By Dean A. Haycock

Special To BioWorld Today

A scrap of peptide, once dismissed by biologists as inert and uninteresting, could turn out to be a powerful medicine.

It is easy to understand why scientists dismissed C-peptide as a bit of refuse. Its origins suggest that is exactly what it is.

The 31 amino acids in C-peptide first appear together in proinsulin, a long prohormone from which insulin is formed. The peptide links two chains of the insulin molecule that assume their final three-dimensional relationship only after C-peptide is cut from proinsulin. Insulin, of course, goes on to star as the key molecule in the regulation of glucose uptake into cells. C-peptide, although stored with insulin in secretory granules and released with it into the bloodstream, was considered a hanger-on.

But it now appears to be much more than that. Surprisingly, when administered to diabetic rats, C-peptide prevents or decreases diabetes-induced damage to neurons and blood vessels.

Furthermore, it can exert these effects as either a right-handed or a left-handed molecule. This a feature biochemists rarely see in biological molecules like proteins and peptides,which can have one of two structural forms, or "chirals." Chirals of a peptide are mirror images of each other. Right- and left-handed gloves are the most frequently cited analogy. This structural specificity enables them to interact with other protein structures in a stereospecific manner. This is true for enzymes and their substrates and for ligands and their receptors. But it is not true of C-peptide; both chiral forms possess biological activity. This implies that the C-peptide is involved in some process that does not recognize or require a specific chiral form. Therefore, its unknown mechanism of action, whatever it is, does not depend on a protein receptor or binding site.

These data are presented in "Prevention of vascular and neural dysfunction in diabetic rats by C-peptide," in the July 25 issue of Science.

The paper reports C-peptide cannot substitute for insulin. Given by itself, it can not reduce hyperglycemia or the amount of sugar in the blood.

"We know that the C-peptide does not affect glucose level and some of the other metabolic imbalances that are direct consequences of elevated glucose levels. It is preventing downstream consequences of those imbalances," senior author Joseph Williamson, professor of pathology at Washington University School of Medicine, in St. Louis, told BioWorld Today.

Williamson and his group had no interest in C-peptide until representatives from Eli Lilly and Co., of Indianapolis, asked them to test it in their well-characterized in vivo and in vitro diabetic model systems. The group studies rats with chemically induced diabetes and non-diabetic rats with small chambers containing granulation tissue. Granulation tissue forms at the site of an injury. When new, it is very vascular and it has a granular appearance.

"The vascular changes we see in granulation tissue, in response to elevated glucose levels restricted to the chamber, are exactly the same as we see in the retina and kidney," Williamson said. All these organs are sensitive to diabetic damage.

Lilly scientists asked the St. Louis group to test the peptide because they were not convinced by clinical studies in Sweden showing that C-peptide produced beneficial effects in diabetic humans.

"They were skeptical because the dogma would lead you to think that if C-peptides were to have any effect, then there should be receptors for it. Many people over the years have attempted to find or demonstrate the presence of those kinds of receptors for C-peptide and no one has been able to show them," Williamson said.

"So to our surprise and to theirs," Williamson recalled, "we found a really dramatic effect."

Ironically, an attempt by clinicians at Washington University to replicate the Swedish clinical results in human patients failed to do so.

"I think our studies point to the likelihood that to really show reproducibility and robust effects it would be important to give higher levels [of C-peptide]," Williamson said.

Animal Trials Provided Breakthrough

Despite the uncertain results in humans, C-peptide effects in diabetic rats and in granulation tissue were robust. But the lack of chirality became a concern for both Lilly scientists and for Williamson's group. Critics reasoned if there were no receptors, it must be a nonspecific effect.

Lilly provided a reverse sequence version of the naturally occurring C-peptide (which is like a mirror image). If the naturally occurring peptide worked through a receptor-based mechanism, this version of the molecule should not work, since it would not fit the stereospecific receptor.

"To our amazement, it worked," Williamson recalled.

The next step was to synthesize a peptide containing all the amino acids in C-peptide but connected in a random order. That peptide was ineffective.

"So that was very reassuring to us that the effects we observed were indeed dependent on the structural characteristics of the C-peptide." Williamson said.

Other experiments with synthetic C-peptide confirmed the finding that its effects did not depend on its chirality.

Although the mechanism of these effects remains unknown, the authors suggest that it may work by forming channels or pores in cell membranes and correcting ionic imbalances associated with diabetes. While there are no precedents in humans, there are in the microbial world. Antibiotic peptides such as cecropins and mangainins work by creating channels in bacterial cell walls.

"That is what we are pursuing now, and actually we have some very interesting preliminary data that the C-peptide is indeed altering the intracellular ion balance," Williamson said. In time the cast-off peptide once dismissed by biologists potentially could lead to better treatment for patients with Type I or Type II diabetes, Williamson indicated. *