By David N. Leff

Even the staunchest opponent of gun control would probably look askance at a firearm that went off before anyone pulled the trigger.

Such is the case of a self-activating cell surface receptor, which normally fires only when its trigger — its hormone ligand, leptin — gets pulled. In the Zucker rat, a laboratory rodent well known to cell biologists as "fatty," that receptor is naturally mutated, so the rat's brain can't get the leptin message.

That message tells rodents and other mammals when they've had enough to eat, and that it's time to push away from the table. In this respect, leptin acts something like the stern headmaster of the workhouse when Oliver Twist dared ask for a second helping of gruel.

For lack of leptin, the Zucker rat, instead of boasting the lean, mean, svelte profile of normal rats, is grotesquely obese, double the weight of its wild-type fellows.

In Zucker, the receptor gene that mutates carries the name, fatty, which is analogous to the db gene in one strain of obese mice. These db genes encode the leptin receptor, as does fatty, the rat. It is really the rat equivalent of the db mouse, in terms of what gets mutated.

In another breed of overweight mice, the ob gene expresses leptin itself.

Today's Proceedings of the National Academy of Sciences (PNAS) tells the tale of this hairless-trigger effect, in an article titled: "Constitutive and impaired signaling of leptin receptors containing the Gln —> Pro extracellular domain fatty mutation." Its senior author is molecular biologist Louis Tartaglia, director of metabolic diseases at Millennium Pharmaceuticals Inc., in Cambridge, Mass.

"What's new in this paper," Tartaglia told BioWorld Today, "is that we've characterized in detail the signaling defects of leptin receptors containing the fatty mutation. It's a very interesting and unusual mutation," he added, "in that it not only results in some defects in receptor signaling, but also in constitutive — self-acting — signaling in the absence of ligand."

Among those signaling defects, Tartaglia explained, is the inability of that receptor to activate certain — but not all — transcription factors, called STATs (signal transducer and activator of transcription). The normal receptor responds to signals from its leptin ligand, and then goes on to regulate various genes.

"STATs," Tartaglia said, "associate directly with the intracellular domains of these membrane-crossing receptors in the cell's cytosol. Then, when activated, they translocate to the nucleus and directly stimulate transcription."

One such transcription factor that it does activate, STAT3, appears to be the most significantly stimulated STAT in the brain's hyperthalamus, which is where leptins and other hormones get in their licks.

Still Uncracked: Fatty's Guilty Secret

"It's not clear for sure," Tartaglia observed, "why fatty rat is fat." He sees one of two possible explanations: "Either the defective signaling, or the constitutive signaling."

Moreover, "It offers the possibility of being one way in which the trait of resistance to leptin can develop, by overstimulation of its receptor during gestation. Leptin resistance," Tartaglia continued, "is thought to be an important component of obesity. One form is an extreme kind of leptin resistance, such as in the db/db mouse, which is defective for signaling. But perhaps too much signaling," he surmised, "such as these constitutive signals during development, might be another reason you could develop leptin resistance."

As reported in PNAS, Tartaglia recounted, "We thought something could be wrong with the signal transducing capabilities of the fatty mutant leptin receptor, in addition to its impact on binding. What we expected to see was dramatically flawed signaling.

"However," he went on, "we got the surprise that what appeared to be the most significant defects in the receptor signaling was the fact that it could signal even in the absence of its leptin ligand."

To reach this finding, the Millennium co-authors transfected hyperthalamic cells with genes expressing both the wild-type and fatty mutant leptin receptor. "Then we assayed for the ability of each to stimulate certain response elements that drive transcription.

"Next, very soon," Tartaglia said, "we want to repeat these in vitro studies in vivo. We'd like to know if we could find evidence for constitutive signaling in live rats, and at what point the pathway has become desensitized, or leptin-resistant."

The team plans to inject leptin directly into the brains of both wild-type and Zucker rats. "This central targeting," Tartaglia explained, "will make sure that there are no issues of transport across the blood-brain barrier. Then we'll compare the amount of STAT activation in the wild-type and fatty rats, in both the presence and absence of leptin."

Pills, Not Needles, To Cross Blood-Brain Barrier

He continued: "When you look at it from a receptor-biologist and drug-development perspective, a single amino-acid substitution, from glutamine to proline, is generating a receptor that is signaling, even in the absence of ligand. We expect that this point mutation will result in a fairly subtle conformational change. Understanding how the receptor gets triggered might help us to develop small molecule drugs that can make a similar structural change, and therefore activate the receptor."

Tartaglia made the point that "there's a lot of excitement in developing small molecules, which can be taken in pill form, to stimulate the receptor. Not only would they be orally available, but would also move freely across the blood-brain barrier, without requiring a specific receptor to transport them across."

Alluding to ongoing clinical trials by Amgen Inc., of Thousand Oaks, Calif., of injectable leptin as an anti-obesity drug, Tartaglia concluded: "There certainly may be reason to believe that leptin may not be a widely used pharmaceutical to treat obesity, because of the difficulties inherent in its injection into the bloodstream, as well as concerns that it may not efficiently cross the blood-brain barrier." *