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Hypothalamic Agouti-Related Protein Messenger Ribonucleic Acid Is Inhibited by Leptin and Stimulated by Fasting¹

Fishberg Center for Neurobiology, Neurobiology of Aging Laboratories, Department of Geriatrics, Mount Sinai School of Medicine, New York, New York 10029

Address all correspondence and requests for reprints to: Charles V. Mobbs, Ph.D., Neurobiology of Aging Laboratories, Box 1639, Mt. Sinai School of Medicine, One Gustave L. Levy Place, New York, New York 10029. E-mail: mobbsc{at}alum.mit.edu

	Abstract

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Agouti-related protein (AGRP) is a homologue of the agouti gene product and, when overexpressed, promotes obesity. Like neuropeptide Y (NPY) messenger RNA (mRNA), AGRP mRNA is produced in the hypothalamic arcuate nucleus and is elevated in leptin-deficient ob/ob and leptin-resistant db/db mice. These data suggest that AGRP mRNA might be affected by leptin and nutritional status in parallel with NPY mRNA. To test this hypothesis, we examined if AGRP mRNA would be, like NPY mRNA, inhibited by leptin injections and stimulated by fasting. AGRP mRNA was elevated in ob/ob mice about 5-fold compared with wild-type controls and was significantly inhibited by leptin treatment, as assessed by Northern blot analysis. In wild-type mice, AGRP mRNA was increased at least 13-fold by a 2-day fast, as assessed both by Northern blot analysis and in situ hybridization. In ad lib fed db/db mice, AGRP mRNA was elevated about 8-fold compared with ad lib fed wild-type controls, and was further increased by fasting in db/db mice. These data suggest that AGRP mRNA and NPY mRNA respond similarly to leptin and fasting.

	Introduction

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IMPAIRED production (1, 2, 3), processing (4), or sensitivity (5, 6) to -MSH is associated with obesity. The physiological significance of these observations is supported by studies showing that -MSH agonists and antagonists can promote satiety and feeding, respectively (7, 8, 9). Furthermore, fasting decreases the hypothalamic expression of POMC (2, 3, 10, 11, 12), the gene which codes for -MSH. Recently, an endogenous antagonist to -MSH, called agouti-related protein (AGRP), was identified and found to be expressed in the hypothalamic arcuate nucleus (13, 14). AGRP is elevated in leptin-deficient ob/ob mice and leptin-insensitive db/db mice (14). Furthermore, overexpression of AGRP in transgenic mice produces obesity (13, 15). However, the regulation of AGRP messenger RNA (mRNA) under more physiological conditions has not yet been reported. Because AGRP promotes anabolic activity, we hypothesized that its regulation would be similar to the regulation of neuropeptide Y (NPY), which also promotes anabolic activity (16). NPY mRNA is inhibited by leptin in ob/ob mice (3, 17) and is stimulated by fasting in wild-type and db/db mice (3). Therefore, we hypothesized that hypothalamic AGRP mRNA would also be inhibited by leptin in ob/ob mice and elevated by fasting in wild-type and db/db mice. Such a result would be consistent with the hypothesis that hypothalamic AGRP plays a physiological role in the regulation of body weight.

	Materials and Methods

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Animals
Male C57Bl/6J, ob/ob (along with control C57Bl/6J), and db/db (along with control C57Bl/KsJ) mice were obtained at 2 months of age from The Jackson Laboratory (Bar Harbor, ME). Mice were individually housed with free access to feed and water under 12-h light, 12-h dark cycle (lights on at 0700 h). All studies had been approved by the appropriate Institutional Animal Review Board. To assess effects of leptin replacement, wild-type and ob/ob mice (n = 5–8 per group) were injected with saline or leptin (1.5 µg/g body wt, ip, twice daily) for 5 days. On day 5, mice were injected in the morning, fasted during the day, and killed just before lights out under anesthesia by CO₂ narcosis. Hypothalamic blocks were removed, frozen on dry ice, and stored at -70 C until RNA extraction and subsequent analysis by Northern blot. To assess effects of fasting in wild-type mice, C57Bl/6J mice (n = 8–9 per group) were fed ad libitum or fasted for 48 h, killed just before lights out, and AGRP mRNA was assessed by Northern blot analysis. To assess effects of fasting in wild-type and db/db mice (n = 4–8 per group), wild-type and db/db mice were fasted either for 48 h or just during the day, as previously described (3). Brain blocks were removed, frozen on dry ice, and stored at -70 C until sectioning by a cryostat for analysis by in situ hybridization. The data for hypothalamic POMC mRNA, NPY mRNA, melanin-concentrating hormone (MCH) mRNA, and leptin mRNA in these mice is reported elsewhere (3).

AGRP probe synthesis
AGRP template was prepared from mouse hypothalamic RNA by RT-PCR with N-terminal primer: 5'-TGACTGCAATGTTGCTGAGTTGTG-3' and C-terminal primer: 5'-TAGGTGCGACTACAGAGGTTCGTG-3'. The amplified fragments were gel-purified, diluted to 50 ng/µl, and stored at -20 C. Single-stranded internally labeled complementary DNA probes (³²P) were produced by amplified primer extension labeling by using only C-terminal primer, using methods as previously described (18).

Northern blot analysis
Total RNA from mediobasal hypothalamus and adipose tissue from gonadal fat pad was extracted in TRIzol (Gibco BRL, Gaithersburg, MD). Three and 7 µg of total RNA were subjected to Northern blot analysis to detect hypothalamic AGRP mRNA and adipose leptin mRNA, respectively. Northern blot analysis was performed as described previously (18). RNA was quantified from samples from individual mice; thus, the analysis entailed no pooling at any time. After AGRP mRNA bands were quantified, membranes were stripped by boiling and hybridized with a ³²P-labeled probe encoding 18S ribosomal RNA. Quantification involved dividing the intensity of the AGRP mRNA band from each individual by the intensity of the signal from the 18S band from that individual, then expressing the means in each study as a percentage of normal controls (fed and/or wild-type).

In situ hybridization
To prepare for in situ hybridization analysis, frozen coronal sections (10 µm thick) through the mouse hypothalamus were cut, fixed in 3% paraformaldehyde in 0.1 M phosphate buffer (pH 7.0) containing 0.03% diethyl pyrocarbonate, dehydrated, and stored at -20 C until use. Sections were processed for in situ hybridization as described previously (3).

Data analysis
The total integrated densities of hybridization signals were determined by computerized densitometric scanning (to quantify in situ hybridization: MCID System, St. Catherine’s, Ontario, Canada) or phosphoimager (to quantify Northern blots: STORM 860, Molecular Dynamics, Inc., Sunnyvale, CA). Statistical analysis was performed by one-way or two-way ANOVA followed by Tukey-Kramer pair-wise comparison, or linear regression analysis.

	Results

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Hypothalamic AGRP mRNA, as assessed by Northern blot analysis, was about 5-fold higher in ob/ob mice than in wild-type controls, normalized to 18S ribosomal RNA (P < 0.05, Fig. 1); the 18S ribosomal band was not itself influenced by genotype (Fig. 1B). The replacement of leptin in ob/ob mice significantly decreased body weight compared with saline-injected ob/ob mice. Changes in body weight during 5-day leptin treatment were +0.9 ± 0.3 g and -4.2 ± 0.5 g in saline and leptin-injected group, respectively (P < 0.05). After leptin injection, AGRP mRNA decreased about 35% in ob/ob mice (P < 0.05, Fig. 1C). In the same experiment, POMC and NPY mRNA were also partially corrected by leptin replacement, as described elsewhere (3). In ob/ob mice, AGRP mRNA was positively correlated with NPY mRNA; this correlation was significant in ob/ob mice injected with saline as well as in ob/ob mice injected with leptin (Fig. 2).

View larger version (39K): [in this window] [in a new window]   Figure 1. Effect of ob/ob genotype and leptin replacement on hypothalamic AGRP mRNA (A) and 18S rRNA (B), as seen on a representative Northern blot. Total RNA (3 µg) from three different individual mice (not pooled) in each group were loaded into each lane. Lanes 1–3, Individual wild-type mice injected with saline. Lanes 4–6, Individual ob/ob mice injected with saline. Lanes 7–9, Individual ob/ob mice injected with leptin (1.5 µg/g body wt, ip, twice per day) for 5 days. C, Quantification of AGRP mRNA levels of all individuals (n = 5–8/group; mean ± SE). Levels for each individual were quantified by dividing the intensity of the AGRP mRNA band by the intensity of the 18S rRNA band for that individual. Means were expressed as percentage of wild-type controls. Groups with different letters are statistically different (P < 0.05).

View larger version (18K): [in this window] [in a new window]   Figure 2. Correlations between AGRP and NPY mRNA in ob/ob mice injected with saline (r = 0.94, P < 0.0005, A) and leptin (r = 0.82, P < 0.05, B). The correlation between AGRP and NPY mRNA in wild-type mice did not achieve statistical significance (r = 0.85, P = 0.07).

View larger version (33K): [in this window] [in a new window]   Figure 3. Effect of fasting on hypothalamic AGRP mRNA (A) and 18S rRNA (B), as observed on a representative Northern blot. Total RNA (3 µg) from three different individual mice (not pooled) in each group were loaded into each lane. Lanes 1–3, Mice fed ab libitum. Lanes 4–6, Mice fasted for 48 h. C, Quantification of AGRP mRNA levels of all individuals (n = 5–8/group; mean ± SE). Levels for each individual were quantified by dividing the intensity of the AGRP mRNA band by the intensity of the 18S rRNA band for that individual. Means were expressed as percentage of fed controls. *, Significant difference from fed mice (P < 0.05).

View larger version (32K): [in this window] [in a new window]   Figure 4. Effect of fasting and db/db genotype on hypothalamic AGRP mRNA as assessed by in situ hybridization. AGRP mRNA levels (mean ± SE, n = 4–8/group) were expressed as percentage of fed, wild-type controls. Groups with different letters are statistically different (P < 0.05).

	Discussion

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However, the reduction of leptin is unlikely to be the sole mechanism by which AGRP mRNA is increased during fasting, because AGRP mRNA was also elevated by fasting in db/db mice, which, due to a mutation in the leptin receptor (23), are resistant to the effects of leptin (24). NPY mRNA, like AGRP mRNA, is also increased by fasting in db/db mice (3). Infusion of insulin directly into the brain inhibits NPY mRNA (25), and fasting decreases both insulin and glucose (18). Therefore, it has been hypothesized that part of the mechanism by which fasting increases NPY mRNA is through a reduction in plasma insulin that accompanies fasting. Furthermore, fasting stimulates NPY mRNA in diabetic rats, even though insulin is not decreased by fasting in these rats (26). These results led to the suggestion that both insulin and glucose have independent inhibitory effects on NPY mRNA, and fasting leads to increased NPY mRNA in part by reducing both insulin and glucose (26). Taken together, these data suggest that reduction of leptin, insulin, and glucose with fasting may all contribute to the mechanism by which AGRP mRNA increases during fasting.

The present studies, demonstrating that hypothalamic AGRP mRNA is elevated in the fasting state, support the hypothesis (14) that the AGRP protein product may play a role in the physiological regulation of body weight. AGRP was identified as a homologous protein of the agouti gene product, and AGRP acts as an antagonist for MC4-R and MC3-R receptors (13, 14, 27). Impairments in these receptor systems produce obese phenotypes (5, 6). An overexpression of the AGRP gene in mice leads to an obese phenotype similar to that observed in agouti mice (13, 15). Taken together with the present studies, these results suggest that AGRP exerts a physiologically significant chronic anabolic effect that contributes to the full expression of the fasting-induced phenotype. Conversely, expression of POMC, the gene coding for -MSH, is reduced by fasting (2, 3). These results suggest that AGRP and -MSH serve as opposing elements in the regulation of body weight. A plausible mechanism would involve antagonism by AGRP of the catabolic action of -MSH acting through the MC4-R or MC3-R receptors because these receptors are expressed in hypothalamic sites consistent with a metabolic role (28). Such a mechanism would suggest that AGRP, acting through melanocortinergic receptors, serves to enhance body weight, and -MSH similarly serves to decrease body weight. The function of MCH may be similar to the function of AGRP (29). Nevertheless, MCH has not been demonstrated to act through known melanocortinergic receptors, and further work will be necessary to demonstrate the receptors and physiological context by which these peptides may interact.

Although the present study demonstrated that AGRP is inhibited by leptin, the physiological significance of this observation remains to be determined. Leptin also inhibits NPY mRNA, but mice deficient for NPY gene show normal metabolic responses to leptin (30). Agouti mice usually exhibit elevated expression of leptin (18) and are resistant to effects of leptin (31). Nevertheless, agouti mice lacking a functional leptin gene are fully responsive to leptin, and the agouti and obese alleles act additively to produce obesity and hyperinsulinemia (32). Thus, to the extent that AGRP and the agouti gene act through similar mechanisms, these data suggest that the physiological effects of leptin on body weight may be largely independent of both NPY and AGRP. Whether the effects of insulin and/or glucose on body weight are independent of NPY and the melanocortin pathway remains to be assessed. Nevertheless, current evidence supports the hypothesis that NPY and AGRP exert similar anabolic effects and are responsive to similar physiological signals reflecting nutritional status.

	Acknowledgments

 
We thank Dr. Hugo Bergen and Dr. James Roberts for useful discussions, and Steven Kleopoulos for help with the in situ hybridization.

	Footnotes

 
¹ This work was supported by a grant from the National Institutes of Health DK-50110–01.

Received June 10, 1998.

	References

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				E. Keen-Rhinehart and T. J. Bartness Peripheral ghrelin injections stimulate food intake, foraging, and food hoarding in Siberian hamsters Am J Physiol Regulatory Integrative Comp Physiol, March 1, 2005; 288(3): R716 - R722. [Abstract] [Full Text] [PDF]

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				S. G. Bouret and R. B. Simerly Minireview: Leptin and Development of Hypothalamic Feeding Circuits Endocrinology, June 1, 2004; 145(6): 2621 - 2626. [Abstract] [Full Text] [PDF]

				K. L.J. Ellacott and R. D. Cone The Central Melanocortin System and the Integration of Short- and Long-term Regulators of Energy Homeostasis Recent Prog. Horm. Res., January 1, 2004; 59(1): 395 - 408. [Abstract] [Full Text]

				K. A. Takahashi, J. L. Smart, H. Liu, and R. D. Cone The Anorexigenic Fatty Acid Synthase Inhibitor, C75, Is a Nonspecific Neuronal Activator Endocrinology, January 1, 2004; 145(1): 184 - 193. [Abstract] [Full Text] [PDF]

				T. M. Mizuno, K. A. Kelley, G. M. Pasinetti, J. L. Roberts, and C. V. Mobbs Transgenic Neuronal Expression of Proopiomelanocortin Attenuates Hyperphagic Response to Fasting and Reverses Metabolic Impairments in Leptin-Deficient Obese Mice Diabetes, November 1, 2003; 52(11): 2675 - 2683. [Abstract] [Full Text] [PDF]

				K. R. Hansen, S. M. Krasnow, M. A. Nolan, G. S. Fraley, J. W. Baumgartner, D. K. Clifton, and R. A. Steiner Activation of the Sympathetic Nervous System by Galanin-Like Peptide--A Possible Link between Leptin and Metabolism Endocrinology, November 1, 2003; 144(11): 4709 - 4717. [Abstract] [Full Text] [PDF]

				J. M. Cerda-Reverter and R. E. Peter Endogenous Melanocortin Antagonist in Fish: Structure, Brain Mapping, and Regulation by Fasting of the Goldfish Agouti-Related Protein Gene Endocrinology, October 1, 2003; 144(10): 4552 - 4561. [Abstract] [Full Text] [PDF]

				M. R. Ricci and B. E. Levin Ontogeny of diet-induced obesity in selectively bred Sprague-Dawley rats Am J Physiol Regulatory Integrative Comp Physiol, September 1, 2003; 285(3): R610 - R618. [Abstract] [Full Text] [PDF]

				G. Segal-Lieberman, R. L. Bradley, E. Kokkotou, M. Carlson, D. J. Trombly, X. Wang, S. Bates, M. G. Myers Jr., J. S. Flier, and E. Maratos-Flier Melanin-concentrating hormone is a critical mediator of the leptin-deficient phenotype PNAS, August 19, 2003; 100(17): 10085 - 10090. [Abstract] [Full Text] [PDF]

				G. Li, C. V. Mobbs, and P. J. Scarpace Central Pro-opiomelanocortin Gene Delivery Results in Hypophagia, Reduced Visceral Adiposity, and Improved Insulin Sensitivity in Genetically Obese Zucker Rats Diabetes, August 1, 2003; 52(8): 1951 - 1957. [Abstract] [Full Text] [PDF]

				M. A. COWLEY, R. D. CONE, P. ENRIORI, I. LOUISELLE, S. M. WILLIAMS, and A. E. EVANS Electrophysiological Actions of Peripheral Hormones on Melanocortin Neurons Ann. N.Y. Acad. Sci., June 1, 2003; 994(1): 175 - 186. [Abstract] [Full Text] [PDF]

S. M. Krasnow, G. S. Fraley, S. M. Schuh, J. W. Baumgartner, D. K. Clifton, and R. A. Steiner A Role for Galanin-Like Peptide in the Integration of Feeding, Body Weight Regulation, and Reproduction in the Mouse Endocrinology, March 1, 2003; 144(3): 813 - 822. [Abstract] [Full Text] [PDF]