The novel GLP-1-gastrin dual agonist ZP3022 improves glucose homeostasis and increases ß-cell mass without affecting islet number in db/db mice.

Antidiabetic treatments aiming to preserve or even to increase ß-cell mass are currently gaining increased interest. Here we investigated the effect of chronic treatment with the novel glucagon-like peptide-1 (GLP-1)-gastrin dual agonist ZP3022 (HGEGTFTSDLSKQMEEEAVRLFIEWLKN-8Ado-8Ado-YGWLDF-NH2) on glycemic control, ß-cell mass and proliferation, and islet number. Male db/db mice were treated with ZP3022, liraglutide, or vehicle for 2, 4, or 8 weeks, with terminal assessment of hemoglobin A1c, basal blood glucose, and plasma insulin concentrations. Pancreata were removed for immunohistochemical staining and stereological quantification of ß-cell mass, islet numbers, proliferation, and apoptosis. Treatment with ZP3022 or liraglutide led to a significant improvement in glycemic control. ZP3022 treatment resulted in a sustained increase in ß-cell mass after 4 and 8 weeks of treatment, whereas the effect of liraglutide was transient. The expansion in ß-cell mass observed in the ZP3022-treated mice appeared to be driven by increased ß-cell proliferation in existing islets rather than by formation of new islets, as mean islet mass increased but the number of islets remained constant. Our data demonstrate that the GLP-1-gastrin dual agonist ZP3022 causes a sustained improvement in glycemic control accompanied by an increase in ß-cell mass, increased proliferation, and increased mean islet mass. The results highlight that the GLP-1-gastrin dual agonist increases ß-cell mass more than liraglutide and that dual agonists could potentially be developed into a new class of antidiabetic treatments.

Beta-arrestin-based Bret2 screening assay for the "non"-beta-arrestin binding CB1 receptor.

CB1 receptor (CB1R) antagonists have been demonstrated to be effective in treating obesity and related disorders. This study has been focused on establishing a beta-arrestin 2-based screening assay for the CB1R using BRET2 technology. When the existing BRET2 screening platform was applied to the CB1R, the authors discovered that the receptor interacted weakly with beta-arrestin 2, resulting in unsatisfactory assay performance. To enhance the beta-arrestin binding capacity, they replaced the C-terminal tail of the CB1R with tails from either the V2 or BRS3 receptors, both of which interact strongly with beta-arrestin 2. Using this chimeric approach, the authors screened a small compound library and identified 21 antagonist and inverse agonist hits with IC50 and EC50 values ranging from 0.3 nM to 7.5 microM. Both primary and secondary screening were performed with Z'>0.5, suggesting that the assay is a robust and cost-effective alternative to existing cell-based assays.

Nuclear export inhibitors and kinase inhibitors identified using a MAPK-activated protein kinase 2 redistribution screen.

Redistribution (BioImage) A/S, Søborg, Denmark) is a novel high-throughput screening technology that monitors translocation of specific protein components of intracellular signaling pathways within intact mammalian cells, using green fluorescent protein as a tag. A single Redistribution assay can be used to identify multiple classes of compounds that act at, or upstream of, the level of the protein target used in the primary screening assay. Such compounds may include both conventional and allosteric enzyme inhibitors, as well as protein-protein interaction modulators. We have developed a series of Redistribution assays to discover and characterize compounds that inhibit tumor necrosis factor-alpha biosynthesis via modulation of the p38 mitogen-activated protein kinase (MAPK) pathway. A primary assay was designed to identify low-molecular-weight compounds that inhibit the activation-dependent nuclear export of the p38 kinase substrate MAPK-activated protein kinase 2 (MK2). Hits from the primary screen were categorized, using secondary assays, either as direct inhibitors of MK2 nuclear export, or as inhibitors of the upstream p38 MAPK pathway. Activity profiles are presented for a nuclear export inhibitor, and a compound that structurally and functionally resembles a known p38 kinase inhibitor. These results demonstrate the utility of Redistribution technology as a pathway screening method for the identification of diverse and novel compounds that are active within therapeutically important signaling pathways.

Characterisation of age-dependent beta cell dynamics in the male db/db mice.

AIM: To characterise changes in pancreatic beta cell mass during the development of diabetes in untreated male C57BLKS/J db/db mice. METHODS: Blood samples were collected from a total of 72 untreated male db/db mice aged 5, 6, 8, 10, 12, 14, 18, 24 and 34 weeks, for measurement of terminal blood glucose, HbA1c, plasma insulin, and C-peptide. Pancreata were removed for quantification of beta cell mass, islet numbers as well as proliferation and apoptosis by immunohistochemistry and stereology. RESULTS: Total pancreatic beta cell mass increased significantly from 2.1 ± 0.3 mg in mice aged 5 weeks to a peak value of 4.84 ± 0.26 mg (P < 0.05) in 12-week-old mice, then gradually decreased to 3.27 ± 0.44 mg in mice aged 34 weeks. Analysis of islets in the 5-, 10-, and 24-week age groups showed increased beta cell proliferation in the 10-week-old animals whereas a low proliferation is seen in older animals. The expansion in beta cell mass was driven by an increase in mean islet mass as the total number of islets was unchanged in the three groups. CONCLUSIONS/INTERPRETATION: The age-dependent beta cell dynamics in male db/db mice has been described from 5-34 weeks of age and at the same time alterations in insulin/glucose homeostasis were assessed. High beta cell proliferation and increased beta cell mass occur in young animals followed by a gradual decline characterised by a low beta cell proliferation in older animals. The expansion of beta cell mass was caused by an increase in mean islet mass and not islet number.