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The identification of LSN3318839, a positive allosteric modulator of the glucagon-like peptide-1 receptor (GLP-1R), is described. LSN3318839 increases the potency and efficacy of the weak metabolite GLP-1(9-36)NH2to become a full agonist at the GLP-1R and modestly potentiates the activity of the highly potent full-length ligand, GLP-1(7-36)NH2. LSN3318839 preferentially enhances G protein-coupled signaling by the GLP-1R over β-arrestin recruitment. Ex vivoexperiments show that the combination of GLP-1(9-36)NH2and LSN3318839 produces glucose-dependent insulin secretion similar to that of GLP-1(7-36)NH2. Under nutrient-stimulated conditions that release GLP-1, LSN3318839 demonstrates robust glucose lowering in animal models alone or in treatment combination with sitagliptin. From a therapeutic perspective, the biological properties of LSN3318839 support the concept that GLP-1R potentiation is sufficient for reducing hyperglycemia.

Drugs that promote the association of protein complexes are an emerging therapeutic strategy. We report discovery of a G protein-coupled receptor (GPCR) ligand that stabilizes an active state conformation by cooperatively binding both the receptor and orthosteric ligand, thereby acting as a ‘molecular glue’. LSN3160440 is a positive allosteric modulator of the GLP-1R optimized to increase the affinity and efficacy of GLP-1(9-36), a proteolytic product of GLP-1(7-36). The compound enhances insulin secretion in a glucose-, ligand- and GLP-1R-dependent manner. Cryo-electron microscopy determined the structure of the GLP-1R bound to LSN3160440 in complex with GLP-1 and heterotrimeric Gs. The modulator binds high in the helical bundle at an interface between TM1 and TM2, allowing access to the peptide ligand. Pharmacological characterization showed strong probe dependence of LSN3160440 for GLP-1(9-36) versus oxyntomodulin that is driven by a single residue. Our findings expand protein–protein modulation drug discovery to uncompetitive, active state stabilizers for peptide hormone receptors.

Class B G-protein-coupled receptors are major targets for the treatment of chronic diseases, including diabetes and obesity1. Structures of active receptors reveal peptide agonists engage deep within the receptor core, leading to an outward movement of extracellular loop 3 and the tops of transmembrane helices 6 and 7, an inward movement of transmembrane helix 1, reorganization of extracellular loop 2 and outward movement of the intracellular side of transmembrane helix 6, resulting in G-protein interaction and activation2–6. Here we solved the structure of a non-peptide agonist, TT-OAD2, bound to the glucagon-like peptide-1 (GLP-1) receptor. Our structure identified an unpredicted non-peptide agonist-binding pocket in which reorganization of extracellular loop 3 and transmembrane helices 6 and 7 manifests independently of direct ligand interaction within the deep transmembrane domain pocket. TT-OAD2 exhibits biased agonism, and kinetics of G-protein activation and signalling that are distinct from peptide agonists. Within the structure, TT-OAD2 protrudes beyond the receptor core to interact with the lipid or detergent, providing an explanation for the distinct activation kinetics that may contribute to the clinical efficacy of this compound series. This work alters our understanding of the events that drive the activation of class B receptors.

Bariatric surgery, such as vertical sleeve gastrectomy (VSG), causes high rates of type 2 diabetes remission and remarkable increases in postprandial glucagon-like peptide-1 (GLP-1) secretion. GLP-1 plays a critical role in islet function by potentiating glucose-stimulated insulin secretion; however, the mechanisms remain incompletely defined. Therefore, we applied a murine VSG model to an inducible β cell-specific GLP-1 receptor (GLP-1R) knockout mouse model to investigate the role of the β cell GLP-1R in islet function. Our data show that loss of β cell GLP-1R signaling decreases α cell GLP-1 expression after VSG. Furthermore, we find a β cell GLP-1R-dependent increase in α cell expression of the prohormone convertase required for the production of GLP-1 after VSG. Together, the findings herein reveal two concepts. First, our data support a paracrine role for α cell-derived GLP-1 in the metabolic benefits observed after VSG. Second, we have identified a role for the β cell GLP-1R as a regulator of α cell proglucagon processing.

In August 2016, several leaders in glucagon biology gathered for the European Association for the Study of Diabetes Hagedorn Workshop in Oxford, England. A key point of discussion focused on the need for basal insulin to allow for the therapeutic benefit of glucagon blockade in the treatment of diabetes. Among the most enlightening experimental results presented were findings from studies in which glucagon receptor-deficient mice were administered streptozotocin to destroy pancreatic β cells or had undergone diphtheria toxin-induced β cell ablation. This article summarizes key features of the discussion as a consensus was reached. Agents that antagonize glucagon may be of great benefit for the treatment of diabetes; however, sufficient levels of basal insulin are required for their therapeutic efficacy.

Vertical sleeve gastrectomy (VSG) produces high rates of type 2 diabetes remission; however, the mechanisms responsible for this remain incompletely defined. Glucagon-like peptide-1 (GLP-1) is a gut hormone that contributes to the maintenance of glucose homeostasis and is elevated after VSG. VSG-induced increases in postprandial GLP-1 secretion have been proposed to contribute to the glucoregulatory benefits of VSG; however, previous work has been equivocal. In order to test the contribution of enhanced β-cell GLP-1 receptor (GLP-1R) signaling we used a β-cell-specific tamoxifen-inducible GLP-1R knockout mouse model. Male β-cell-specific Glp-1rβ-cell+/+wild type (WT) and Glp-1rβ-cell−/−knockout (KO) littermates were placed on a high-fat diet for 6 weeks and then switched to high-fat diet supplemented with tamoxifen for the rest of the study. Mice underwent sham or VSG surgery after 2 weeks of tamoxifen diet and were fed ad libitum postoperatively. Mice underwent oral glucose tolerance testing at 3 weeks and were euthanized at 6 weeks after surgery. VSG reduced body weight and food intake independent of genotype. However, glucose tolerance was only improved in VSG WT compared with sham WT, whereas VSG KO had impaired glucose tolerance relative to VSG WT. Augmentation of glucose-stimulated insulin secretion during the oral glucose tolerance test was blunted in VSG KO compared with VSG WT. Therefore, our data suggest that enhanced β-cell GLP-1R signaling contributes to improved glucose regulation after VSG by promoting increased glucose-stimulated insulin secretion.

Glucagon-like peptide-1 (GLP-1) is an incretin hormone that regulates glucose homeostasis. Because of their direct stimulation of insulin secretion from pancreatic β cells, GLP-1 receptor (GLP-1R) agonists are now important therapeutic options for the treatment of type 2 diabetes. To better understand the mechanisms that control the insulinotropic actions of GLP-1, affinity purification and mass spectrometry (AP-MS) were employed to uncover potential proteins that functionally interact with the GLP-1R. AP-MS performed on Chinese hamster ovary cells or MIN6 β cells, both expressing the human GLP-1R, revealed 99 proteins potentially associated with the GLP-1R. Three novel GLP-1R interactors (PGRMC1, Rab5b, and Rab5c) were further validated through co-immunoprecipitation/immunoblotting, fluorescence resonance energy transfer, and immunofluorescence. Functional studies revealed that overexpression of PGRMC1, a novel cell surface receptor that associated with liganded GLP-1R, enhanced GLP-1-induced insulin secretion (GIIS) with the most robust effect. Knockdown of PGRMC1 in β cells decreased GIIS, indicative of positive interaction with GLP-1R. To gain insight mechanistically, we demonstrated that the cell surface PGRMC1 ligand P4-BSA increased GIIS, whereas its antagonist AG-205 decreased GIIS. It was then found that PGRMC1 increased GLP-1-induced cAMP accumulation. PGRMC1 activation and GIIS induced by P4-BSA could be blocked by inhibition of adenylyl cyclase/EPAC signaling or the EGF receptor–PI3K signal transduction pathway. These data reveal a dual mechanism for PGRMC1-increased GIIS mediated through cAMP and EGF receptor signaling. In conclusion, we identified several novel GLP-1R interacting proteins. PGRMC1 expressed on the cell surface of β cells was shown to interact with the activated GLP-1R to enhance the insulinotropic actions of GLP-1.

The glucagon-like peptide-1 receptor (GLP-1R) is a major therapeutic target for the treatment of type 2 diabetes due to its role in glucose homeostasis. Despite the availability of peptide-based GLP-1R drugs for treatment of this disease, there is great interest in developing small molecules that can be administered orally. The GLP-1R system is complex, with multiple endogenous and clinically used peptide ligands that exhibit different signaling biases at this receptor. This study revealed that small molecule ligands acting at this receptor are differentially biased to peptide ligands and also from each other with respect to the signaling pathways that they activate. Furthermore, allosteric small molecule ligands were also able to induce bias in signaling mediated by orthosteric ligands. This was dependent on both the orthosteric and allosteric ligand as no two allosteric-orthosteric ligand pairs could induce the same signaling profile. We highlight the need to profile compounds across multiple signaling pathways and in combination with multiple orthosteric ligands in systems such as the GLP-1R where more than one endogenous ligand exists. In the context of pleiotropical coupling of receptors and the interplay of multiple pathways leading to physiologic responses, profiling of small molecules in this manner may lead to a better understanding of the physiologic consequences of biased signaling at this receptor. This could enable the design and development of improved therapeutics that have the ability to fine-tune receptor signaling, leading to beneficial therapeutic outcomes while reducing side effect profiles.

Identifying novel mechanisms to enhance glucagon-like peptide-1 (GLP-1) receptor signaling may enable nascent medicinal chemistry strategies with the aim of developing new orally available therapeutic agents for the treatment of type 2 diabetes mellitus. Therefore, we tested the hypothesis that selectively modulating the low-affinity GLP-1 receptor agonist, oxyntomodulin, would improve the insulin secretory properties of this naturally occurring hormone to provide a rationale for pursuing an unexplored therapeutic approach. Signal transduction and competition binding studies were used to investigate oxyntomodulin activity on the GLP-1 receptor in the presence of the small molecule GLP-1 receptor modulator, 4-(3-benzyloxyphenyl)-2-ethylsulfinyl-6-(trifluoromethyl)pyrimidine (BETP). In vivo, the intravenous glucose tolerance test characterized oxyntomodulin-induced insulin secretion in animals administered the small molecule. BETP increased oxyntomodulin binding affinity for the GLP-1 receptor and enhanced oxyntomodulin-mediated GLP-1 receptor signaling as measured by activation of the α subunit of heterotrimeric G protein and cAMP accumulation. In addition, oxyntomodulin-induced insulin secretion was enhanced in the presence of the compound. BETP was pharmacologically characterized to induce biased signaling by oxyntomodulin. These studies demonstrate that small molecules targeting the GLP-1 receptor can increase binding and receptor activation of the endogenous peptide oxyntomodulin. The biased signaling engendered by BETP suggests that GLP-1 receptor mobilization of cAMP is the critical insulinotropic signaling event. Because of the unique metabolic properties of oxyntomodulin, identifying molecules that enhance its activity should be pursued to assess the efficacy and safety of this novel mechanism.

G protein-coupled receptors (GPCRs) are the largest family of cell surface receptors and a key drug target class. Recently, allosteric drugs that can cobind with and modulate the activity of the endogenous ligand(s) for the receptor have become a major focus of the pharmaceutical and biotechnology industry for the development of novel GPCR therapeutic agents. This class of drugs has distinct properties compared with drugs targeting the endogenous (orthosteric) ligand-binding site that include the ability to sculpt cellular signaling and to respond differently in the presence of discrete orthosteric ligands, a behavior termed “probe dependence.” Here, using cell signaling assays combined with ex vivo and in vivo studies of insulin secretion, we demonstrate that allosteric ligands can cause marked potentiation of previously “inert” metabolic products of neurotransmitters and peptide hormones, a novel consequence of the phenomenon of probe dependence. Indeed, at the muscarinic M2receptor and glucagon-like peptide 1 (GLP-1) receptor, allosteric potentiation of the metabolites, choline and GLP-1(9–36)NH2, respectively, was ∼100-fold and up to 200-fold greater than that seen with the physiological signaling molecules acetylcholine and GLP-1(7–36)NH2. Modulation of GLP-1(9–36)NH2was also demonstrated in ex vivo and in vivo assays of insulin secretion. This work opens up new avenues for allosteric drug discovery by directly targeting modulation of metabolites, but it also identifies a behavior that could contribute to unexpected clinical outcomes if interaction of allosteric drugs with metabolites is not part of their preclinical assessment.

Glucagon is the key counter-regulatory hormone that opposes the action of insulin. In states of relative hypoglycaemia, glucagon acts to increase blood glucose by stimulating hepatic glycogen breakdown and gluconeogenesis to achieve euglycaemia. Type 2 diabetes is characterised by inappropriate regulation of hepatic glucose production, which is due, at least in part, to an imbalance in the bihormonal relationship between plasma levels of glucagon and insulin. The glucose-lowering effects of glucagon peptide antagonists and antiglucagon neutralising antibodies first demonstrated the potential of glucagon receptor (GCGR) antagonism as a treatment for hyperglycaemia. In recent years, the development of GCGR antisense oligonucleotides and small molecular weight GCGR antagonists have been pursued as possible therapeu-tic agents to target glucagon action as a treatment for Type 2 diabe-tes.

The pituitary gland is a complex endocrine organ secreting hormones that regulate a wide array of vertebrate physiological processes, including growth, lactation, metabolic homeostasis, reproduction, water balance, and the stress response. In the mature organ, specialized cells that have a common origin in the early ectoderm release their characteristic products into the bloodstream. Together, the embryological processes that commit the hormone-secreting cells to their specific fates and the clinical and agricultural relevance of understanding pituitary function have defined pituitary development as an excellent model system for the study of the genetic cascades that guide cell determination and differentiation. Recently, many genes that regulate pituitary development have been identified. These genes encode transcription factors and signaling proteins and often are expressed in temporally controlled, pituitary-specific or pituitary-restricted patterns. Further, dominant and recessive mutations in these genes are associated with compound pituitary diseases in human patients and animal models. The advance of genome projects will facilitate approaches to understand the genetic mechanisms that regulate the activation of pituitary regulatory genes and to discover how the function of the encoded transcription factors is precisely regulated by intrinsic and extrapituitary signals. Gene array and differential screening approaches will enable the identification of direct and downstream target genes of pituitary transcription factors. Protein interaction screens will identify regulatory proteins required for pituitary gene control. Characterization of the pathways that coordinate pituitary development will direct treatment of pediatric and adult pituitary diseases, guide genetic counseling of families with hereditary conditions affecting the pituitary, and may allow embryonic manipulation to improve productivity in the meat industry.

The LHX3 LIM homeodomain transcription factor is required for pituitary development and motor neuron specification. TheLhx3gene encodes two isoforms, LHX3a and LHX3b, that differ in their amino-terminal sequences. Humans and mice with defective Lhx3genes are deficient in gonadotrope, lactotrope, somatotrope, and thyrotrope pituitary cells. We show that, whereas Lhx3bis highly expressed in theseLhx3-dependent cell types, high levels ofLhx3aexpression are restricted to α glycoprotein subunit-expressing thyrotropes and gonadotropes. Cross-species comparison reveals the LHX3b-specific domain is more conserved than the LHX3a-specific domain. We demonstrate that the LHX3b-specific domain is a transferable inhibitor that reduces gene activation and DNA binding by homeodomain proteins. In addition, we identify a novel LHX3 protein (M2-LHX3) and determine that this molecule is generated by an internal translation initiation codon. The LHX3a- and LHX3b-specific coding sequences regulate differential usage of this internal start codon. Further, we identify the major activation domain of LHX3 in the carboxyl terminus of the molecule. M2-LHX3 is active because it retains this domain and binds DNA better than LHX3a or LHX3b. Other LIM homeodomain genes, including Lhx4, generate similar truncated proteins. These studies describe how transcriptional regulatory genes can generate multiple functional proteins.

The Lhx3 LIM homeodomain transcription factor is critical for pituitary gland formation and specification of the anterior pituitary hormone-secreting cell types. Two mutations in LHX3, a missense mutation changing a tyrosine to a cysteine and an intragenic deletion that results in a truncated protein lacking the DNA-binding homeodomain, have been identified in humans. These mutations were identified in patients with retarded growth and combined pituitary hormone deficiency and also abnormal neck and cervical spine development. For both the LHX3a and LHX3b isoforms, we compared the ability of wild type and mutant LHX3 proteins to trans-activate pituitary genes, bind DNA recognition elements, and interact with partner proteins. The tyrosine missense mutation inhibits the ability of LHX3 to induce transcription from selected target genes but does not prevent DNA binding and interaction with partner proteins such as NLI and Pit-1. Mutant LHX3 proteins lacking a homeodomain do not bind DNA and do not induce transcription from pituitary genes. These studies demonstrate that mutations in the LHX3 isoforms impair their gene regulatory functions and support the hypothesis that defects in the LHX3gene cause complex pituitary disease in humans.

Leptin, the product of theobgene, is expressed exclusively in adipose tissue. However, adipocyte cell lines, such as 3T3-L1 adipocytes, have generally been reported to express extremely low levels of leptin mRNA. We compared 3T3-L1's to the closely related line 3T3-F442A, and to another murine adipocyte line, TA1. TA1 cells, when differentiated by indomethacin/insulin treatment, express leptin at levels greater than those of 3T3-L1 adipocytes differentiated by the traditional methylisobutylxanthine/dexamethasone/insulin protocol. However, when 3T3-L1's are differentiated in the presence of indomethacin/insulin their expression levels of leptin increase dramatically. 3T3-F442A preadipocytes also express high levels of leptin when differentiated in the presence of T3and insulin, but when differentiated in the presence of indomethacin/insulin, expression levels drop precipitously. These changes in leptin mRNA and protein expression are not reflected by changes in CCAAT/enhancer binding protein-α (c/EBPα), peroxizomal proliferator activated receptor-γ (PPARγ), lipoprotein lipase (LPL), fatty-acid binding protein aP2 or uncoupling protein-2 (UCP2) mRNA levels, and suggest a mechanism unique to the leptin gene.

An adipocyte factor transducing the quantity of adipose to a hormonal message that targets the hypothalamus has been postulated for many years. These findings indicate that adipose tissue performs important biological functions beyond storage of fat. In fact, data indicate that adipose tissue functions very much like other endocrine tissues, releasing a hormone named leptin into the circulatory system to relay a message to its targets. Although there may be more than one target tissue for leptin, the best is the hypothalamus. Because most types of human and murine obesity are accompanied by elevated leptin levels and the concentrations of this hormone correlate well with degree of adiposity, it is thought that leptin may serve as a fuel gage for the hypothalamus. When sufficient fuel is present or when lipid is being synthesized and stored in adipose, leptin levels rise and reduce further feeding behavior. In addition, a signal of abundant fuel reserve serves as a message that energy can be expended for anabolism of protein and other biological processes such as growth, repair, and reproduction. Our data, together with those reported by others, indicate that leptin inhibits the hypothalamic-pituitary-adrenal axis and thus antagonizes the catabolic glucocorticoids. A review of data available also supports that leptin stimulates the hypothalamic-growth hormone axis and, hence, promotes protein synthesis. Energy must be expended to support this anabolic state and data indicate the energy is derived from oxidative metabolism of fuel that is stimulated by the hypothalamic-pituitary-thyroid axis. In conclusion, each of these neuroendocrine axes require information of the fuel supply to function efficiently and this data can be at least partially supplied by leptin.

C/EBP-α binds a C/EBP consensus site in the leptin promoter and activates transcriptionin vitro.We assessed adipose tissue expression of C/EBP-α, leptin and β-actin in Sprague Dawley rats under conditions that modulate leptin mRNA abundance in order to study the relationship between leptin and C/EBP-α expression patterns. During acute fasting, which decreased the level of leptin and β-actin mRNA, C/EBP-α mRNA expression was unaltered. In leptin-treated and pair-fed animals, C/EBP-α mRNA was unaltered compared toad libitumfed controls, while leptin and β-actin mRNA expression was again decreased. These results indicate that changes in the level of leptin gene expression are not directly associated with changes in the level of C/EBP-α abundance.

Regulation of obesegene (ob) expression in ob/oband db/dbmice and in cultured rat adipocytes was examined. It has been demonstrated that exogenous human OB protein (leptin) treatment reduces food intake and weight gain, as well as insulin, glucose, and corticosterone levels in ob/obmice. In the present report we show that leptin treatment down-regulates endogenous adipose obmRNA. However, treatment of isolated rat adipocytes with 100 ng/ml human or murine leptin had no direct effect on expression of endogenous obmRNA, suggesting that leptin may be able to down-regulate its own expression by an indirect, non-autocrine mechanism. Glucocorticoids increased both obmRNA levels and secreted leptin levels in vitro. Conversely, agents that increase intracellular cAMP, such as β-adrenergic agonists or Bt2cAMP itself, decreased obmRNA expression and leptin secretion. Therefore, increased glucocorticoid levels and decreased sympathetic neural activity may contribute to the elevated obmRNA expression observed in genetically obese, hyperglucocorticoid rodents. Furthermore, leptin might regulate its own expression through a feedback mechanism involving the hypothalamic pituitary axis.