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Molecular and cellular correlates of the developmental acquisition of mechanisms modulating ingestive behavior
Physiology & Behavior 82 (2004) 145 – 147
Molecular and cellular correlates of the developmental acquisition of mechanisms modulating ingestive behavior Streamson C. Chua Jr.* Division of Molecular Genetics, Department of Pediatrics, Columbia University, 1150 St. Nicholas Avenue, New York, NY 10032, USA Received 13 January 2004; accepted 2 April 2004
Abstract Postnatal development in most mammals is accompanied by the acquisition of controls of ingestion. In rodents, the initial and default controller appears to be gastric stretch. In the second week of life, rat pups acquire the ability to sense the presence of nutrients within the gut and appropriately modulate ingestion. In the third week of life, rat pups start to become weaned from the dam’s milk and begin independent ingestion. There have been strong indications that neuropeptide Y is a stimulator of ingestion in adults, although there was very little information in pups. Dr. Gerard Smith initiated a series of studies that provide strong evidence to indicate that hypothalamic neuropeptide Y (NPY) neurons are strong candidates for providing the ability of preweaning rat pups to modulate ingestion according to caloric intake. Moreover, the studies also suggest that the overactivity of hypothalamic NPY neurons presage the onset of hyperphagia in syndromes associated with defects in leptin signaling. D 2004 Elsevier Inc. All rights reserved. Keywords: Development; Ingestion; Neuropeptide Y; Nutrient sensing
1. Introduction This is a tribute to the outstanding qualities of Gerard P. Smith as a mentor and a collaborator, written from the point of view of a molecular biologist. Gerry is a fountain of knowledge for much of the past history regarding studies of appetitive behavior. His internal databank is tremendously enhanced by strong opinions based on broad, integrated views of the field. Notwithstanding his own strong opinions, Gerry always urged me to do my own reading with some pointers to the relevant reports so that I could formulate my own understanding about a given area. All of these points have made me place a great degree of significance to Gerry’s opinions, and he has served as a test platform for some of my ideas and projects.
2. The search for a stimulator of ingestion during development Gerry has been a wonderful person to have as a collaborator, especially on projects that have stimulated both of * Tel.: +1-212-851-5314; fax: +1-212-851-5306. E-mail address: [email protected] (S.C. Chua Jr.). 0031-9384/$ – see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.physbeh.2004.04.040
our interests. One of the fascinating aspects of ingestive behavior is the gradual acquisition of the controls of ingestion and its neuronal and molecular correlates. Gerry pointed out (to me) fascinating studies about the gradual accumulation of regulatory signals in developing rodents. Early studies had shown that rat pups are controlled by gastric fill in the first week of life and that the regulation of ingestion by nutrients began to develop in the second week of life. Gerry enlisted Timothy Kowalski, then a post doc at the Bourne Laboratory, to examine the possibility that hypothalamic neurons that use neuropeptide Y (NPY) as a neurotransmitter could play a role in this developmental program. Because Gerry had been working on cholecystokinin as a negative modulator, it was natural that he would seek out a positive stimulator of ingestion. This project resulted in a series of reports that provided supportive evidence that NPYergic neurons within the arcuate nucleus of the hypothalamus could play an important role [1– 5], summarized in Fig. 1. First was the demonstration that hypothalamic NPY neurons responded to food deprivation of rat pups as early as Postnatal Day 2 by increasing NPY mRNA concentrations. This observation was concordant with many prior studies that showed that NPY mRNA was increased in many states of negative energy balance in adult animals [6,7]. Furthermore, this observation could be inter-
146
S.C. Chua Jr. / Physiology & Behavior 82 (2004) 145–147
Fig. 1. Developmental program of the acquisition of controls of ingestion. The first line shows the major developmental stages at Weeks 1 (ingestion controlled mainly by gastric fill), 2 (acquisition of nutrient sensing), and 3 (development of independent ingestion). Shading increases with the gradual overlay of more regulatory modes. The second bar shows that arcuate NPY mRNA is regulated by deprivation as early as Postnatal Day 2. The third bar shows the regulation of NPY expression by leptin, as revealed by analysis of LEPR-deficient rats. The fourth bar shows the development of hyperphagia in LEPR-deficient rats. At Postnatal Days 12 and 15, fa/fa pups show hyperphagia, while 18-day fa/fa pups show hyperphagia with decreased latency to feeding.
preted by extending the concept of stimulation –secretion coupling in neurons to engage neurotransmitter synthesis. One would expect that sustained release of neuropeptides would require the ongoing synthesis of the necessary precursors, neuropeptide mRNA and the preproneuropeptide. This was followed by the observation that the ingestion and infusion of milk (but not water) reversed the increase in hypothalamic NPY mRNA as early as Postnatal Day 2. These findings suggested that arcuate NPY neurons could respond to ingested nutrients. The regulation of the NPY neuron as early as Postnatal Day 2 would certainly be a strong qualification for an important regulator of ingestive behavior.
3. Development of hyperphagia in a model of genetic obesity dependent upon NPY overexpression To extend these findings, Gerry and Tim wanted to utilize a model that had been shown to be reliant upon increased NPY signaling to produce hyperphagia and obesity. They resorted to using rat pups that were deficient in the leptin receptor. These rats carried the original fatty mutation, which is a point mutation that alters the normal glutamine at Position 269 to a proline [8,9]. This amino acid substitution alters the conformation of the mutant receptor such that it is unable to be transported to the cell surface, effectively producing an LEPR-null phenotype for cells homozygous for the fatty mutation. We, and many other groups, had shown that NPY mRNA was elevated in LEPR-deficient mice and rats, as adults [7,10]. Moreover, obese leptin-null mice were less hyperphagic and less obese when made deficient for NPY by the introduction of an Npy knockout allele [11]. There-
fore, the stage was set with a model of obesity that was dependent upon the NPY overexpression for the obese phenotype. We showed that LEPR-deficient pups had elevated concentrations of arcuate nucleus NPY mRNA by Postnatal Day 5. This increase in Npy gene expression was observed prior to the onset of hyperphagia in LEPRnull pups at Postnatal Day 12, consonant with the initial premise that the regulation of NPY expression is a critical component in the acquisition of nutritive regulation of ingestion. However, these results have to be tempered by the following observations: (a) outbred adult mice homozygous for a knockout allele of the Npy gene apparently have normal ingestive behavior [12]; (b) inbred C57BL/6J mice without any NPY are mildly obese and show deficits in deprivation-induced hyperphagia [13]; and (c) mice homozygous for knockout alleles of the various Npy receptor genes (Y1 [14,15], Y2 [16 –19], and Y4 [20]) exhibit mild obesity. Much further work will probably be necessary to fully resolve the functions of hypothalamic NPY neurons. Nevertheless, the work done by Gerry has laid a foundation and a model for the molecular analysis of the developmental program for ingestive behavior control mechanisms. The prospect of correlating molecular and cellular mechanims to the developmental controls of feeding looks highly promising at the current time. Experimental manipulations provided by genetic technologies, such as transgenesis and viral vectors and single-cell and single-channel recordings, permit a wide window into the development of the neural network that controls and modulates behavior. A newer mode of investigation, one based on chemically identified neurons and their synaptic inputs and outputs, will add greatly to the investigations based on whole organismal experimentation.
S.C. Chua Jr. / Physiology & Behavior 82 (2004) 145–147
Finally, Gerry has been enormously generous in his support of junior and senior investigators, both for scientists within the Bourne Laboratory as well as scientists outside of his immediate influence. I was amazed at the continuous stream of visiting investigators that was hosted by Gerry and Jim Gibbs, Gerry’s long-time partner in science. The collegial atmosphere at the Bourne Laboratory was engendered by benevolence coupled to a spirited but objectively based search for scientific truth, all bound together by scientific integrity.
References [1] Kowalski TJ, Ster AM, Smith GP. Increased hypothalamic neuropeptide Y expression in deprived preweanling rats is reversed by intragastric infusion of milk. Physiol Behav 2002;75(3):425 – 32. [2] Kowalski TJ, Houpt TA, Jahng J, Okada N, Liu SM, Chua Jr, SC, et al. Neuropeptide Y overexpression in the preweanling Zucker (fa/ fa) rat. Physiol Behav 1999;67(4):521 – 5. [3] Kowalski TJ, Ster AM, Smith GP. Ontogeny of hyperphagia in the Zucker (fa/fa) rat. Am J Physiol 1998;275(4 Pt 2):R1106 – 9. [4] Kowalski TJ, Houpt TA, Jahng J, Okada N, Chua SC, Smith GP. Ontogeny of neuropeptide Y expression in response to deprivation in lean Zucker rat pups. Am J Physiol 1998;275(2 Pt 2):R466 – 70. [5] Ster AM, Kowalski TJ, Dube MG, Kalra SP, Smith GP. Decreased hypothalamic concentration of neuropeptide Y correlates with onset of hyperphagia in fa/fa rats on postnatal day 12. Physiol Behav 2003;78(4 – 5):517 – 20. [6] Chua Jr, SC, Leibel RL, Hirsch J. Food deprivation and age modulate neuropeptide gene expression in the murine hypothalamus and adrenal gland. Brain Res Mol Brain Res 1991;9(1 – 2):95 – 101. [7] Chua Jr, SC, Brown AW, Kim J, Hennessey KL, Leibel RL, Hirsch J. Food deprivation and hypothalamic neuropeptide gene expression: Effects of strain background and the diabetes mutation. Brain Res Mol Brain Res 1991;11(3 – 4):291 – 9. [8] Chua Jr SC, White DW, Wu-Peng XS, Liu SM, Okada N, Kershaw EE, et al. Phenotype of fatty due to Gln269Pro mutation in the leptin receptor (Lepr). Diabetes 1996;45(8):1141 – 3.
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[9] White DW, Wang DW, Chua Jr, SC, Morgenstern JP, Leibel RL, Baumann H, et al. Constitutive and impaired signaling of leptin receptors containing the Gln ! Pro extracellular domain fatty mutation. Proc Natl Acad Sci U S A 1997;94(20):10657 – 62. [10] Jhanwar-Uniyal M, Chua SC. Critical effects of aging and nutritional state on hypothalamic neuropeptide Y and galanin gene expression in lean and genetically obese Zucker rats. Brain Res Mol Brain Res 1993;19(3):195 – 202. [11] Erickson JC, Hollopeter G, Palmiter RD. Attenuation of the obesity syndrome of ob/ob mice by the loss of neuropeptide Y. Science 1996;274(5293):1704 – 7. [12] Erickson JC, Clegg KE, Palmiter RD. Sensitivity to leptin and susceptibility to seizures of mice lacking neuropeptide Y. Nature 1996; 381(6581):415 – 21. [13] Segal-Lieberman G, Trombly DJ, Juthani V, Wang X, Maratos-Flier E. NPYablation in C57BL/6 mice leads to mild obesity and to an impaired refeeding response to fasting. Am J Physiol Endocrinol Metabol 2003;284(6):E1131 – 9. [14] Kushi A, Sasai H, Koizumi H, Takeda N, Yokoyama M, Nakamura M. Obesity and mild hyperinsulinemia found in neuropeptide Y-Y1 receptor-deficient mice. Proc Natl Acad Sci U S A 1998;95(26): 15659 – 64. [15] Pralong FP, Gonzales C, Voirol MJ, Palmiter RD, Brunner HR, Gaillard RC, et al. The neuropeptide Y Y1 receptor regulates leptin-mediated control of energy homeostasis and reproductive functions. FASEB J 2002;16(7):712 – 4. [16] Lee EW, Grant DS, Movafagh S, Zukowska Z. Impaired angiogenesis in neuropeptide Y (NPY)-Y2 receptor knockout mice. Peptides 2003;24(1):99 – 106. [17] Sainsbury A, Schwarzer C, Couzens M, Herzog H. Y2 receptor deletion attenuates the type 2 diabetic syndrome of ob/ob mice. Diabetes 2002;51(12):3420 – 7. [18] Sainsbury A, Schwarzer C, Couzens M, Fetissov S, Furtinger S, Jenkins A, et al. Important role of hypothalamic Y2 receptors in body weight regulation revealed in conditional knockout mice. Proc Natl Acad Sci U S A 2002;99(13):8938 – 43. [19] Baldock PA, Sainsbury A, Couzens M, Enriquez RF, Thomas GP, Gardiner EM, et al. Hypothalamic Y2 receptors regulate bone formation. J Clin Invest 2002;109(7):915 – 21. [20] Sainsbury A, Schwarzer C, Couzens M, Jenkins A, Oakes SR, Ormandy CJ, et al. Y4 receptor knockout rescues fertility in ob/ob mice. Genes Dev 2002;16(9):1077 – 88.
Molecular and cellular correlates of the developmental acquisition of mechanisms modulating ingestive behavior Streamson C. Chua Jr.* Division of Molecular Genetics, Department of Pediatrics, Columbia University, 1150 St. Nicholas Avenue, New York, NY 10032, USA Received 13 January 2004; accepted 2 April 2004
Abstract Postnatal development in most mammals is accompanied by the acquisition of controls of ingestion. In rodents, the initial and default controller appears to be gastric stretch. In the second week of life, rat pups acquire the ability to sense the presence of nutrients within the gut and appropriately modulate ingestion. In the third week of life, rat pups start to become weaned from the dam’s milk and begin independent ingestion. There have been strong indications that neuropeptide Y is a stimulator of ingestion in adults, although there was very little information in pups. Dr. Gerard Smith initiated a series of studies that provide strong evidence to indicate that hypothalamic neuropeptide Y (NPY) neurons are strong candidates for providing the ability of preweaning rat pups to modulate ingestion according to caloric intake. Moreover, the studies also suggest that the overactivity of hypothalamic NPY neurons presage the onset of hyperphagia in syndromes associated with defects in leptin signaling. D 2004 Elsevier Inc. All rights reserved. Keywords: Development; Ingestion; Neuropeptide Y; Nutrient sensing
1. Introduction This is a tribute to the outstanding qualities of Gerard P. Smith as a mentor and a collaborator, written from the point of view of a molecular biologist. Gerry is a fountain of knowledge for much of the past history regarding studies of appetitive behavior. His internal databank is tremendously enhanced by strong opinions based on broad, integrated views of the field. Notwithstanding his own strong opinions, Gerry always urged me to do my own reading with some pointers to the relevant reports so that I could formulate my own understanding about a given area. All of these points have made me place a great degree of significance to Gerry’s opinions, and he has served as a test platform for some of my ideas and projects.
2. The search for a stimulator of ingestion during development Gerry has been a wonderful person to have as a collaborator, especially on projects that have stimulated both of * Tel.: +1-212-851-5314; fax: +1-212-851-5306. E-mail address: [email protected] (S.C. Chua Jr.). 0031-9384/$ – see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.physbeh.2004.04.040
our interests. One of the fascinating aspects of ingestive behavior is the gradual acquisition of the controls of ingestion and its neuronal and molecular correlates. Gerry pointed out (to me) fascinating studies about the gradual accumulation of regulatory signals in developing rodents. Early studies had shown that rat pups are controlled by gastric fill in the first week of life and that the regulation of ingestion by nutrients began to develop in the second week of life. Gerry enlisted Timothy Kowalski, then a post doc at the Bourne Laboratory, to examine the possibility that hypothalamic neurons that use neuropeptide Y (NPY) as a neurotransmitter could play a role in this developmental program. Because Gerry had been working on cholecystokinin as a negative modulator, it was natural that he would seek out a positive stimulator of ingestion. This project resulted in a series of reports that provided supportive evidence that NPYergic neurons within the arcuate nucleus of the hypothalamus could play an important role [1– 5], summarized in Fig. 1. First was the demonstration that hypothalamic NPY neurons responded to food deprivation of rat pups as early as Postnatal Day 2 by increasing NPY mRNA concentrations. This observation was concordant with many prior studies that showed that NPY mRNA was increased in many states of negative energy balance in adult animals [6,7]. Furthermore, this observation could be inter-
146
S.C. Chua Jr. / Physiology & Behavior 82 (2004) 145–147
Fig. 1. Developmental program of the acquisition of controls of ingestion. The first line shows the major developmental stages at Weeks 1 (ingestion controlled mainly by gastric fill), 2 (acquisition of nutrient sensing), and 3 (development of independent ingestion). Shading increases with the gradual overlay of more regulatory modes. The second bar shows that arcuate NPY mRNA is regulated by deprivation as early as Postnatal Day 2. The third bar shows the regulation of NPY expression by leptin, as revealed by analysis of LEPR-deficient rats. The fourth bar shows the development of hyperphagia in LEPR-deficient rats. At Postnatal Days 12 and 15, fa/fa pups show hyperphagia, while 18-day fa/fa pups show hyperphagia with decreased latency to feeding.
preted by extending the concept of stimulation –secretion coupling in neurons to engage neurotransmitter synthesis. One would expect that sustained release of neuropeptides would require the ongoing synthesis of the necessary precursors, neuropeptide mRNA and the preproneuropeptide. This was followed by the observation that the ingestion and infusion of milk (but not water) reversed the increase in hypothalamic NPY mRNA as early as Postnatal Day 2. These findings suggested that arcuate NPY neurons could respond to ingested nutrients. The regulation of the NPY neuron as early as Postnatal Day 2 would certainly be a strong qualification for an important regulator of ingestive behavior.
3. Development of hyperphagia in a model of genetic obesity dependent upon NPY overexpression To extend these findings, Gerry and Tim wanted to utilize a model that had been shown to be reliant upon increased NPY signaling to produce hyperphagia and obesity. They resorted to using rat pups that were deficient in the leptin receptor. These rats carried the original fatty mutation, which is a point mutation that alters the normal glutamine at Position 269 to a proline [8,9]. This amino acid substitution alters the conformation of the mutant receptor such that it is unable to be transported to the cell surface, effectively producing an LEPR-null phenotype for cells homozygous for the fatty mutation. We, and many other groups, had shown that NPY mRNA was elevated in LEPR-deficient mice and rats, as adults [7,10]. Moreover, obese leptin-null mice were less hyperphagic and less obese when made deficient for NPY by the introduction of an Npy knockout allele [11]. There-
fore, the stage was set with a model of obesity that was dependent upon the NPY overexpression for the obese phenotype. We showed that LEPR-deficient pups had elevated concentrations of arcuate nucleus NPY mRNA by Postnatal Day 5. This increase in Npy gene expression was observed prior to the onset of hyperphagia in LEPRnull pups at Postnatal Day 12, consonant with the initial premise that the regulation of NPY expression is a critical component in the acquisition of nutritive regulation of ingestion. However, these results have to be tempered by the following observations: (a) outbred adult mice homozygous for a knockout allele of the Npy gene apparently have normal ingestive behavior [12]; (b) inbred C57BL/6J mice without any NPY are mildly obese and show deficits in deprivation-induced hyperphagia [13]; and (c) mice homozygous for knockout alleles of the various Npy receptor genes (Y1 [14,15], Y2 [16 –19], and Y4 [20]) exhibit mild obesity. Much further work will probably be necessary to fully resolve the functions of hypothalamic NPY neurons. Nevertheless, the work done by Gerry has laid a foundation and a model for the molecular analysis of the developmental program for ingestive behavior control mechanisms. The prospect of correlating molecular and cellular mechanims to the developmental controls of feeding looks highly promising at the current time. Experimental manipulations provided by genetic technologies, such as transgenesis and viral vectors and single-cell and single-channel recordings, permit a wide window into the development of the neural network that controls and modulates behavior. A newer mode of investigation, one based on chemically identified neurons and their synaptic inputs and outputs, will add greatly to the investigations based on whole organismal experimentation.
S.C. Chua Jr. / Physiology & Behavior 82 (2004) 145–147
Finally, Gerry has been enormously generous in his support of junior and senior investigators, both for scientists within the Bourne Laboratory as well as scientists outside of his immediate influence. I was amazed at the continuous stream of visiting investigators that was hosted by Gerry and Jim Gibbs, Gerry’s long-time partner in science. The collegial atmosphere at the Bourne Laboratory was engendered by benevolence coupled to a spirited but objectively based search for scientific truth, all bound together by scientific integrity.
References [1] Kowalski TJ, Ster AM, Smith GP. Increased hypothalamic neuropeptide Y expression in deprived preweanling rats is reversed by intragastric infusion of milk. Physiol Behav 2002;75(3):425 – 32. [2] Kowalski TJ, Houpt TA, Jahng J, Okada N, Liu SM, Chua Jr, SC, et al. Neuropeptide Y overexpression in the preweanling Zucker (fa/ fa) rat. Physiol Behav 1999;67(4):521 – 5. [3] Kowalski TJ, Ster AM, Smith GP. Ontogeny of hyperphagia in the Zucker (fa/fa) rat. Am J Physiol 1998;275(4 Pt 2):R1106 – 9. [4] Kowalski TJ, Houpt TA, Jahng J, Okada N, Chua SC, Smith GP. Ontogeny of neuropeptide Y expression in response to deprivation in lean Zucker rat pups. Am J Physiol 1998;275(2 Pt 2):R466 – 70. [5] Ster AM, Kowalski TJ, Dube MG, Kalra SP, Smith GP. Decreased hypothalamic concentration of neuropeptide Y correlates with onset of hyperphagia in fa/fa rats on postnatal day 12. Physiol Behav 2003;78(4 – 5):517 – 20. [6] Chua Jr, SC, Leibel RL, Hirsch J. Food deprivation and age modulate neuropeptide gene expression in the murine hypothalamus and adrenal gland. Brain Res Mol Brain Res 1991;9(1 – 2):95 – 101. [7] Chua Jr, SC, Brown AW, Kim J, Hennessey KL, Leibel RL, Hirsch J. Food deprivation and hypothalamic neuropeptide gene expression: Effects of strain background and the diabetes mutation. Brain Res Mol Brain Res 1991;11(3 – 4):291 – 9. [8] Chua Jr SC, White DW, Wu-Peng XS, Liu SM, Okada N, Kershaw EE, et al. Phenotype of fatty due to Gln269Pro mutation in the leptin receptor (Lepr). Diabetes 1996;45(8):1141 – 3.
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[9] White DW, Wang DW, Chua Jr, SC, Morgenstern JP, Leibel RL, Baumann H, et al. Constitutive and impaired signaling of leptin receptors containing the Gln ! Pro extracellular domain fatty mutation. Proc Natl Acad Sci U S A 1997;94(20):10657 – 62. [10] Jhanwar-Uniyal M, Chua SC. Critical effects of aging and nutritional state on hypothalamic neuropeptide Y and galanin gene expression in lean and genetically obese Zucker rats. Brain Res Mol Brain Res 1993;19(3):195 – 202. [11] Erickson JC, Hollopeter G, Palmiter RD. Attenuation of the obesity syndrome of ob/ob mice by the loss of neuropeptide Y. Science 1996;274(5293):1704 – 7. [12] Erickson JC, Clegg KE, Palmiter RD. Sensitivity to leptin and susceptibility to seizures of mice lacking neuropeptide Y. Nature 1996; 381(6581):415 – 21. [13] Segal-Lieberman G, Trombly DJ, Juthani V, Wang X, Maratos-Flier E. NPYablation in C57BL/6 mice leads to mild obesity and to an impaired refeeding response to fasting. Am J Physiol Endocrinol Metabol 2003;284(6):E1131 – 9. [14] Kushi A, Sasai H, Koizumi H, Takeda N, Yokoyama M, Nakamura M. Obesity and mild hyperinsulinemia found in neuropeptide Y-Y1 receptor-deficient mice. Proc Natl Acad Sci U S A 1998;95(26): 15659 – 64. [15] Pralong FP, Gonzales C, Voirol MJ, Palmiter RD, Brunner HR, Gaillard RC, et al. The neuropeptide Y Y1 receptor regulates leptin-mediated control of energy homeostasis and reproductive functions. FASEB J 2002;16(7):712 – 4. [16] Lee EW, Grant DS, Movafagh S, Zukowska Z. Impaired angiogenesis in neuropeptide Y (NPY)-Y2 receptor knockout mice. Peptides 2003;24(1):99 – 106. [17] Sainsbury A, Schwarzer C, Couzens M, Herzog H. Y2 receptor deletion attenuates the type 2 diabetic syndrome of ob/ob mice. Diabetes 2002;51(12):3420 – 7. [18] Sainsbury A, Schwarzer C, Couzens M, Fetissov S, Furtinger S, Jenkins A, et al. Important role of hypothalamic Y2 receptors in body weight regulation revealed in conditional knockout mice. Proc Natl Acad Sci U S A 2002;99(13):8938 – 43. [19] Baldock PA, Sainsbury A, Couzens M, Enriquez RF, Thomas GP, Gardiner EM, et al. Hypothalamic Y2 receptors regulate bone formation. J Clin Invest 2002;109(7):915 – 21. [20] Sainsbury A, Schwarzer C, Couzens M, Jenkins A, Oakes SR, Ormandy CJ, et al. Y4 receptor knockout rescues fertility in ob/ob mice. Genes Dev 2002;16(9):1077 – 88.