In search for the bioactive conformation of glucagon, "positional cyclization scanning" was used to determine secondary structures of glucagon required for maximal interaction with the glucagon receptor. Because glucagon is flexible in nature, its bioactive conformation is not known except for an amphiphilic helical conformation at the C-terminal region. To understand the conformational requirement for the N-terminal region that appears to be essential for signal transduction, a series of glucagon analogues conformationally constrained by disulfide or lactam bridges have been designed and synthesized. The conformational restrictions via disulfide bridges between cysteine i and cysteine i + 5, or lactam bridges between lysine i and glutamic acid i + 4, were applied to induce and stabilize certain corresponding secondary structures. The results from the binding assays showed that all the cyclic analogues with disulfide bridges bound to the receptor with significantly reduced binding affinities compared to their linear counterparts. On the contrary, glucagon analogues containing lactam bridges, in particular, c[Lys(5), Glu(9)]glucagon amide (10) and c[Lys(17), Glu(21)]glucagon amide (14), demonstrated more than 7-fold increased receptor binding affinities than native glucagon. These results suggest that the bioactive conformation of glucagon may adopt a helical conformation at the N-terminal region as well as the C-terminal region, which was not evident from earlier biophysical studies of glucagon.

In pursuit of truncated glucagon analogues that can interact with the glucagon receptor with substantial binding affinity, 23 truncated glucagon analogues have been designed and synthesized. These truncated analogues consist of several fragments of glucagon with 11 or 12 amino acid residues (1-4), conformationally constrained analogues containing the sequence of the middle region of glucagon (5-15), and truncated analogues containing the sequence of the C-terminal region (16-23). Biological assays of these analogues showed that the truncated glucagon analogues with the sequence of the C-terminal region possess significantly better binding affinity compared to the truncated analogues with the sequence of the middle region, and these analogues (17-23) demonstrated potent antagonistic activity (pA(2) values between 6.5 and 7.5). On the basis of these results, it can be suggested that glucagon interacts with its receptor with two hydrophobic patches located in the middle and the C-terminal regions of glucagon, and both hydrophobic patches are necessary for significant receptor recognition. These two hydrophobic binding motifs, located in two different regions of glucagon, appear to be the reason why the earlier attempts to obtain truncated analogues with good binding affinity did not result in any success. Long peptide hormones such as glucagon seem to require more than one binding pocket on the receptors for maximal interaction.

We examined the functional role of glycine at position 4 in the potent glucagon antagonist [desHis(1), Glu(9)]glucagon amide, by substituting the L- and D-enantiomers of alanine and leucine for Gly(4) in this antagonist. The methyl and isobutyl side-chain substituents were introduced to evaluate the preference shown by the glucagon receptor, if any, for the orientation of the N-terminal residues. The L-amino acids demonstrated only slightly better receptor recognition than the D-enantiomers. These results suggest that the Gly(4) residue in glucagon antagonists may be exposed to the outside of the receptor. The enhanced binding affinities of analogs 1 and 3 compared with the parent antagonist, [desHis(1), Glu(9)]glucagon amide, may have resulted from the strengthened hydrophobic patch in the N-terminal region and/or the increased propensity for a helical conformation due to the replacement of alanine and leucine for glycine. Thus, as a result of the increased receptor binding affinities, antagonist activities of analogs 1-4 were increased 10-fold compared with the parent antagonist, [desHis(1), Glu(9)]glucagon amide. These potent glucagon antagonists have among the highest pA(2) values of any glucagon analogs reported to date.

The current study examined the effects of radish leaves powder on the excretion of fecal triglyceride, and sterol and hepatic UDP-glucuronyl transferase (UDPGT) activity in rats fed hypercholesterolemic diet. Male Sprague-Dawley rats weighing 100±10 g were randomly assigned to normal control group (N group), normal diet with 5% radish leaves powder supplemented group (NR) and hypercholesterolemic groups, which were subdivided into radish leaves powder free diet group (HC) and 2.5% (HRL), 5% (HRM), and 10% (HRH) radish leaves powder supplemented groups. The experimental diets were fed ad libitum for 4 weeks. Fecal weights and water contents were significantly increased in all radish leaves powder supplemented groups (NR, HRL, HRM, and HRH) than that of N and HC groups. Fecal total lipid contents including fecal neutral and acidic sterols in radish leaves powder supplemented groups were higher than those of the HC group, and especially that of HRH group was the highest among all experimental groups. Hepatic UDPGT activity of HRH group was 38% higher than that of HC group. Excretions of fecal bile acid were increased 2.3 and 2.7 folds in HRM and HRH groups compared with that of HC group. And neutral sterol, coprostanol, and coprostanone contents of them were higher in radish leaves supplemented groups than in HC group. These results suggest that radish leaves may act as potential substitute for a dietary fiber capable of improving a gastrointestinal function and lipid metabolism.

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