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Induction of Neuropeptide Y Gene Expression in the Dorsal Medial Hypothalamic Nucleus in Two Models of the Agouti Obesity Syndrome

Vollum Institute for Advanced Biomedical Research (R.A.K., R.D.C.) Oregon Health Sciences University Portland, Oregon 97201-3098
Millennium Pharmaceuticals, Inc. (D.H., C.A.L.) Cambridge, Massachusetts 02139
Oregon Regional Primate Research Center (R.B.S.) Beaverton, Oregon 97006

	ABSTRACT

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Dominant mutations at the agouti locus induce several phenotypic changes in the mouse including yellow pigmentation (phaeomelanization) of the coat and adult-onset obesity. Nonpigmentary phenotypic changes associated with the agouti locus are due to ectopic expression of the agouti-signaling protein (ASP), and the pheomelanizing effects on coat color are due to ASP antagonism of -MSH binding to the melanocyte MC1 receptor. Recently it has been demonstrated that pharmacological antagonism of hypothalamic melanocortin receptors or genetic deletion of the melanocortin 4 receptor (MC4-R) recapitulates aspects of the agouti obesity syndrome, thus establishing that chronic disruption of central melanocortinergic signaling is the cause of agouti-induced obesity. To learn more about potential downstream effectors involved in these melanocortinergic obesity syndromes, we have examined expression of the orexigenic peptides galanin and neuropeptide Y (NPY), as well as the anorexigenic POMC in lethal yellow (A^y), MC4-R knockout (MC4-RKO), and leptin-deficient (ob/ob) mice. No significant changes in galanin or POMC gene expression were seen in any of the obese models. In situ hybridizations using an antisense NPY probe demonstrated that in obese A^y mice, arcuate nucleus NPY mRNA levels were equivalent to that of their C57BL/6J littermates. However, NPY was expressed at high levels in a new site, the dorsal medial hypothalamic nucleus (DMH). Expression of NPY in the DMH was also seen in obese MC4-RKO homozygous (-/-) mice, but not in lean heterozygous (±) or wild type (+/+) control mice. This identifies the DMH as a brain region that is functionally altered by the disruption of melanocortinergic signaling and suggests that this nucleus, possibly via elevated NPY expression, may have an etiological role in the melanocortinergic obesity syndrome.

	INTRODUCTION

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Dominant alleles of the agouti locus induce most notably a yellow coat color in the mouse, whereas the most dominant mutations (e.g. A^y or A^vy) produce additional phenotypic changes including adult-onset obesity, increased linear growth, and hyperinsulinemia (for review, see Refs. 1 and 2). The cloning of the murine agouti gene (3) predicts a 108-amino acid secreted protein that is normally expressed in skin during postnatal days 3 to 5, corresponding to the time during which pheomelanin deposition occurs in the developing hair shaft. In contrast, the dominant lethal yellow (A^y) allele of agouti results in constitutive and ubiquitous expression of agouti-signaling protein (ASP) as a consequence of a promoter rearrangement (4). The obesity and coat color phenotypes of the A^y animal are recapitulated by transgenic mice having a ß-actin promoter driving ubiquitous ASP expression (5). However, transgenic mice using a keratin-14 promoter to direct skin-specific expression of ASP have only the yellow coat color phenotype, demonstrating that agouti acts locally in a paracrine manner (6) and that expression outside of the skin is required for the induction of obesity.

Experiments by Lu et al. (7) demonstrated that ASP is a competitive antagonist of the melanocortin-1 receptor expressed in skin melanocytes (8, 9). The melanocortin-1 receptor is normally stimulated by -MSH to increase intracellular cAMP levels, inducing tyrosinase activity, and eumelanin or brown/black pigment synthesis. One hypothesis for the nonpigmentary phenotypes seen in A^y mice is that aberrant expression of ASP in the central nervous system antagonizes related hypothalamic melanocortin receptors (7). This model was originally proposed based upon evidence that ASP is also a competitive antagonist of the highly related melanocortin-4 receptor (MC4-R) (7), which is expressed in regions of the hypothalamus important for the control of feeding behavior (10). Strong support for this model comes from two recent studies. First, central administration of the -MSH analog SHU9119, a high-affinity antagonist of the neural MC3 and MC4 receptors, significantly stimulates feeding in mice, whereas an agonist potently inhibited feeding in several murine models of hyperphagia (11). Second, ablation of functional MC4 receptors by targeted disruption of the MC4-R gene in mice recapitulates several aspects of the agouti obesity syndrome (12).

One possible effect of losing MC4-R activity, either by genetic deletion or chronic antagonism by ASP, may be perturbation of normal hypothalamic signaling. Neuropeptide Y (NPY) (13) and galanin (14) are two neuropeptides present in high concentrations in the hypothalamus that, when injected into the brain, stimulate feeding. Recent experiments have shown that hypothalamic NPY levels are elevated in other models of rodent obesity, including the ob/ob mouse (15) and the Zucker fatty rat (16), as well as in streptozocin-induced diabetic rats (17) and in mice given hypothalamic lesions via gold-thioglucose treatment (18). Therefore it seemed possible that NPY or possibly galanin synthesis may be stimulated in A^y or MC4-R deficient mice. Alternatively, melanocortinergic neurons may respond to loss of normal MC4-R function by compensatory changes in the synthesis of the melanocortin ligands encoded by the preprohormone gene POMC. In this report, NPY, galanin, and POMC gene expression were examined as a function of the A^y or MC4-RKO obesity syndromes.

	RESULTS

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NPY Gene Expression but Not Galanin or POMC Is Altered in Lethal Yellow (A^y) Mice
Representative autoradiograms of anatomically matched coronal brain sections [at the level of the dorsal medial hypothalamic nucleus (DMH)] obtained after in situ hybridizations with NPY, galanin, and POMC antisense probes are shown in Fig. 1. The upper panel shows the hypothalamic NPY mRNA expression for C57BL/6J (C57) mice (left), C57Bl/6J-A^y (A^y) mice (middle), and C57Bl/6J-Lep^ob (ob/ob) mice (right). Although the hybridization intensity for NPY gene expression in the hypothalamic arcuate nucleus (ARC) of the ob/ob mouse was clearly elevated when compared with the control C57 mouse, there was no visible difference in NPY gene expression in the ARC of the A^y mouse. Furthermore, NPY expression in the cerebral cortex, not associated with feeding behavior, was also similar in all three animal models. However, in the A^y mouse a population of intensely labeled neurons in the DMH was identified (with minor labeling of the lateral hypothalamic area), whereas hypothalamic expression of NPY in the ob/ob mouse was restricted to the ARC only. No comparable differences in either galanin (middle) or POMC (bottom) mRNA expression levels were observed between the C57, A^y, and ob/ob mice (Fig. 1).

View larger version (51K): [in this window] [in a new window]   Figure 1. NPY, Galanin, and POMC mRNA Expression in C57BL/6J, Obese C57BL/6J-Ay, and Obese C57BL/6J-Lepob Mice Representative autoradiograms of coronal brain sections through the DMH are shown after hybridizations with prepro-NPY (upper), galanin (middle), and POMC (lower) antisense probes. NPY expression in the control C57BL/6J animal (left) was limited to the ARC and the cerebral cortex (CTX). Hypothalamic NPY expression is elevated in the ARC of the C57BL/6J-Lepob animal (right), whereas the C57BL/6J-Ay animal (middle) has normal ARC expression but elevated expression in the DMH. Hybridization signals for galanin and POMC mRNA indicate similar expression patterns in each mouse strain, with galanin in both the ARC and DMH, whereas POMC is restricted to the ARC.

View larger version (93K): [in this window] [in a new window]   Figure 2. Elevated DMH NPY Gene Expression Is Uniquely Associated with the Obese State in C57BL/6J-Ay Mice Shown are dark-field photomicrographs of coronal hypothalamic sections (mid- DMH level) of young nonobese (3 wk, 15 g), adult nonobese (12 wk, 28 g), and obese (6 mo, 55 g) C57BL/6J-Ay mice (panels A, B, and C, respectively) in addition to an obese (58 g) C57BL/6J-Lepob animal (panel D) after in situ hybridizations with a prepro-NPY probe. Arrowheads indicate DMH neurons highly expressing NPY mRNA in the obese C57BL/6J-Ay animal (panel C).

View larger version (23K): [in this window] [in a new window]   Figure 3. NPY mRNA Levels in the ARC (left) and DMH (right) of C57BL/6J, C57BL/6J-Lepob, C57BL/6J-Ay Nonobese, and C57BL/6J-Ay Obese Mice In the ARC, NPY mRNA levels of leptin-deficient (C57BL/6J-Lepob) mice, but not C57BL/6J-Ay animals, were significantly elevated as compared with C57BL/6J control mice (P < 0.03). In the DMH, measurable NPY mRNA levels above background were found only in obese C57BL/6J-Ay mice, which is highly significant when compared with C57BL/6J control mice (P < 0.0001). Absorbancies (relative NPY signal intensity) of the autoradiographic images on Cronex x-ray film were measured as described (see Materials and Methods) and analyzed using GraphPad Prism software.

View larger version (63K): [in this window] [in a new window]   Figure 4. Obese Melanocortin-4 Receptor (MC4-R) Deficient Mice Express NPY in the DMH Representative autoradiograms of coronal brain sections through the DMH are shown after in situ hybridization with prepro-NPY for wild type (+/+), heterozygous MC4-R deficient (±), and homozygous MC4-R deficient (-/-) mice in panels A, B, and C, respectively. Hypothalamic expression of NPY is restricted to the ARC in the nonobese wild type and heterozygous MC4-R-deficient mice.

	DISCUSSION

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In the present experiments, both male and female A^y mice weighing more than 55 g exhibited elevated NPY mRNA expression in DMH neurons; younger A^y mice weighing less than 30 g displayed normal hypothalamic NPY expression in ARC neurons and no expression in the DMH (Fig. 2). Furthermore, nonobese mice that have a single functional MC4-R allele (heterozygous MC4-R knockout) do not express NPY aberrantly. The expression of NPY in the DMH is not associated with all models of obesity, however, because obese ob/ob mice did not display any detectable NPY expression in the DMH. Therefore, activation of NPY expression in DMH neurons is correlated specifically with the melanocortinergic obesity syndromes that derive from a loss of MC4-R activity.

Expression of NPY mRNA in the DMH has not been reported previously in other mouse or rat models of obesity; however, there are some reports of altered NPY peptide levels in this nucleus associated with feeding status (19, 20, 21). In these experiments, fluctuations in NPY peptide levels in the DMH are probably reflecting the activation of NPY-ergic neurons that originate in the ARC and project to the DMH (22). Interestingly, the only reported activation of NPY-ergic neurons originating in the DMH is in lactating rats that also display a characteristic elevation of NPY mRNA in the DMH nucleus (23) in response to suckling, which may reflect an activation of a feeding pathway in response to an increased nutritional need.

A simplified model proposing a role for NPY in both the ob/ob and melanocortinergic obesity models is shown schematically in Fig. 5. The disruption of leptin signaling in the ob/ob or db/db mouse results in an elevation of NPY gene expression in the ARC, perhaps directly via leptin receptors in the ARC. This enhanced release of NPY is responsible for a significant portion of the phenotype in the ob/ob animal, as supported by recent observations on attenuation of the obesity syndrome by deletion of the NPY gene from the ob/ob mouse (24).

View larger version (36K): [in this window] [in a new window]   Figure 5. Model of Neuroendocrine Circuitry in the Melanocortinergic Obesity Syndrome Normal POMC signaling from ARC neurons to the DMH via -MSH is disrupted by either 1) ectopic expression of ASP, a competitive antagonist of -MSH, or 2) genetic deletion of the melanocortin-4 receptor (MC4-R). Loss of MC4-R activity results in the aberrant expression of NPY in DMH neurons that project to the PVN. Excess NPY in the PVN may then mediate component(s) of the agouti obesity syndrome.

Induction of NPY in the DMH is clearly an informative marker of the melanocortinergic obesity syndromes and identifies the DMH as a possible downstream target of POMC neurons in their effects on feeding and metabolism. Although NPY gene induction does not appear to precede obesity in the A^y model, it may be an etiological factor in melanocortinergic obesity models. Support for this comes from several lines of research. First, although POMC neurons within the ARC project throughout the brain, immunostaining with an antibody directed to -MSH shows that a major projection of arcuate POMC neurons is to the DMH. Second, in situ hybridization studies have demonstrated that one of the major sites of MC4-R expression is in the anterior part of the DMH (10). Moreover, recent experiments by Thompson et al. (25) indicate that DMH neurons project primarily within the hypothalamus, with direct innervation of the PVN, known to express NPY5 receptors (26). However, whether the DMH neurons that project to the PVN and release NPY also express MC4 receptors is unknown.

While deletion of the NPY gene does not produce an overt feeding behavior disorder (27), genetic crossing of NPY deficiency into leptin-deficient mice attenuates the hyperphagia of ob/ob mice approximately 50%, demonstrating a role for elevated NPY in the pathophysiology of obesity resulting from the absence of leptin (24). We propose that a component of the hyperphagia, altered metabolism, increased linear growth, or reduced fertility seen in melanocortinergic obesity syndromes may be due to altered function of the DMH, and perhaps specifically due to elevated release of DMH-derived NPY at the PVN. Ultimately, deletion of the NPY gene from the A^Y mouse may be used to determine the role of elevated NPY expression in the melanocortinergic obesity syndrome.

	MATERIALS AND METHODS

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Animals
Control C57Bl/6J mice, C57Bl/6J-A^y heterozygous mice, and C57Bl/6J-Lep^ob mice were maintained under a 12-h light, 12-h dark cycle at constant temperature, and provided food (Purina mouse chow) and water ad libitum. C57Bl/6J-A^y mice in groups of three animals each consisting of young nonobese (3 wk, 15–17 g), adult nonobese (12 wk, 25–28 g), and adult obese (6 mo, 55–62 g) mice were compared with aged matched control C57Bl/6J mice (15–45 g, one to three animals each), and three obese C57Bl/6J-Lep^ob mice (6 mo, 58–60 g). Anesthetized (avertin) animals were killed between 1500 and 1700 before lights out via cardiac puncture and perfused with saline (20 ml) and then 50 ml of ice-cold fixation buffer (4% paraformaldehyde in borate buffer, pH 9.5). Whole brains were rapidly removed and then postfixed overnight in 10% sucrose/fixative buffer. Blocked hypothalamic sections were frozen in powdered dry ice and then stored at -80 C until sectioned. A detailed description of the MC4-R gene deletion mouse is described by Huszar et al. (12). For the MC4-R deletion studies, littermates consisting of a male and a female mouse from each genotype, homozygous (-/-) MC4-R-deficient (55–60 g), heterozygous (±), and homozygous (+/+) wild type (25–30 g) were treated as above.

In Situ Hybridization
Probe Synthesis.
Antisense NPY was prepared by linearizing the plasmid pBLNPY-1, which contains 511 bp of the rat NPY gene (kindly provided by Dr. Susan Smith) with FspI. Antisense POMC was prepared by linearizing the plasmid mPOMCE3ribo with NcoI, which contains exon 3 of the mouse POMC gene (kindly provided by Dr. Malcolm Low). Antisense galanin was prepared from a 680-bp rat galanin cDNA plasmid provided by Dr. Robert Steiner, after HindIII linearization. [³⁵S]cRNA probes were prepared by transcribing 1 µg of each linearized DNA with T3 polymerase (NPY), or T7 polymerase (POMC and galanin) for 1 h at 37 C in a reaction containing [³⁵S]UTP (NEN, Boston, MA) using a commercially available in vitro transcription kit (Promega, Madison, WI).

Hypothalamic brain blocks were mounted on a frozen stage and serially sectioned into four series of 20-µm slices with a sliding microtome. Sections were prepared and processed for in situ hybridization as previously described (18). Sections were hybridized for 20 h at 58 C with ³⁵S-labeled probes (5 x 10⁶ cpm/ml in 65% formamide, 0.26 M NaCl, 1.3x Denhardt’s solution, 1.3 mM EDTA, 13% dextran sulfate, 13 mM Tris, pH 8). Sections were then digested with RNase (20 µg/ml) for 30 min at 37 C and then desalted in a series of washes from 4x SSC (0.15 M NaCl, 0.1 M Na₃C₆H₅O₇·2 H₂O) 1 mM dithiothreitol to a final stringency of 0.1x SSC/1 mM dithiothreitol at 65 C for 30 min. Sections were dehydrated in ascending ethanol, vacuum dried at room temperature for 30 min, and then exposed to Dupont Cronex film (Dupont, Wilmington, DE) for several days. Dried slides were then dipped in NTB-2 emulsion (Kodak, Rochester, NY) and developed after 6 days.

Quantification of Autoradiograms.
The absorbancies of the autoradiographic images on Cronex x-ray film were measured by using a Macintosh-based image analysis system and NIH Image software obtained from NIH. The film was illuminated with a ChromaPro 45 light source, which provided even illumination, and the image was captured with a Dage MTI 70 series video camera equipped with a Newvicon tube and interfaced with a Scion image capture board. The mean absorbancies of the autoradiographic images on Cronex x-ray film over the ARC or DMH (12 h exposure) at the same level from each brain were measured. The mean absorbance over a large irregularly-shaped region adjacent to the ARC or DMH, that did not contain specific hybridization, was also measured on each section and used to calculate mean background density, which was subtracted from the absorbance measurement of signals over the ARC or DMH. Commercially available ¹⁴[C] autoradiographic standards were exposed to each x-ray film along with experimental material. The mean absorbance of an interactively defined region over each standard was measured; these measurements confirmed the linearity of the film’s responsiveness, as well as the consistency of signal detection across films. The mean absorbancies of the auroradiographic images recorded over the ARC or DMH all fell within the linear range of the standard values. A two-way ANOVA test was used to test for significant differences in levels of POMC, galanin, or NPY mRNA hybridization among the treatment groups in each experiment. A P value of less than 0.05 was defined as significant.

	ACKNOWLEDGMENTS

 
The authors would like to thank Malcolm Low and Guibao Gu for helpful discussions; Meigan Crabtree, Michele Zee, and Denise O’Leary for technical assistance; and June Shiigi for graphic support.

	FOOTNOTES

 
Address requests for reprints to: Roger D. Cone, Vollum Institute for Advanced Biomedical Research, Oregon Health Sciences University, 3181 Southwest Sam Jackson Park Road, Portland, Oregon 97201-3098.

This work is supported by NIH Grants [NIDDK and NICHD to R.D.C., NIDDK to R.A.K., and NICHD to R.B.S.], and by Millenium Pharmaceuticals, Inc., (to D.H. and C.A.L.).

Received for publication December 11, 1996. Revision received January 31, 1997. Accepted for publication February 4, 1997.

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