"Ghrelin has a lot of interesting activities besides growth hormone release, such as [regulation of] feeding, energy homeostasis, and the cardiovascular system, says Kangawa, "and that attracts researchers from many fields." Lilly's article was the "first to show that ghrelin plays an important role in energy balance, and that [this effect] may be even more important than its effect on growth hormone," says Mark Heiman, Lilly's research adviser in its endocrine division.
|Courtesy of Kenji Kangawa|
The Hunt Begins
Subsequently, researchers began making nonpeptide derivatives of Bowers' molecules, which they hoped would be orally active growth hormone stimulators. They called these derivatives growth-hormone secretagogues (GHS) to distinguish them from Bowers' peptides. Other investigators studied the structure of GHRH, which was found to be nothing like that of Bowers' peptides, suggesting that they act on a receptor other than GHRH.
Companies such as Eli Lilly became interested in GHS for its potential use in the human growth hormone market, but confusion mounted as Heiman's team noticed anomalies early in the preclinical stage. With long-term administration of growth-hormone-releasing peptides and GHSs, the team expected to see an increase in growth rate and a decrease in fat content in the test animals. Instead, they saw "an increase in adiposity and an increase in food intake, and in fact when we injected [the animals] peripherally we saw increase in food intake that we had never seen by peripheral injection of anything. Injecting into the brain, yes, but to have something work peripherally was really rare."
The researchers needed a better understanding of these observations, and in 1996 a team at Merck and Co. announced the identity of the GHS receptor (GHS-R).3,4 The discovery presented a serious challenge: The pituitary gland is the obvious place to look for a receptor that regulates growth hormone, but the receptor was actually far more abundant in the hypothalamus, in areas that regulate energy balance. "This was confusing to most in the field, but ... there were explanations that it regulated growth hormone at the hypothalamic rather than the pituitary level," says Heiman. "The location of the receptor [in the hypothalamus] wasn't so consistent with stimulation of growth hormone [as it was] with regulation of energy balance." Yet, the natural ligand remained elusive, and the conflicting preclinical observations still puzzled them.
Finding the Answers
|Courtesy of Kenji Kangawa|
Once again, the target was not where it was expected to be. Kangawa's team initiated a screen of peripheral tissue extracts and finally detected GHS-R-mediated activity in stomach extracts. Despite this discovery, questions still remained. Ghrelin has a fatty acid modification that complicates purification and identification, so Kangawa's expertise in organic chemistry proved crucial at this point. He analyzed GHS-R using mass spectrometry and HPLC (high-performance liquid chromatography), and then gained confirmation of the structure by independent chemical synthesis.
When Lilly's Heiman and his postdoc, Matthias Tschöp, spotted the paper by Kangawa's team (published just a few months before the Lilly paper), they knew they had found the endogenous ligand. Even though the subject of Kangawa's paper was ghrelin's effect on growth hormone, the Lilly team recognized that ghrelin's existence in the stomach implicated a role in energy balance, perhaps explaining the confusing preclinical findings. Peptide chemist David Smiley set to work synthesizing ghrelin, and the group tested it in rats with a focus on studying energy balance. "We even did some of the experiments in growth-hormone-deficient animals, just to prove that it's not acting through growth hormone," says Heiman.
The Lilly team found that when the animals fasted, ghrelin levels increased. To make sure that the effect was not physical, they distended the rats' stomachs yet saw no change in ghrelin levels. But when they filled the rats' stomachs with a dextrose solution, ghrelin levels dropped. GHS-R also exists in hypothalamic neurons and in the brainstem, leading the team to hypothesize that ghrelin "is a way for the hypothalamus to know the content of the stomach," thus helping it to gauge the body's energy needs, says Heiman.
The importance of the energy-balance observation was underscored soon afterward when researchers at Hammersmith Hospital in London gave volunteers a fixed-calorie breakfast, followed a few hours later by an all-you-can-eat lunch. The volunteers, who had received a dose of ghrelin, overate by as much as 28%.5 Another group monitored ghrelin levels in volunteers over time and found that ghrelin levels rose before a meal and dropped soon afterward.6
Kangawa and his colleagues went on to show that ghrelin is synthesized by X/A-like cells in the stomach7 and that it may play a role in regulating the cardiovascular system.8 He also suspects that ghrelin's fatty acid modification may offer more clues about energy homeostasis. "A peptide hormone modified with such a fatty acid has never been identified previously," says Kangawa. "Bio-synthesis of ghrelin in the stomach is increased by fasting. Fatty acid modification of ghrelin is essential for its activation. Free fatty acid level [in serum] is increased in a fasting state. I wonder if there are some relationships among [them]."
These discoveries and the fact that rat ghrelin differs from human ghrelin by only two amino acids makes this ligand and its receptor intriguing pharmaceutical targets. Heiman would not give details about Eli Lilly's intentions, but "you can use your imagination and see both sides: There are cases where individuals have a wasting disease and they need calories, and [you get] the opposite effect if there is too much ghrelin."
|Courtesy of Mark Heiman|
In retrospect, says Heiman, the signs were there all along that the endogenous ligand for GHS-R had an effect on energy balance. "We could go through the literature and cite [others] making statements that the only side-effect [of GHS] was an increase in appetite. We just had a keen focus on effects other than growth hormone ... and centered on the idea that maybe there's a different role for these GHSs and growth-hormone-releasing peptides."
Kangawa agrees that ghrelin plays a role in growth hormone release and energy homeostasis as well as a variety of other processes, while Heiman and his colleagues tend to downplay its role in growth hormone regulation. The differing views reemphasize the ongoing uncertainty. "Many good scientists [still] believe that the main role of ghrelin is [regulation of] growth hormone," says Heiman. "We'll see how that changes over time."
1. M. Kojima, H. Hosoda, Y. Date, M. Nakazato, H. Matsu, K. Kangawa, "Ghrelin is a growth-hormone-releasing aylated peptide from stomach," Nature, 402:656-60, 1999. (Cited in 266 papers)
2. M. Tschöp, D.L. Smiley, M.L. Heiman, "Ghrelin induces adiposity in rodents," Nature, 407:908-13, 2000. (Cited in 92 papers)
3. K.K. McKee et al., "Molecular analysis of rat pituitary and hypothalamic growth hormone secretagogue receptors," Molecular Endocrinology, 11:415-23, 1997.
4. A.D. Howard et al., "A receptor in pituitary and hypothalamus that functions in growth hormone release," Science, 273:974-7, 1996.
5. A.M. Wren et al., "Ghrelin enhances appetite and increases food intake in humans," Journal of Clinical Endocrinology Metabolism, 86:5992-6, 2001.
6. D.E. Cummings et al., "A preprandial rise in plasma ghrelin levels suggests a role in meal initiation in humans," Diabetes, 50:1714-9, 2001.
7. Y. Date et al., "Ghrelin, a novel growth hormone-releasing acylated peptide, is synthesized in a distinct endocrine cell type in the gastrointestinal tracts of rats and humans," Endocrinology, 141:4255-61, 2000.
8. N. Nagaya et al., "Hemodynamic, renal, and hormonal effects of ghrelin infusion in patients with chronic heart failure," Journal of Clinical Endocrinology Metabolism, 86:5854-9, 2001.