Liraglutide is an acylated glucagon-like peptide-1 (GLP-1) analogue that binds to serum albumin in vivo and is approved for once-daily treatment of diabetes as well as obesity. The aim of the present studies was to design a once weekly GLP-1 analogue by increasing albumin affinity and secure full stability against metabolic degradation. The fatty acid moiety and the linking chemistry to GLP-1 were the key features to secure high albumin affinity and GLP-1 receptor (GLP-1R) potency and in obtaining a prolonged exposure and action of the GLP-1 analogue. Semaglutide was selected as the optimal once weekly candidate. Semaglutide has two amino acid substitutions compared to human GLP-1 (Aib(8), Arg(34)) and is derivatized at lysine 26. The GLP-1R affinity of semaglutide (0.38 ± 0.06 nM) was three-fold decreased compared to liraglutide, whereas the albumin affinity was increased. The plasma half-life was 46.1 h in mini-pigs following i.v. administration, and semaglutide has an MRT of 63.6 h after s.c. dosing to mini-pigs. Semaglutide is currently in phase 3 clinical testing.

Mammalian circadian rhythms generated in the hypothalamic suprachiasmatic nuclei are entrained to the environmental light/dark cycle via a monosynaptic pathway, the retinohypothalamic tract (RHT). We have shown previously that retinal ganglion cells containing pituitary adenylate cyclase-activating polypeptide (PACAP) constitute the RHT. Light activates the RHT via unknown photoreceptors different from the classical photoreceptors located in the outer retina. Two types of photopigments, melanopsin and the cryptochromes (CRY1 and CRY2), both of which are located in the inner retina, have been suggested as "circadian photopigments." In the present study, we cloned rat melanopsin photopigment cDNA and produced a specific melanopsin antibody. Using in situ hybridization histochemistry combined with immunohistochemistry, we demonstrate that the distribution of melanopsin was identical to that of the PACAP-containing retinal ganglion cells. Colocalization studies using the specific melanopsin antibody and/or cRNA probes in combination with PACAP immunostaining revealed that melanopsin was found exclusively in the PACAP-containing retinal ganglion cells located at the surface of somata and dendrites. These data, in conjunction with published action spectra analyses and work in retinally degenerated (rd/rd/cl) mutant mice, suggest that melanopsin is a circadian photopigment located in retinal ganglion cells projecting to the biological clock.

A novel isoform of human elongation factor‐1α (EF‐1α2) has been characterised. It shows a high similarity to other EF‐1α proteins, especially to a rat EF‐1α variant and it has all the characteristics of a functional EF‐1α protein. The pattern of expression of both EF‐1α2 and EF‐1α was analysed in different human tissues. This showed that the two proteins were differentially expressed, EF‐1α2 was expressed in brain, heart, skeletal muscle and in the transformed cell lines AMA and K14, but was undetectable in other tissues and in both primary and transformed human fibroblasts. EF‐1α was expressed in brain, placenta, lung, liver, kidney, pancreas and in all the cell lines that we have analysed but barely detectable in heart and skeletal muscle.

Glucagon-like peptide-1 (GLP-1) and exendin-4 (Ex4) are homologous peptides with established potential for treatment of type 2 diabetes. They bind and activate the pancreatic GLP-1 receptor (GLP-1R) with similar affinity and potency and thereby promote insulin secretion in a glucose-dependent manner. GLP-1R belongs to family B of the seven transmembrane G-protein coupled receptors. The N-terminal extracellular domain (nGLP-1R) is a ligand binding domain with differential affinity for Ex4 and GLP-1: low affinity for GLP-1 and high affinity for exendin-4. The superior affinity of nGLP-1R for Ex4 was previously explained by an additional interaction between nGLP-1R and the C-terminal Trp-cage of Ex4. In this study we have combined biophysical and pharmacological approaches thus relating structural properties of the ligands in solution to their relative binding affinity for nGLP-1R. We used both a tracer competition assay and ligand-induced thermal stabilization of nGLP-1R to measure the relative affinity of full length, truncated, and chimeric ligands for soluble refolded nGLP-1R. The ligands in solution and the conformational consequences of ligand binding to nGLP-1R were characterized by circular dichroism and fluorescence spectroscopy. We found a correlation between the helical content of the free ligands and their relative binding affinity for nGLP-1R, supporting the hypothesis that the ligands are helical at least in the segment that binds to nGLP-1R. The Trp-cage of Ex4 was not necessary to maintain a superior helicity of Ex4 compared to GLP-1. The results suggest that the differential affinity of nGLP-1R is explained almost entirely by divergent residues in the central part of the ligands: Leu10-Gly30 of Ex4 and Val16-Arg36 of GLP-1. In view of our results it appears that the Trp-cage plays only a minor role for the interaction between Ex4 and nGLP-1R and for the differential affinity of nGLP-1R for GLP-1 and Ex4.