By Dean A. Haycock
Special To BioWorld Today
The identification of a couple of simple molecules that accumulate in the brain during hunger and that can stimulate feeding in well-fed animals is a welcome discovery in a field known for its complexity.
But the successful strategy that led to the identification of this new family of feeding regulators may be just as interesting as the discovery itself.
The new molecules on the feeding and energy homeostasis scene are named after the Greek word for appetite, orexis. Orexins represent a newly identified family of neuropeptides located in the lateral hypothalamus, long a center of feeding control interest in the brain.
The strategy used to find orexins is an impressive example of reverse pharmacology.
Instead of having a well-characterized messenger molecule in hand and seeking its receptor, scientists from the University of Texas and London-based SmithKline Beecham plc reversed the procedure. They chose an array of "orphan receptors," receptors with no known ligands, and searched for molecules that interacted with them.
The work is detailed in "Orexins and orexin receptors: A family of hypothalamic neuropeptides and G protein-coupled receptors that regulate feeding behavior," in the Feb. 20, 1998, issue of Cell.
Orphan Receptors Exposed By Genomics Studies
Genomics studies have been turning up many orphan receptors revealed by their cDNA sequences and lack of known ligands. The majority of these are G protein-coupled cell surface receptors (GPCRs). The researchers narrowed the list of orphan GPCRs down to about 50.
GPCRs use molecules called G proteins to convey the message received by a receptor from the outside to the inside of a cell. Small peptides used as signaling molecules in the nervous system and peptide hormones all seem to bind to GPCRs. Thus orphan GPCRs present biotechnology companies with a wealth of potentially rewarding therapeutic targets.
Masashi Yanagisawa, an investigator at the Howard Hughes Medical Institute and a professor in the department of molecular genetics at the University of Texas Southwestern Medical Center, in Dallas, and his collaborators systematically searched for endogenous peptides that bound to and elicited responses from orphan GPCRs.
"We specifically collected the receptors that looked like they would have peptide ligands," Yanagisawa said.
They began by generating 50 or so stable, transfected cell lines, each of which expressed an orphan GPCR cDNA. These cells were used as reporter cells lines, ready to respond when exposed to a peptide that interacted with the orphan receptor they expressed. The test materials came from a variety of different tissue types which had been processed using a separation procedure called high-resolution HPLC.
While testing several fractions from rat brain extracts, the researchers saw they produced a large increase in levels of internal calcium, one of the receptor-mediated responses the scientists monitored.
The peptides they found, orexin-A and -B, are not structurally similar to other regulatory peptides. Both are derived from the same, larger precursor molecule called prepro-orexin by the action of proteolytic enzymes. The peptides are found in humans, bovines, rats and mice.
"You can easily imagine all mammals at the very least have it," Yanagisawa said. "This isn't published yet, but we have actually cloned a Xenopus [African clawed toad] version as well."
In mice, mRNA for the orexin precursor molecule is found in a part of the brain that has long been associated with the regulation of eating, the lateral hypothalamus. It was this localization that suggested to the researchers that their peptide hits might be related to the regulation of feeding behavior.
This suspicion was confirmed when they demonstrated that a bolus of orexin administered directly into the brain through a pre-implanted catheter stimulated food consumption in a dose-dependent way. At the two-hour time point, a low (3 nanomoles) and a high (30 nanomoles) dose of orexin-A caused animals to eat approximately six and 10 times, respectively, as much food as controls. Between two and four hours after administration, both doses simulated food intake by threefold.
This stimulation pointed to a role for these neuropeptides in the central regulation of feeding.
To further examine the physiological regulatory role of orexins, the researchers measured levels of orexin's precursor mRNA in the hypothalamus of fed and fasted rats. After two days of fasting, hypothalamic prepro-orexin mRNA increased by 2.4 times compared with fed animals.
Orexins May Yield Obesity Drugs
In a minireview titled "Obesity and the hypothalamus: novel peptides for new pathways," in the same issue of Cell, Jeffrey Flier, of Beth Israel Deaconess Medical Center and Harvard Medical School, in Boston, and Eleftheria Maratos-Flier, of the Joslin Diabetes Center and Harvard Medical School, describe the work by Yanagisawa and his co-authors as "a technical tour de force."
Although it is too early to know where orexins belong in the complicated physiological control system that regulates feeding behavior, orexin peptides and their receptors provide exciting new targets for drug developers interested in treating diseases such as eating disorders and diabetes.
Future projects for the Texas group will include experiments designed to explore possible links between orexin-containing neurons and glucose levels in the blood.
"We will artificially render animals hypoglycemic by simply injecting, for example, insulin. You can trace back to the brain to see if those orexin-containing neurons are activated or not," Yanagisawa told BioWorld Today.
Efforts in Texas are going ahead "at full speed," according to Yanagisawa, to knock out orexin and orexin receptor genes in mice, while researchers at SmithKline Beecham want to discover small molecules to block or activate the orexin receptor.
"We are hoping that orexin may give us the first molecular entity [to be associated with] the long-described function of the lateral hypothalamus as a feeding center. Certainly the characteristics of orexin fits in perfectly in that scheme," Yanagisawa said. *