Galanin (1-13)-Bradykinin (2-9) amide

Although galanin has been shown to increase insulin sensitivity in skeletal muscle of rats, there is no literature available about the effect of galanin on Glucose Transporter 4 (GLUT4) translocation from intracellular membrane pools to plasma membranes in adipocytes of type 2 diabetic rats. In the present study M35, a galanin antagonist was used to elucidate whether exercise-induced galanin release increased GLUT4 translocation in adipocytes of streptozotocin-induced diabetic rats. The present findings showed that plasma galanin levels after swimming training in all four trained groups were higher compared with each sedentary control. M35 treatment had an inhibitory effect on glucose infusion rates in the euglycemic-hyperinsulinemic clamp test and GLUT4 mRNA expression levels in adipocytes. Moreover, M35 treatment reduced GLUT4 concentration in both plasma membranes and total cell membranes. The ratios of GLUT4 contents in plasma membranes to total cell membranes in four drug groups were lower compared with each control. These data demonstrate a beneficial role of endogenous galanin to transfer GLUT4 from internal stores to plasma membranes in adipocytes of type 2 diabetic rats. Galanin plays a significant role in regulation of glucose metabolic homeostasis and is an important hormone relative to diabetes.

The chimeric peptide M35 [galanin(1-13)-bradykinin(2-9) amide] is a high-affinity galanin receptor ligand acting as a galanin receptor antagonist in the rat spinal cord, rat hippocampus and isolated mouse pancreatic islets. We have radiolabelled M35 and performed equilibrium binding studies with [125I]M35 on the rat pancreatic beta-cell line Rin m 5F, whereby we show the existence of high-affinity binding site (KD = 0.9 +/- 0.1 nM) with a Bmax of 72 +/- 3 fmol/mg protein. Galanin displaces [125I]M35 with the same affinity (KD = 1 nM) as it displaces [125I]galanin. Displacement of [125I]galanin by M35 from Rin m 5F cell membranes shows the presence of two binding sites for M35 with KD-values of 0.3 +/- 0.1 nM and 0.52 +/- 0.03 microM, respectively. The GTP- and pertussis toxin-sensitivity of M35 binding to Rin m 5F membranes shows that binding of [125I]M35 is almost completely abolished by the presence of GTP or after pertussis toxin treatment of the cells, indicating an agonist-like binding of M35 to the galanin receptors. M35 has a dual effect on the galanin mediated inhibition of forskolin stimulated cyclic AMP production in Rin m 5F cells: at low concentrations M35 antagonises the effect of galanin, whereas at concentrations above 10 nM M35 acts as a galanin receptor agonist. These agonist-like effects of galanin and M35 are not additive, thus the mixed agonist/antagonist properties arise from the chimeric nature of M35[galanin(1-13)-bradykinin(2-9)amide] acting solely at galanin receptors.

The three cloned galanin receptors show a higher affinity for galanin than for galanin N-terminal fragments. Galanin fragment (1-15) binding sites were discovered in the rat Central Nervous System, especially in dorsal hippocampus, indicating a relevant role of galanin fragments in central galanin communication. The hypothesis was introduced that these N-terminal galanin fragment preferring sites are formed through the formation of GalR1-GalR2 heteromers which may play a significant role in mediating galanin fragment (1-15) signaling. In HEK293T cells evidence for the existence of GalR1-GalR2 heteroreceptor complexes were obtained with proximity ligation and BRET(2) assays. PLA positive blobs representing GalR1-GalR2 heteroreceptor complexes were also observed in the raphe-hippocampal system. In CRE luciferase reporter gene assays, galanin (1-15) was more potent than galanin (1-29) in inhibiting the forskolin-induced increase of luciferase activity in GalR1-GalR2 transfected cells. The inhibition of CREB by 50nM of galanin (1-15) and of galanin (1-29) was fully counteracted by the non-selective galanin antagonist M35 and the selective GalR2 antagonist M871. These results suggested that the orthosteric agonist binding site of GalR1 protomer may have an increased affinity for the galanin (1-15) vs galanin (1-29) which can lead to its demonstrated increase in potency to inhibit CREB vs galanin (1-29). In contrast, in NFAT reporter gene assays galanin (1-29) shows a higher efficacy than galanin (1-15) in increasing Gq/11 mediated signaling over the GalR2 of these heteroreceptor complexes. This disbalance in the signaling of the GalR1-GalR2 heteroreceptor complexes induced by galanin (1-15) may contribute to depression-like actions since GalR1 agonists produce such effects.