Understanding the regulation of islet cell mass has important implications for the discovery of regenerative therapies for diabetes. The liver plays a central role in metabolism and the regulation of endocrine cell number, but liver-derived factors that regulate α-cell and β-cell mass remain unidentified. We propose a nutrient-sensing circuit between liver and pancreas in which glucagon-dependent control of hepatic amino acid metabolism regulates α-cell mass. We found that glucagon receptor inhibition reduced hepatic amino acid catabolism, increased serum amino acids, and induced α-cell proliferation in an mTOR-dependent manner. In addition, mTOR inhibition blocked amino-acid-dependent α-cell replication ex vivo and enabled conversion of α-cells into β-like cells in vivo. Serum amino acids and α-cell proliferation were increased in neonatal mice but fell throughout postnatal development in a glucagon-dependent manner. These data reveal that amino acids act as sensors of glucagon signaling and can function as growth factors that increase α-cell proliferation.

Members of the class B family of G protein-coupled receptors (GPCRs) bind peptide hormones and have causal roles in many diseases, ranging from diabetes and osteoporosis to anxiety. Although peptide, small-molecule, and antibody inhibitors of these GPCRs have been identified, structure-based descriptions of receptor antagonism are scarce. Here we report the mechanisms of glucagon receptor inhibition by blocking antibodies targeting the receptor's extracellular domain (ECD). These studies uncovered a role for the ECD as an intrinsic negative regulator of receptor activity. The crystal structure of the ECD in complex with the Fab fragment of one antibody, mAb1, reveals that this antibody inhibits glucagon receptor by occluding a surface extending across the entire hormone-binding cleft. A second antibody, mAb23, blocks glucagon binding and inhibits basal receptor activity, indicating that it is an inverse agonist and that the ECD can negatively regulate receptor activity independent of ligand binding. Biochemical analyses of receptor mutants in the context of a high-resolution ECD structure show that this previously unrecognized inhibitory activity of the ECD involves an interaction with the third extracellular loop of the receptor and suggest that glucagon-mediated structural changes in the ECD accompany receptor activation. These studies have implications for the design of drugs to treat class B GPCR-related diseases, including the potential for developing novel allosteric regulators that target the ECDs of these receptors.T he glucagon receptor (GCGR) is a member of the class B G protein-coupled receptor (GPCR) family (1) that mediates the activity of glucagon, a pancreatic islet-derived peptide hormone that plays a central role in the pathophysiology of diabetes (2). Several GCGR antagonists that improve glycemic control in animal models of diabetes and diabetic patients have been described (3-8). Although biochemical studies of glucagon and GCGR mutants have facilitated the mapping of some elements that contribute to glucagon binding (4, 9-12), the molecular mechanisms of GCGR activation and inhibition remain largely unknown because there are currently no high-resolution structures of GCGR. The current model for activation class B GPCRs proposes a tethering mechanism whereby the C-terminal half of the peptide ligand first binds a large extracellular domain (ECD), thereby enabling a high-affinity interaction of the N-terminal half of the ligand with a cleft formed by the transmembrane α-helical bundle (13,14), termed the juxtamembrane (JM) domain. This interaction induces a structural change in the transmembrane and intracellular face of the receptor that enables G protein coupling, likely similar to that described for the activated form of the β-adrenergic receptor (15). Recent structural studies of several class B GPCR ECDs and ECD-ligand complexes support this model (16)(17)(18)(19)(20)(21). Glucagon likely interacts with GCGR in a similar fashion to the interaction of other peptide ligands with class B GPC...

To assess the molecular changes associated with pancreatic ␤-cell dysfunction occurring during the onset of type 2 diabetes, we profiled pancreatic islet mRNAs from diabetic male and high-fat-fed female Zucker diabetic fatty (ZDF) rats and their nondiabetic lean counterparts on custom islet-specific oligonucleotide arrays. The most prominent changes in both the male and female models of type 2 diabetes were increases in the mRNAs encoding proteases and extracellular matrix components that are associated with tissue remodeling and fibrosis. The mRNAs for metalloproteinase (MMP)-2, -12, and -14 were sharply increased with the onset of islet dysfunction and diabetes. Zymography of islet extracts revealed a concurrent, >10-fold increase in MMP-2 protease activity in islets from 9-week-old male ZDF rats. Treatment of female ZDF rats receiving a diabetogenic diet with PD166793, a broad-spectrum MMP inhibitor, substantially prevented diabetes. The effect of this compound was due in part to marked ␤-cell expansion. These studies indicate that MMPs contribute to islet fibrosis and insulin insufficiency in ZDF rats. Class-targeted protease inhibitors should be explored for their potential therapeutic utility in preservation of ␤-cell mass in type 2 diabetes. Diabetes 54:2612-2619, 2005

Background: Allosteric regulators of GPCRs provide unique pharmacological properties.Results: The mechanism of allosteric inhibition of the glucagon receptor by an antibody, which is uniquely sensitive to a naturally occurring G40S mutation, is detailed.Conclusion: Allosteric sites on the glucagon receptor extracellular domain regulate receptor activity.Significance: Mechanisms of allosteric regulation of GPCRs aid discovery of drugs with improved selectivity.

Patterns of gene expression have been well documented during embryogenesis for the Drosophila melanogaster trunk segments. The same is not the case for the terminal segments. Here, gene expression patterns are followed during embryogenesis in the caudal segments (A8-A10 and the anal plate), with special attention paid to the novel regulation of engrailed (en). Chosen for this study are the pair-rule genes even-skipped (eve), fushi tarazu (ftz), runt (run), hairy (h), paired (prd) and odd-skipped (odd), and the segment polarity gene (en). The results demonstrate a progressive and coupled translocation of gene expression distally for all genes studied, suggesting that the most posterior segments are determined later than trunk segments.