Introduction

A^y/a mice have yellow hair, adult-onset obesity, hyperinsulinaemia and impaired glucose tolerance.¹ This phenotype is recapitulated by ectopic expression of agouti cDNA in transgenic mice.^{2, 3} Agouti protein is a high-affinity antagonist at the melanocortin receptor-1 (MC1-R) and MC4-R.⁴ Agouti protein antagonises MC1-Rs within the skin to produce yellow pigmentation, and in A^y/a mice, antagonises MC4-Rs within the central nervous system (CNS), causing hyperphagia and obesity.⁴ Consistent with this, MC4-R knockout mice exhibit a similar obesity syndrome, yet do not have yellow fur.⁵ Agouti-related protein (AgRP), an endogenous homologue of agouti protein, selectively antagonises MC3- and MC4-Rs.⁶ Overexpression of AgRP in transgenic mice produces an obese phenotype similar to that of MC4-R knockout or A^y/a mice.^{6, 7}

AgRP is coexpressed with neuropeptide Y (NPY), another orexigenic signal, in neurones of the hypothalamic arcuate nucleus.^{8, 9} Pro-opiomelanocortin (POMC), the precursor of the endogenous MC3- and MC4-R agonist alpha-melanocyte-stimulating hormone (-MSH), is expressed in a separate population of arcuate neurons.⁹ Leptin, the adipocyte-derived hormone, acts as a satiety factor to regulate feeding,¹⁰ and both arcuate POMC neurones¹¹ and AgRP neurones¹² express the active form of the leptin receptor, Ob-R mRNA. Leptin is thought to regulate energy balance by influencing these two neuronal populations. Obese A^y/a mice have reduced arcuate AgRP mRNA expression compared to pre-obese A^y/a mice.¹³ NPY mRNA expression in pre-obese A^y/a mice is restricted to the arcuate nucleus; however, in obese A^y/a mice, NPY mRNA is also expressed in the dorsomedial nucleus (DMN).¹⁴

Mature obese A^y/a mice are hyperleptinemic, due to an increased fat mass and are reported to be insensitive to exogenous, peripheral leptin.¹⁵ Furthermore, young, lean A^y/a mice, which have normal circulating leptin, are also resistant to exogenous leptin.¹⁶ Melanocortinergic blockade by agouti protein in these mice therefore appears to antagonise the CNS effects of leptin. However, these conclusions of leptin resistance in A^y/a mice are based on chronic studies of peripheral leptin administration, with daily measurements of body weight as the end point.^{15, 16} Detailed analyses of food intake might provide further evidence for this hypothesis.

Recently, gut hormones have been implicated in the regulation of energy balance. Ghrelin, produced by the stomach,¹⁷ has a potent orexigenic action when administered peripherally^{18, 19} or directly into the CNS.^{18, 20, 21, 22} Hypothalamic NPY and AgRP may be important in mediating the orexigenic effects of ghrelin. Ghrelin stimulates NPY release from hypothalamic explants in vitro²³ and co-administration of a Y1 or Y5 NPY receptor antagonist attenuates the orexigenic effect of centrally injected ghrelin.²¹ Similarly, co-administration of anti-AgRP IgG and ghrelin into the CNS diminishes ghrelin's orexigenic effects.²¹

PYY_3–36 is produced by the endocrine cells of the gastrointestinal mucosa.²⁴ Recently, we have demonstrated that administration of PYY_3–36 both peripherally and into the arcuate nucleus inhibits food intake in rodents by modulating NPY and melanocortin systems within the arcuate nucleus.²⁵ PYY_3–36 inhibits NPY/AgRP neurones via arcuate Y2 receptors, subsequently disinhibiting POMC neurones to cause a reduction in food intake.²⁵

Since ghrelin increases feeding via upregulation of hypothalamic NPY and AgRP, we hypothesised that in obese A^y/a mice, with altered hypothalamic expression of these neuropeptides, peripheral ghrelin would produce different feeding responses compared to wild-type (WT) controls. Similarly, altered levels of hypothalamic NPY and AgRP mRNA in obese A^y/a mice could result in resistance to peripheral PYY_3–36. Conversely, in pre-obese A^y/a mice, with normal hypothalamic NPY and AgRP gene expression, ghrelin or PYY_3–36 administration should produce similar feeding responses to age-matched WT mice. Therefore, we examined the effects of peripheral injection of PYY_3–36 and ghrelin on feeding in adult pre-obese (7–8 weeks) and obese (14–15 weeks) A^y/a mice. To examine whether the ectopic expression of agouti protein in A^y/a mice results in complete MC4-R inhibition in vivo, as well as in vitro,⁴ we also examined the effects of -MSH and leptin on food intake in pre-obese and obese A^y/a mice.

Methods

Materials

The following compounds were used: NDP--MSH, human PYY_3–36 and human ghrelin (all BACHEM (UK) Ltd.), and purified recombinant mouse leptin (R & D Systems, MN, USA).

Animals

C57BL/6J (WT) and C57BL/6J-A^y (A^y/a) mice were purchased from Jackson Laboratories, ME, USA. A breeding colony was established and all mice used in these studies were littermates derived from this colony. Mice were maintained in individual cages under controlled temperature (21–23°C) and light conditions (lights on 07:00–19:00 h) with ad libitum access to food (RM3 diet, SDS UK Ltd.) and water unless otherwise stated. Animal procedures were approved by the British Home Office Animals (Scientific Procedures) Act, 1986.

Feeding studies

Prior to feeding studies, mice received a sham intraperitoneal (i.p.) injection of 100 l phosphate-buffered saline (PBS) to acclimatise them to the procedure. Experiments were separated by at least 3 days. All peptides were diluted in PBS (10 mM, pH 7.0) to concentrations that allowed delivery of the dose in 100 l. Mice were fasted for 24 h prior to administration of anorectic peptides (-MSH, PYY_3–36 and leptin). Ghrelin was administered in the non-fasted state. All injections were administered i.p. and given in the early light-phase (08:00–09:00 h). Doses were chosen using calculations based on previously published studies: NDP--MSH; 0.2 mol/kg body weight,²⁶ PYY_3–36; 0.02 mol/kg body weight,²⁵ leptin; 2 mol/kg body weight²⁷ and ghrelin 0.2 mol/kg body weight.¹⁸ Following injection, each mouse was returned to its home cage with a pre-weighed amount of chow. Food intake was measured at 1, 2, 4, 8 and 24 h post-injection.

For feeding studies, A^y/a mice were studied at two different time points; firstly, in early adulthood (pre-obese cohort; 7–8 weeks) and secondly, aged 14–15 weeks (obese cohort). Control age-matched WT littermates were also studied at these time points.

Plasma leptin, ghrelin and PYY measurement

Two cohorts of non-fasted A^y/a and WT mice were studied, aged 7–8 weeks (pre-obese cohort) and 14–15 weeks (obese cohort), respectively. Mice were killed in the early-light-phase by inhalation of 99% CO₂ until respiration had just ceased. Whole blood was collected by cardiac puncture and mixed with 0.06 mg aprotinin (Bayer, Haywards Heath, UK) to prevent peptide degradation. Plasma was separated by centrifugation, frozen and stored at -20°C until radioimmunoassay (RIA).

All samples were assayed in duplicate. Mouse plasma leptin immunoreactivity (IR) was measured in 50 l samples using a commercial RIA according to the manufacturer's instructions (Linco Research, St Charles, MO, USA). Ghrelin-like IR was measured with a specific and sensitive RIA. The antibody used (SC-10368 G102 Santa Cruz, CA, USA) recognises both octanoyl and des octanoyl ghrelin and does not crossreact with any known gastrointestinal or pancreatic peptide hormones. The antibody was used at a final dilution of 1:50 000. The ¹²⁵I-labelled ghrelin was prepared using Bolton and Hunter reagent²⁸ (Amersham International, UK) and purified by reversed phase-high pressure liquid chromotography (RP-HPLC) using a linear gradient from 10 to 40% acetonitrile, 0.05% TFA over 90 min. The specific activity of ghrelin label was 48 Bq/fmol. The assay was performed in a total volume of 0.7 ml of 0.06 M phosphate buffer (pH=7.2) containing 0.3% BSA and incubated for 3 days at 4°C before separation of antibody-bound label by dextran-coated charcoal. The assay detected changes of 25 pmol/l of plasma ghrelin with 95% confidence limit. The intra-assay coefficient of variation (at 5 fmol addition) was 10.5%. PYY-like IR was measured using a specific and sensitive RIA, as previously described.²⁹

Statistical analysis

Results are given as means.e.m. For feeding studies, comparisons were made between groups using one-way ANOVA with post hoc Fisher's Least Significant Difference method (Systat, Evanston, IL, USA) (Studies 2–5) or an unpaired Student's two-tailed t-test (Study 1 and Study 6). The value of P<0.05 was taken as the level of significance.

Results

Study 1. Effects of fasting on food intake in pre-obese and obese A^y/a mice

Pre-obese A^y/a and WT litter mates were of similar body weight (19.30.8 (pre-obese A^y/a) vs 18.00.3 g (WT), n=11–12 per group). In the mature obese cohort, A^y/a mice were significantly heavier than age-matched WT controls (32.61.2 (obese A^y/a) vs 23.60.3 g (WT), P<0.0001, n=11–12 per group).

To study the effects of fasting in A^y/a mice, a 24 h fast and subsequent refeed for 24 h, without peptide administration, was performed. The refeeding profile of pre-obese A^y/a mice following a 24 h fast was similar to that of WT littermates (Figure 1a). In contrast, 24 h-fasted obese A^y/a and WT mice exhibited very different refeeding profiles. Fasted, obese A^y/a mice ate 27% less than WT mice for the first 8 h after reintroduction of food (Figure 1b). The effects of this fast remained evident at the end of the dark phase, 24 h after the reintroduction of food, with obese A^y/a mice consuming 13% less food than WT mice (Figure 1b). This altered refeeding profile in obese A^y/a mice following fasting is important to consider when interpreting the effects of anorectic peptides (PYY_3–36, -MSH and leptin) on food intake in obese A^y/a mice, since these peptides were administered in the fasted state.

Figure 1.

Study 1. Effects of fasting on food intake in pre-obese and obese A^y/a mice. Cumulative food intake in (a) pre-obese and (b) obese A^y/a mice (n=11–12 per group) compared to age-matched WT mice following a 24 h fast. ^**P<0.005 and ^***P<0.0005 vs WT mice.

Full figure and legend (22K)

Study 2. Effects of PYY_3–36 on food intake in pre-obese and obese A^y/a mice

PYY_3–36 suppressed food intake 2 h post-injection in fasted pre-obese A^y/a mice and WT controls (Figure 2a). At 24 h post-injection, there was no difference in food intake between all four groups. PYY_3–36 also reduced 2 h food intake in fasted, obese A^y/a mice and WT mice (Figure 2b). PYY_3–36 continued to inhibit feeding in obese A^y/a mice up to 24 h post-injection (obese A^y/a mice (0–24 h): 5.70.3 (PYY_3–36) vs 6.60.1 g (saline), P<0.0005). Consistent with Study 1, fasted obese A^y/a controls receiving an ip saline injection had significantly reduced food intake at 24 h compared to saline-injected WT controls ((0–24 h): 6.60.1 (obese A^y/a mice) vs 7.10.1 g (WT mice), P<0.0005).

Figure 2.

Study 2. Effects of peripheral PYY_3–36 (0.02 mol/kg body weight) on 0–2 h food intake in (a) pre-obese (n=5–6 per group) and (b) obese A^y/a mice (n=5–6 per group) ^aP<0.0005 vs saline-injected WT mice, ^bP<0.005 vs saline-injected pre-obese A^y/a mice, ^cP<0.005 vs saline-injected WT mice, ^dP<0.005 vs saline-injected WT mice,^eP<0.0005 vs saline-injected obese A^y/a mice and ^fP<0.0005 vs PYY_3–36-injected WT mice.

Full figure and legend (20K)

Study 3. Effects of -MSH on food intake in pre-obese and obese A^y/a mice

Studying the pre-obese cohort, -MSH reduced food intake at 2 h post-injection, in both genotypes (Figure 3a). Food intake was unchanged in all four groups at 24 h post-injection. In fasted, obese A^y/a mice, 2 h food intake was also suppressed by -MSH compared to saline (Figure 3b). There was a significant difference in food intake between fasted, saline-injected obese A^y/a and WT controls until 8 h post-injection (0–8 h: 6.40.2 (obese A^y/a mice) vs 6.90.4 (WT mice), P<0.005). At 24 h post-injection, food intake was similar in all the groups.

Figure 3.

Study 3. Effects of peripheral -MSH (0.2 mol/kg body weight) on 0–2 h food intake in (a) pre-obese (n=5–6 per group) and (b) obese A^y/a mice (n=5–6 per group). ^aP<0.0005 vs saline-injected WT mice, ^bP<0.05 vs saline-injected pre-obese A^y/a mice, ^cP<0.005 vs saline-injected WT mice, ^dP<0.05 vs saline-injected WT mice,^eP<0.0005 vs saline-injected obese A^y/a mice.

Full figure and legend (19K)

Study 4. Effects of leptin on food intake in pre-obese and obese A^y/a mice

Leptin inhibited food intake 2 h post-injection in pre-obese A^y/a and WT mice (Figure 4a). At 24 h post-injection, food intake was similar in all four groups. In the obese cohort, leptin also suppressed feeding 2 h post-injection (Figure 4b). At 24 h post-injection, food intake in the leptin-treated obese A^y/a mice remained suppressed compared to saline-injected obese A^y/a mice (obese A^y/a mice (0–24 h): 5.20.2 (leptin) vs 6.40.3 g (saline), P<0.005). Consistent with our previous studies, an i.p. saline injection significantly suppressed food intake in fasted, obese A^y/a mice compared to WT controls at 8, but not 24 h (0–8 h: 2.50.1 (obese A^y/a mice) vs 3.10.1 g (WT mice), P<0.05).

Figure 4.

Study 4. Effects of peripheral leptin (2 mol/kg body weight) on 0–2 h food intake in (a) pre-obese (n=5–6 per group) and (b) obese (n=5–7 per group) A^y/a mice. ^aP<0.005 vs saline-injected WT mice, ^bP<0.0005 vs saline-injected pre-obese A^y/a mice, ^cP<0.005 vs saline-injected WT mice, ^dP<0.0005 vs saline-injected WT mice,^eP<0.0005 vs saline-injected obese A^y/a mice and ^fP<0.0005 vs leptin-injected WT mice.

Full figure and legend (20K)

Study 5. Effects of ghrelin on food intake in pre-obese and obese A^y/a mice

Our previous work in rodents has demonstrated that i.p. ghrelin stimulates feeding at 1 h post-injection, but not at subsequent time points.¹⁸ Consistent with this, ghrelin increased food intake 1 h post-injection in freely feeding WT mice of both pre-obese and obese cohorts (Figure 5a and b). This stimulatory effect on feeding was not observed in either cohort at further time-points. However, ghrelin did not significantly alter feeding in either pre-obese or obese non-fasted A^y/a mice (Figure 5a and b) at any time point. Notably, since ghrelin or saline injections were administered in the fed state, food intake was similar in obese A^y/a mice and WT littermates following an i.p. saline injection.

Figure 5.

Study 5. Effects of ghrelin (0.2 mol/kg body weight) on 0–1 h food intake in (a) pre-obese (n=5 per group) and (b) obese (n=5–6 per group) A^y/a mice. ^aP<0.05 vs saline-injected WT mice.

Full figure and legend (19K)

Study 6. Plasma leptin, ghrelin and PYY concentrations in pre-obese and obese A^y/a mice

Consistent with previous findings,^{30, 31} plasma leptin was significantly increased in obese A^y/a mice compared to age-matched WT controls (Figure 6). However, in pre-obese A^y/a mice, plasma leptin was five times higher than in WT controls (Figure 6). Therefore, pre-obese mice, despite having the same body weight as WT controls, are likely to have increased body adiposity.

Figure 6.

Study 6. Plasma leptin (n=7–13 per genotype), ghrelin (n=5–14 per genotype) and PYY (n=15–19 per genotype) in pre-obese and obese A^y/a mice. ^*P<0.05, ^**P<0.005, and ^***P<0.0005 vs WT mice.

Full figure and legend (54K)

In both pre-obese and obese A^y/a mice, plasma ghrelin concentration was significantly reduced compared to age-matched WT controls (Figure 6). There was no difference in plasma ghrelin between pre-obese and obese A^y/a mice.

Plasma PYY was similar in fasted pre-obese A^y/a mice compared to WT controls (Figure 6). However, plasma PYY in fasted obese A^y/a mice was increased by almost 50% compared to age-matched WT controls (Figure 6).

Discussion

These studies show that obese A^y/a mice are more sensitive to the effects of fasting than WT mice or pre-obese A^y/a mice. It has previously been reported that obese A^y/a mice react with a disproportionate inhibition of feeding following stresses such as restraint, handling and i.p. injection, although the cause of this is unknown.³² In the current studies, fasting and i.p. injection may be sufficient stresses to reduce feeding in obese A^y/a mice. I.p. saline-injected fasted obese A^y/a mice consistently ate less than WT controls, although this effect did not occur in the fed state. Therefore, it is likely that the stress of a 24 h fast, rather than that of an i.p. saline injection, inhibits feeding in obese A^y/a mice. Interestingly, fasted pre-obese A^y/a mice displayed similar refeeding profiles to WT controls. The inhibition of food intake in fasted obese A^y/a mice following administration of PYY_3–36, -MSH or leptin does not merely reflect this differential stress response as food intake was significantly reduced compared to saline-injected obese A^y/a controls.

This is the first report of the effects of peripherally administered PYY_3–36 and ghrelin on food intake in A^y/a mice. Our studies demonstrate that PYY_3–36 reduces food intake in both pre-obese and obese A^y/a mice. Hypothalamic arcuate NPY mRNA levels in pre-obese and obese A^y/a mice are similar to those of WT controls, although NPY mRNA expression is elevated in the DMN of obese A^y/a mice.¹⁴ Our results are consistent with previous studies proposing that PYY_3–36 acts via arcuate NPY neurones. A further interpretation of our findings is that PYY_3–36 reduces feeding independently of DMN NPY pathways. However, it must be noted that Kesterson et al¹⁴ observed elevated DMN NPY mRNA levels in older and heavier obese A^y/a mice than those in the current study. Interestingly, plasma PYY levels in fasted obese A^y/a mice were significantly elevated. This contrasts with recent findings that obese humans have low plasma PYY.²⁹ However, obesity in A^y/a mice is secondary to abnormal hypothalamic signalling, whereas obesity in humans is multifactorial. An elevation in plasma PYY may be an appropriate compensatory signal to the CNS to attempt to normalise energy homeostasis. This is in accordance with pre-obese A^y/a mice having normal plasma PYY levels.

PYY_3–36 inhibits NPY/AgRP neurones, disinhibiting POMC neurones and therefore stimulating -MSH release.²⁵ In A^y/a mice, agouti protein competitively antagonises MC4-Rs and hence acts downstream of POMC neurones. However, this antagonism did not alter the anorectic actions of exogenous PYY_3–36 in A^y/a mice. Similarly, peripheral injection of either -MSH or leptin, both of which exert their effects through activation of CNS MC3- and MC4-Rs,³³ reduced feeding in pre-obese and obese A^y/a mice. These findings may reflect sufficient melanocortinergic antagonism by agouti protein in A^y/a mice to block the effects of endogenous, but not exogenous PYY_3–36, -MSH and leptin. Alternatively, PYY_3–36, -MSH and leptin may reduce food intake through a mechanism involving an alternative receptor not antagonised by agouti protein, such as the MC3-R.⁴ A possible role for the MC3-R in the regulation of food intake is also supported by the observation that obese A^y/a mice have reduced food intake following CNS injection of MTII, a MC3-R and MC4-R agonist.³⁴

Our findings that pre-obese and obese A^y/a mice are sensitive to peripheral leptin is in contrast to previous studies.^{15, 16, 27} However, in several of these studies, 'leptin resistance' was demonstrated by measuring daily body weight.^{15, 16} A study of feeding responses of obese A^y/a mice to peripheral leptin concentrated on average daily food intake and involved smaller animal numbers than the current study.²⁷ Similarly, the effects of peripheral -MSH on energy balance in A^y/a mice are limited to studying body weight rather than food intake.²⁶ Our data show that the effects of peripheral -MSH and leptin on food intake in pre-obese A^y/a mice are short-lived, with no effects on 24 h food intake. Therefore, these short-lived alterations in feeding may not influence daily body weight. However, in obese A^y/a mice, the anorectic effects of leptin are observed at 24 h post-injection. In support of our findings, chronic peripheral administration of leptin in transgenic mice ectopically expressing agouti protein transiently reduced daily food intake without a simultaneous reduction in body weight.³⁵ Hence, chronic leptin administration to A^y/a mice may acutely reduce feeding without altering body weight.

Conversely, we have observed that both pre-obese and obese A^y/a mice are resistant to the orexigenic effects of peripherally injected ghrelin. Arcuate NPY neurones, rather than those at other hypothalamic sites, appear to mediate the actions of peripheral ghrelin on feeding.^{36, 37} However, there are prominent arcuate NPY-containing efferents projecting to the DMN³³ and hence, the elevated DMN NPY gene expression in obese A^y/a mice could prevent a further increase in NPY mRNA, contributing to their insensitivity to ghrelin. Since pre-obese A^y/a mice have normal NPY mRNA expression in these nuclei,¹⁴ yet are also resistant to ghrelin, altered NPY circuits may not be responsible for ghrelin insensitivity in pre-obese A^y/a mice. Nevertheless, normal DMN NPY gene expression does not exclude abnormal DMN NPY neuronal function.

Owing to the insensitivity of both pre-obese and obese A^y/a mice to peripheral ghrelin, AgRP, rather than NPY, could be important in mediating ghrelin's orexigenic effects. AgRP is an important mediator in ghrelin-induced feeding,²¹ and NPY-deficient mice have a normal feeding response to peripherally administered ghrelin.²⁰ Obese A^y/a mice, similar in age to those in the current study, have reduced arcuate AgRP mRNA expression¹³ and peripheral ghrelin may be unable to sufficiently upregulate AgRP to stimulate feeding. However, pre-obese A^y/a mice have similar arcuate AgRP mRNA levels to WT littermates,¹³ yet are also insensitive to peripheral ghrelin. Alternatively, in pre-obese and obese A^y/a mice, chronic antagonism of hypothalamic MC4-Rs by agouti protein could prevent a further upregulation of AgRP by ghrelin.

Resistance of A^y/a mice to exogenous peripheral ghrelin administration is not secondary to elevated circulating ghrelin, since we have shown that both pre-obese and obese A^y/a mice have low plasma ghrelin. This is consistent with previous studies of murine³⁸ and human,^{29, 39, 40, 41, 42} models of obesity. A reduction in plasma ghrelin in human obesity is believed to be compensatory rather than causative. Resistance to ghrelin in obesity would be another compensatory mechanism to prevent further over-eating. The observed reduction in plasma ghrelin concentrations in pre-obese and obese A^y/a mice could be secondary to their increased plasma leptin levels, since leptin has been shown to negatively regulate ghrelin.⁴³ Although a previous study has shown that pre-obese A^y/a mice do not have elevated plasma leptin,¹⁶ this described much younger mice than those used in the current study.

In conclusion, we have shown that pre-obese and obese A^y/a mice are sensitive to the anorectic effects of peripheral PYY_3–36. This is consistent with our recent findings that obese humans are sensitive to exogenous PYY_3–36.²⁹ In contrast, neither pre-obese nor obese A^y/a mice respond to the orexigenic peptide ghrelin, despite having low circulating plasma ghrelin. In obese A^y/a mice, the mechanism underlying resistance to ghrelin is unclear, but could involve altered hypothalamic NPY and/or AgRP signalling. Antagonism of CNS MC4-Rs by ectopic agouti expression could also inhibit ghrelin-induced feeding. This supports a role for the melanocortin system in mediating ghrelin's effects. Since PYY_3–36 inhibits feeding in A^y/a mice, the MC4-R may not be essential for the anorectic effects of this peptide. Our data show that obesity in the A^y/a mouse is associated with ghrelin resistance and hence it would be interesting to study the effects of peripheral ghrelin in obese humans. However, the responsiveness of obese A^y/a mice to PYY_3–36 supports this peptide as a potential antiobesity agent.²⁹

References

1. Wolff GL, Roberts DW, Mountjoy KG. Physiological consequences of ectopic agouti gene expression: the yellow obese mouse syndrome. Physiol Genomics 1999; 1: 151–163. | PubMed |
2. Klebig ML, Wilkinson JE, Geisler JG, Woychik RP. Ectopic expression of the agouti gene in transgenic mice causes obesity features of type ii diabetes and yellow fur. Proc Natl Acad Sci USA 1995; 92: 4728–4732. | Article | PubMed | ChemPort |
3. Perry WL, Hustad CM, Swing DA, Jenkins NA, Copeland NG. A transgenic mouse assay for agouti protein activity. Genetics 1995; 140: 267–274. | PubMed | ChemPort |
4. Lu D, Willard D, Patel IR, Kadwell S, Overton L, Kost T, Luther M, Chen W, Woychik RP, Wilkison WO, Cone RD. Agouti protein is an antagonist of the melanocyte-stimulating-hormone receptor. Nature 1994; 371: 799–802. | Article | PubMed | ISI | ChemPort |
5. Huszar D, Lynch CA, Fairchild-Huntress V, Dunmore JH, Fang Q, Berkemeier LR, Gu W, Kesterson RA, Boston BA, Cone RD, Smith FJ, Campfield LA, Burn P, Lee F. Targeted disruption of the melanocortin-4 receptor results in obesity in mice. Cell 1997; 88: 131–141. | Article | PubMed | ISI | ChemPort |
6. Ollmann MM, Wilson BD, Yang YK, Kerns JA, Chen Y, Gantz I, Barsh GS. Antagonism of central melanocortin receptors in vitro and in vivo by agouti-related protein. Science 1997; 278: 135–138. | Article | PubMed | ISI | ChemPort |
7. Graham M, Shutter JR, Sarmiento U, Sarosi I, Stark KL. Overexpression of agrt leads to obesity in transgenic mice. Nat Genet 1997; 17: 273–274. | Article | PubMed | ISI | ChemPort |
8. Broberger C, Johansen J, Johansson C, Schalling M, Hokfelt T. The neuropeptide Y/Agouti gene-related protein (AGRP) brain circuitry in normal, anorectic, and monosodium glutamate-treated mice. Proc Natl Acad Sci USA 1998; 95: 15043–15048. | Article | PubMed | ChemPort |
9. Hahn TM, Breininger JF, Baskin DG, Schwartz MW. Coexpression of Agrp and NPY in fasting-activated hypothalamic neurons. Nat Neurosci 1998; 1: 271–272. | Article | PubMed | ISI | ChemPort |
10. Zhang Y, Proenca R, Maffei M, Barone M, Leopold L, Friedman JM. Positional cloning of the mouse obese gene and its human homologue. Nature 1994; 372: 425–432. | Article | PubMed | ISI | ChemPort |
11. Cheung CC, Clifton DK, Steiner RA. Proopiomelanocortin neurons are direct targets for leptin in the hypothalamus. Endocrinology 1997; 138: 4489–4492. | Article | PubMed | ISI | ChemPort |
12. Mercer JG, Hoggard N, Williams LM, Lawrence CB, Hannah LT, Morgan PJ, Trayhurn P. Coexpression of leptin receptor and preproneuropeptide Y MRNA in arcuate nucleus of mouse hypothalamus. J Neuroendocrinol 1996; 8: 733–735. | Article | PubMed | ISI | ChemPort |
13. Tsuruta Y, Yoshimatsu H, Hidaka S, Kondou S, Okamoto K, Sakata T. Hyperleptinemia in A(y)/a mice upregulates arcuate cocaine- and amphetamine-regulated transcript expression. Am J Physiol Endocrinol Metab 2002; 282: E967–E973. | PubMed | ISI | ChemPort |
14. Kesterson RA, Huszar D, Lynch CA, Simerly RB, Cone RD. Induction of neuropeptide Y gene expression in the dorsal medial hypothalamic nucleus in two models of the agouti obesity syndrome. Mol Endocrinol 1997; 11: 630–637. | Article | PubMed | ISI | ChemPort |
15. Halaas JL, Boozer C, Blair-West J, Fidahusein N, Denton DA, Friedman JM. Physiological response to long-term peripheral and central leptin infusion in lean and obese mice. Proc Natl Acad Sci USA 1997; 94: 8878–8883. | Article | PubMed | ChemPort |
16. Wilson BD, Bagnol D, Kaelin CB, Ollmann MM, Gantz I, Watson SJ, Barsh GS. Physiological and anatomical circuitry between agouti-related protein and leptin signaling. Endocrinology 1999; 140: 2387–2397. | Article | PubMed | ISI | ChemPort |
17. Kojima M, Hosoda H, Date Y, Nakazato M, Matsuo H, Kangawa K. Ghrelin is a growth-hormone-releasing acylated peptide from stomach. Nature 1999; 402: 656–660. | Article | PubMed | ISI | ChemPort |
18. Wren AM, Small CJ, Ward HL, Murphy KG, Dakin CL, Taheri S, Kennedy AR, Roberts GH, Morgan DG, Ghatei MA, Bloom SR. The novel hypothalamic peptide ghrelin stimulates food intake and growth hormone secretion. Endocrinology 2000; 141: 4325–4328. | Article | PubMed | ISI | ChemPort |
19. Wren AM, Small CJ, Abbott CR, Dhillo WS, Seal LJ, Cohen MA, Batterham RL, Taheri S, Stanley SA, Ghatei MA, Bloom SR. Ghrelin causes hyperphagia and obesity in rats. Diabetes 2001; 50: 2540–2547. | Article | PubMed | ISI | ChemPort |
20. Tschop M, Smiley DL, Heiman ML. Ghrelin induces adiposity in rodents. Nature 2000; 407: 908–913. | Article | PubMed | ISI | ChemPort |
21. Nakazato M, Murakami N, Date Y, Kojima M, Matsuo H, Kangawa K, Matsukura S. A role for ghrelin in the central regulation of feeding. Nature 2001; 409: 194–198. | Article | PubMed | ISI | ChemPort |
22. Shintani M, Ogawa Y, Ebihara K, Aizawa-Abe M, Miyanaga F, Takaya K, Hayashi T, Inoue G, Hosoda K, Kojima M, Kangawa K, Nakao K. Ghrelin, an endogenous growth hormone secretagogue, is a novel orexigenic peptide that antagonizes leptin action through the activation of hypothalamic neuropeptide Y/Y1 receptor pathway. Diabetes 2001; 50: 227–232. | Article | PubMed | ISI | ChemPort |
23. Wren AM, Small CJ, Fribbens CV, Neary NM, Ward HL, Seal LJ, Ghatei MA, Bloom SR. The hypothalamic mechanisms of the hypophysiotropic action of ghrelin. Neuroendocrinology 2002; 76: 316–324. | Article | PubMed | ISI | ChemPort |
24. Tatemoto K. Isolation and characterization of peptide YY (PYY), a candidate gut hormone that inhibits pancreatic exocrine secretion. Proc Natl Acad Sci USA 1982; 79: 2514–2518. | PubMed |
25. Batterham RL, Cowley MA, Small CJ, Herzog H, Cohen MA, Dakin CL, Wren AM, Brynes AE, Low MJ, Ghatei MA, Cone RD, Bloom SR. Gut hormone PYY(_3–36) physiologically inhibits food intake. Nature 2002; 418: 650–654. | Article | PubMed | ISI | ChemPort |
26. Zemel MB, Moore JW, Moustaid N, Kim JH, Nichols JS, Blanchard SG, Parks DJ, Harris C, Lee FW, Grizzle M, James M, Wilkison WO. Effects of a potent melanocortin agonist on the diabetic/obese phenotype in yellow mice. Int J Obes Relat Metab Disord 1998; 22: 678–683. | Article | PubMed |
27. Boston BA, Blaydon KM, Varnerin J, Cone RD. Independent and additive effects of central POMC and leptin pathways on murine obesity. Science 1997; 278: 1641–1644. | Article | PubMed | ISI | ChemPort |
28. Bolton AE, Hunter WM. The labelling of proteins to high specific radioactivities by conjugation to a 125I-containing acylating agent. Biochem J 1973; 133: 529–539. | PubMed | ISI | ChemPort |
29. Batterham RL, Cohen MA, Ellis SM, Le Roux CW, Withers DJ, Frost GS, Ghatei MA, Bloom SR. Inhibition of food intake in obese subjects by peptide YY_3–36. N Engl J Med 2003; 349: 941–948. | Article | PubMed | ISI | ChemPort |
30. Maffei M, Halaas J, Ravussin E, Pratley RE, Lee GH, Zhang Y, Fei H, Kim S, Lallone R, Ranganathan S. Leptin levels in human and rodent: measurement of plasma leptin and Ob RNA in obese and weight-reduced subjects. Nat Med 1995; 1: 1155–1161. | Article | PubMed | ISI | ChemPort |
31. Correia ML, Haynes WG, Rahmouni K, Morgan DA, Sivitz WI, Mark AL. The concept of selective leptin resistance: evidence from agouti yellow obese mice. Diabetes 2002; 51: 439–442. | PubMed | ISI | ChemPort |
32. De Souza J, Butler AA, Cone RD. Disproportionate inhibition of feeding in A(y) mice by certain stressors: a cautionary note. Neuroendocrinology 2000; 72: 126–132. | Article | PubMed | ISI | ChemPort |
33. Kalra SP, Dube MG, Pu S, Xu B, Horvath TL, Kalra PS. Interacting appetite-regulating pathways in the hypothalamic regulation of body weight. Endocr Rev 1999; 20: 68–100. | Article | PubMed | ISI | ChemPort |
34. Fan W, Boston BA, Kesterson RA, Hruby VJ, Cone RD. Role of melanocortinergic neurons in feeding and the agouti obesity syndrome. Nature 1997; 385: 165–168. | Article | PubMed | ISI | ChemPort |
35. Harris RB, Mitchell TD, Mynatt RL. Leptin responsiveness in mice that ectopically express agouti protein. Physiol Behav 2002; 75: 159–167. | Article | PubMed |
36. Hewson AK, Dickson SL. Systemic administration of ghrelin in-duces Fos and Egr-1 proteins in the hypothalamic arcuate nucleus of fasted and fed rats. J Neuroendocrinol 2000; 12: 1047–1049. | Article | PubMed | ISI | ChemPort |
37. Wang L, Saint-Pierre DH, Tache Y. Peripheral ghrelin selectively increases Fos expression in neuropeptide Y-synthesizing neurons in mouse hypothalamic arcuate nucleus. Neurosci Lett 2002; 325: 47–51. | PubMed | ISI | ChemPort |
38. Ariyasu H, Takaya K, Hosoda H, Iwakura H, Ebihara K, Mori K, Ogawa Y, Hosoda K, Akamizu T, Kojima M, Kangawa K, Nakao K. Delayed short-term secretory regulation of ghrelin in obese animals: evidenced by a specific RIA for the active form of ghrelin. Endocrinology 2002; 143: 3341–3350. | Article | PubMed | ISI | ChemPort |
39. Tschop M, Weyer C, Tataranni PA, Devanarayan V, Ravussin E, Heiman ML. Circulating ghrelin levels are decreased in human obesity. Diabetes 2001; 50: 707–709. | Article | PubMed | ISI | ChemPort |
40. Cummings DE, Clement K, Purnell JQ, Vaisse C, Foster KE, Frayo RS, Schwartz MW, Basdevant A, Weigle DS. Elevated plasma ghrelin levels in prader willi syndrome. Nat Med 2002; 8: 643–644. | Article | PubMed | ISI | ChemPort |
41. Shiiya T, Nakazato M, Mizuta M, Date Y, Mondal MS, Tanaka M, Nozoe S, Hosoda H, Kangawa K, Matsukura S. Plasma ghrelin levels in lean and obese humans and the effect of glucose on ghrelin secretion. J Clin Endocrinol Metab 2002; 87: 240–244. | Article | PubMed | ISI | ChemPort |
42. Haqq AM, Farooqi IS, O'Rahilly S, Stadler DD, Rosenfeld RG, Pratt KL, LaFranchi SH, Purnell JQ. Serum ghrelin levels are inversely correlated with body mass index, age, and insulin concentrations in normal children and are markedly increased in prader-willi syndrome. J Clin Endocrinol Metab 2003; 88: 174–178. | Article | PubMed | ISI | ChemPort |
43. Barazzoni R, Zanetti M, Stebel M, Biolo G, Cattin L, Guarnieri G. Hyperleptinemia prevents increased plasma ghrelin concentration during short-term moderate caloric restriction in rats. Gastroenterology 2003; 124: 1188–1192. | Article | PubMed |

Acknowledgements

We thank K Murphy, K Smith and A Wren for their helpful comments while reading this manuscript. NMM is a Wellcome Trust Clinical Training Fellow. This work is supported by an MRC programme Grant G7811974 (Bloom, Ghatei and Small).