2 transcription factor loss is obesity.

There are both human and rodent models in which loss of prohormone processing results in obesity. For example, the fat/fat mouse carries a mutation in the gene for carboxypeptidase E, which reduces the ability of that animal to package proinsulin, progastrin, procholecystokinin, and POMC in intracellular compartments for processing by PC1 (7). Recently, Berman et al. (45) showed that levels of PC1 protein and mature POMC-derived peptides were also reduced throughout the hypothalamus of fat/fat mice. This reduction appears to be due to an accumulation of immature PC1, rather than a reduction in PC1 mRNA as we have found in the N2KO mice. Human germline mutations affecting PC1-mediated protein processing have also been characterized in obese individuals. Germline mutations in the PC1 gene that affects the amino acid sequence or splicing of the PC1 mRNA lead to abnormally high levels of unprocessed insulin and POMC (46). In addition, mutations in amino acid residues essential for proper PC1-mediated processing of the POMC gene also result in profound obesity in humans (47). Interestingly, mice containing a deletion of the PC1 gene are not obese but rather show growth delays (stunting) possibly due to reduced GHRH (48).

Transcription factors belonging to the bHLH transcription factor family bind to DNA at a motif called the E-box, a 6-bp sequence of DNA represented by CANNTG (49). Analysis of both the human and mouse PC1 and PC2 promoters reveals several potential binding sites for bHLH transcription factors such as Nhlh2. The human and mouse PC2 promoters contain one E-box each, whereas the human and mouse PC1 promoters have three and two E-box motifs, respectively (50, 51, 52). What is remarkable about the human and mouse PC1 promoters is the placement of each of the E-box motifs relative to potential binding sites for the STAT family of transcription factors. STAT transcription factors contain a leucine zipper region and form homo- and heterodimers before binding to DNA and activating transcription (53). Members of the bHLH family also form homo- and heterodimers before binding to DNA (49). We have previously found that Nhlh2 can interact with leucine zipper-containing transcription factors, such as c-Jun and c-Fos (data not shown), suggesting that it may also be able to heterodimerize with STAT transcription factors. In this article, we show that there is more Nhlh2 expression in the rostral POMC neurons, the subset of POMC neurons that also contain leptin receptors and activated STAT3 transcription factor upon leptin stimulation (34). These findings support a role for the Nhlh2 transcription factor either alone or in conjunction with STAT transcription factors, such as STAT3, in the hypothalamic regulation of the PC1 and PC2 genes in response to energy balance changes in the body. Work from two other groups has shown that hypothalamic PC1 expression is affected by leptin administration and energy balance deficit (54) and by gp130 signaling through the Janus kinase (JAK)/STAT pathway (55). Furthermore, the Nillni laboratory, using both in vitro and in vivo approaches, recently showed strong evidence implicating leptin in the regulation of PC1 and PC2 expression and protein biosynthesis and, as a consequence of those regulatory changes, the biosynthesis and further posttranslational processing of pro-TRH (Sanchez, V. C., J. Goldstein, R. Stuart, V. Hovanesian, H. Munzberg, T. Friedman, C. Vaslet, C. Bjorbaek, and E. A. Nillni, submitted for publication). These data further support our hypothesis that PC1 expression can be controlled by the STAT3 and Nhlh2 signaling pathways.

The results presented here identify a novel mechanism of hypothalamic POMC and TRH gene regulation in response to energy requirements by the body. The data demonstrate that posttranslational regulation of gene expression and prohormone processing pathways are critical to the ultimate amount of hypothalamic {alpha}MSH and mature TRH produced and that the level of POMC or prepro-TRH mRNA in neurons does not always reflect the amount of functional neuropeptides present. One published report suggests that the leptin pathway stimulates increased expression of Nhlh2 (56). Our results suggest that changes in energy balance, either by leptin or other pathways, would signal through Nhlh2, resulting in increased transcription of PC1, and coordinately, with the increased POMC and prepro-TRH mRNA induced by food intake or increased circulating leptin (10, 57), would stimulate increased processing of the newly translated prohormones. Activation of both POMC and TRH signal transduction pathways would then balance energy expenditure with intake. Interestingly, {alpha}MSH also has effects on the reproductive axis (58), and N2KO mice are hypogonadal with reduced fertility (2). Thus, regulation of POMC processing via Nhlh2-mediated regulation of PC1 may be a link between the body weight and reproductive axes.

The N2KO mouse was the first neuronal transcription factor knockout mouse with a phenotype of adult-onset obesity. We have shown that the Nhlh2 transcription factor is expressed regionally in the rostral ARC POMC neurons, where we have demonstrated that absence of Nhlh2 reduces expression of the PC1 and PC2 mRNA and {alpha}MSH peptides. In the PVN, we have shown reduced PC1 and PC2 mRNA along with a reduction in both mRNA and peptide for TRH. The phenotype of late-onset obesity in N2KO mice mimics adult-onset obesity in humans. Thus our findings also provide insight into the complexity of human obesity. Overall, this work has direct implications to the understanding of transcriptional and posttranscriptional gene regulation in normal body-weight maintenance and provides several levels at which therapeutic intervention of body-weight control can occur.


   Acknowledgments
 
The authors thank Dr. Sandra Petersen (University of Massachusetts, Amherst) for help setting up dual-label in situ hybridizations, Dr. Nabil Seidah (Clinical Research Institute of Montreal) for the murine cDNAs and antibodies to PC1 and PC2, Dr. W. Scott Young (National Institutes of Health) for the murine cDNA to POMC, Dr. Joel Elmquist (Beth Israel Deaconess Medical Center, Boston, MA) for the murine cDNA to mouse TRH, Dr. Theo Visser (Erasmus University, The Netherlands) for the antibody to rat TRH, and Christopher A. Coyle and Alison Miller Bardwell for excellent technical assistance.


   Footnotes
 
This work was supported by Healey Endowment Grant SL 1-60253, a United States Department of Agriculture, Massachusetts Agricultural Experiment Station Project MAS00860, and NIH Grants R01 DK59903 (to D.J.G.) and R01 DK58148 (to E.A.N.).

Abbreviations: ARC, Arcuate nucleus of the hypothalamus; bHLH, basic helix-loop-helix; DIG, digoxigenin; LH, lateral hypothalamus; MC4-R, melanocortin-4 receptor; N2KO, Nhlh2 knockout mice; Nhlh2, nescient helix-loop-helix 2 protein; PC1, prohormone convertase I; PC2, prohormone convertase II; POMC, proopiomelanocortin; PVN, paraventricular nucleus of the hypothalamus; STAT3, signal transducer and activator of transcription 3.

Received July 3, 2003.

Accepted for publication December 22, 2003.


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E. A. Nillni
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X. Zhu, A. S. Gleiberman, and M. G. Rosenfeld
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J. W. M. Creemers, L. E. Pritchard, A. Gyte, P. Le Rouzic, S. Meulemans, S. L. Wardlaw, X. Zhu, D. F. Steiner, N. Davies, D. Armstrong, et al.
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T. Zimmermann-Belsing and U. Feldt-Rasmussen
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