Customizable Click Biochemistry Strategy for the Design and Preparation of Glucagon-like Peptide-1 Conjugates and Coagonists.

The development of oligomeric glucagon-like peptide-1 (GLP-1) and GLP-1-containing coagonists holds promise for enhancing the therapeutic potential of the GLP-1-based drugs for treating type 2 diabetes mellitus (T2DM). Here, we report a facile, efficient, and customizable strategy based on genetically encoded SpyCatcher-SpyTag chemistry and an inducible, cleavable self-aggregating tag (icSAT) scheme. icSAT-tagged SpyTag-fused GLP-1 and the dimeric or trimeric SpyCatcher scaffold were designed for dimeric or trimeric GLP-1, while icSAT-tagged SpyCatcher-fused GLP-1 and the icSAT-tagged SpyTag-fused GIP were designed for dual GLP-1/GIP (glucose-dependent insulinotropic polypeptide) receptor agonist. These SpyCatcher- and SpyTag-fused protein pairs were spontaneously ligated directly from the cell lysates. The subsequent icSAT scheme, coupled with a two-step standard column purification, resulted in target proteins with authentic N-termini, with yields ranging from 35 to 65 mg/L and purities exceeding 99%. In vitro assays revealed 3.0- to 4.1-fold increased activities for dimeric and trimeric GLP-1 compared to mono-GLP-1. The dual GLP-1/GIP receptor agonist exhibited balanced activity toward the GLP-1 receptor or the GIP receptor. All the proteins exhibited 1.8- to 3.0-fold prolonged half-lives in human serum compared to mono-GLP-1 or GIP. This study provides a generally applicable click biochemistry strategy for developing oligomeric or dual peptide/protein-based drug candidates.

Impact of secretin receptor homo-dimerization on natural ligand binding.

Class B G protein-coupled receptors can form dimeric complexes important for high potency biological effects. Here, we apply pharmacological, biochemical, and biophysical techniques to cells and membranes expressing the prototypic secretin receptor (SecR) to gain insights into secretin binding to homo-dimeric and monomeric SecR. Spatial proximity between peptide and receptor residues, probed by disulfide bond formation, demonstrates that the secretin N-terminus moves from adjacent to extracellular loop 3 (ECL3) at wild type SecR toward ECL2 in non-dimerizing mutants. Analysis of fluorescent secretin analogs demonstrates stable engagement of the secretin C-terminal region within the receptor extracellular domain (ECD) for both dimeric and monomeric receptors, while the mid-region exhibits lower mobility while docked at the monomer. Moreover, decoupling of G protein interaction reduces mobility of the peptide mid-region at wild type receptor to levels similar to the mutant, whereas it has no further impact on the monomer. These data support a model of peptide engagement whereby the ability of SecR to dimerize promotes higher conformational dynamics of the peptide-bound receptor ECD and ECLs that likely facilitates more efficient G protein recruitment and activation, consistent with the higher observed functional potency of secretin at wild type SecR relative to the monomeric mutant receptor.

Mapping the interaction site for ß-arrestin-2 in the prokineticin 2 receptor.

G protein-coupled receptors (GPCRs) are a family of cell membrane receptors that couple and activate heterotrimeric G proteins and their associated intracellular signalling processes after ligand binding. Although the carboxyl terminal of the receptors is essential for this action, it can also serve as a docking site for regulatory proteins such as the ß-arrestins. Prokineticin receptors (PKR1 and PKR2) are a new class of GPCRs that are able to activate different classes of G proteins and form complexes with ß-arrestins after activation by the endogenous agonists PK2. The aim of this work was to define the molecular determinants within PKR2 that are required for ß-arrestin-2 binding and to investigate the role of ß-arrestin-2 in the signalling pathways induced by PKR2 activation. Our data show that PKR2 binds constitutively to ß-arrestin-2 and that this process occurs through the core region of the receptor without being affected by the carboxy-terminal region. Indeed, a PKR2 mutant lacking the carboxy-terminal amino acids retains the ability to bind constitutively to ß-arrestin-2, whereas a mutant lacking the third intracellular loop does not. Overall, our data suggest that the C-terminus of PKR2 is critical for the stability of the ß-arrestin-2-receptor complex in the presence of PK2 ligand. This leads to the ß-arrestin-2 conformational change required to initiate intracellular signalling that ultimately leads to ERK phosphorylation and activation.

Stimulating intestinal GIP release reduces food intake and body weight in mice.

OBJECTIVE: Glucose dependent insulinotropic polypeptide (GIP) is well established as an incretin hormone, boosting glucose-dependent insulin secretion. However, whilst anorectic actions of its sister-incretin glucagon-like peptide-1 (GLP-1) are well established, a physiological role for GIP in appetite regulation is controversial, despite the superior weight loss seen in preclinical models and humans with GLP-1/GIP dual receptor agonists compared with GLP-1R agonism alone. METHODS: We generated a mouse model in which GIP expressing K-cells can be activated through hM3Dq Designer Receptor Activated by Designer Drugs (DREADD, GIP-Dq) to explore physiological actions of intestinally-released GIP. RESULTS: In lean mice, Dq-stimulation of GIP expressing cells increased plasma GIP to levels similar to those found postprandially. The increase in GIP was associated with improved glucose tolerance, as expected, but also triggered an unexpected robust inhibition of food intake. Validating that this represented a response to intestinally-released GIP, the suppression of food intake was prevented by injecting mice peripherally or centrally with antagonistic GIPR-antibodies, and was reproduced in an intersectional model utilising Gip-Cre/Villin-Flp to limit Dq transgene expression to K-cells in the intestinal epithelium. The effects of GIP cell activation were maintained in diet induced obese mice, in which chronic K-cell activation reduced food intake and attenuated body weight gain. CONCLUSIONS: These studies establish a physiological gut-brain GIP-axis regulating food intake in mice, adding to the multi-faceted metabolic effects of GIP which need to be taken into account when developing GIPR-targeted therapies for obesity and diabetes.

The interplay of glucose-dependent insulinotropic polypeptide in adipose tissue.

Adipose tissue was once known as a reservoir for energy storage but is now considered a crucial organ for hormone and energy flux with important effects on health and disease. Glucose-dependent insulinotropic polypeptide (GIP) is an incretin hormone secreted from the small intestinal K cells, responsible for augmenting insulin release, and has gained attention for its independent and amicable effects with glucagon-like peptide 1 (GLP-1), another incretin hormone secreted from the small intestinal L cells. The GIP receptor (GIPR) is found in whole adipose tissue, whereas the GLP-1 receptor (GLP-1R) is not, and some studies suggest that GIPR action lowers body weight and plays a role in lipolysis, glucose/lipid uptake/disposal, adipose tissue blood flow, lipid oxidation, and free-fatty acid (FFA) re-esterification, which may or may not be influenced by other hormones such as insulin. This review summarizes the research on the effects of GIP in adipose tissue (distinct depots of white and brown) using cellular, rodent, and human models. In doing so, we explore the mechanisms of GIPR-based medications for treating metabolic disorders, such as type 2 diabetes and obesity, and how GIPR agonism and antagonism contribute to improvements in metabolic health outcomes, potentially through actions in adipose tissues.

Motilin, a Novel Orexigenic Factor, Involved in Feeding Regulation in Yangtze Sturgeon (Acipenser dabryanus).

Motilin is a gastrointestinal hormone that is mainly produced in the duodenum of mammals, and it is responsible for regulating appetite. However, the role and expression of motilin are poorly understood during starvation and the weaning stage, which is of great importance in the seeding cultivation of fish. In this study, the sequences of Yangtze sturgeon (Acipenser dabryanus Motilin (AdMotilin)) motilin receptor (AdMotilinR) were cloned and characterized. The results of tissue expression showed that by contrast with mammals, AdMotilin mRNA was richly expressed in the brain, whereas AdMotilinR was highly expressed in the stomach, duodenum, and brain. Weaning from a natural diet of T. Limnodrilus to commercial feed significantly promoted the expression of AdMotilin in the brain during the period from day 1 to day 10, and after re-feeding with T. Limnodrilus the change in expression of AdMotilin was partially reversed. Similarly, it was revealed that fasting increased the expression of AdMotilin in the brain (3 h, 6 h) and duodenum (3 h), and the expression of AdMotilinR in the brain (1 h) in a time-dependent manner. Furthermore, it was observed that peripheral injection of motilin-NH2 increased food intake and the filling index of the digestive tract in the Yangtze sturgeon, which was accompanied by the changes of AdMotilinR and appetite factors expression in the brain (POMC, CART, AGRP, NPY and CCK) and stomach (CCK). These results indicate that motilin acts as an indicator of nutritional status, and also serves as a novel orexigenic factor that stimulates food intake in Acipenser dabryanus. This study lays a strong foundation for the application of motilin as a biomarker in the estimation of hunger in juvenile Acipenser dabryanu during the weaning phase, and enhances the understanding of the role of motilin as a novel regulator of feeding in fish.

A Robust Platform for the Molecular Design of Potent, Protease-Stable, Long-Acting GIP Analogues.

Glucose-dependent insulinotropic peptide (GIP) is a 42-amino acid peptide hormone that regulates postprandial glucose levels. GIP binds to its cognate receptor, GIPR, and mediates metabolic physiology by improved insulin sensitivity, ß-cell proliferation, increased energy consumption, and stimulated glucagon secretion. Dipeptidyl peptidase-4 (DPP4) catalyzes the rapid inactivation of GIP within 6 min in vivo. Here, we report a molecular platform for the design of GIP analogues that are refractory to DPP4 action and exhibit differential activation of the receptor, thus offering potentially hundreds of GIP-based compounds to fine-tune pharmacology. The lead compound from our studies, which harbored a combination of N-terminal alkylation and side-chain lipidation, was equipotent and retained full efficacy at GIPR as the native peptide, while being completely refractory toward DPP4, and was resistant to trypsin. The GIP analogue identified from these studies was further evaluated in vivo and is one of the longest-acting GIPR agonists to date.

Loss of GIPR in LEPR cells impairs glucose control by GIP and GIP:GLP-1 co-agonism without affecting body weight and food intake in mice.

OBJECTIVE: The glucose-dependent insulinotropic polypeptide (GIP) decreases body weight via central GIP receptor (GIPR) signaling, but the underlying mechanisms remain largely unknown. Here, we assessed whether GIP regulates body weight and glucose control via GIPR signaling in cells that express the leptin receptor (Lepr). METHODS: Hypothalamic, hindbrain, and pancreatic co-expression of Gipr and Lepr was assessed using single cell RNAseq analysis. Mice with deletion of Gipr in Lepr cells were generated and metabolically characterized for alterations in diet-induced obesity (DIO), glucose control and leptin sensitivity. Long-acting single- and dual-agonists at GIPR and GLP-1R were further used to assess drug effects on energy and glucose metabolism in DIO wildtype (WT) and Lepr-Gipr knock-out (KO) mice. RESULTS: Gipr and Lepr show strong co-expression in the pancreas, but not in the hypothalamus and hindbrain. DIO Lepr-Gipr KO mice are indistinguishable from WT controls related to body weight, food intake and diet-induced leptin resistance. Acyl-GIP and the GIPR:GLP-1R co-agonist MAR709 remain fully efficacious to decrease body weight and food intake in DIO Lepr-Gipr KO mice. Consistent with the demonstration that Gipr and Lepr highly co-localize in the endocrine pancreas, including the ß-cells, we find the superior glycemic effect of GIPR:GLP-1R co-agonism over single GLP-1R agonism to vanish in Lepr-Gipr KO mice. CONCLUSIONS: GIPR signaling in cells/neurons that express the leptin receptor is not implicated in the control of body weight or food intake, but is of crucial importance for the superior glycemic effects of GIPR:GLP-1R co-agonism relative to single GLP-1R agonism.

Regulation of energy metabolism through central GIPR signaling.

In recent years, significant progress has been made to pharmacologically combat the obesity pandemic, particularly with regard to biochemically tailored drugs that simultaneously target the receptors for glucagon-like peptide-1 (GLP-1) and the glucose-dependent insulinotropic polypeptide (GIP). But while the pharmacological benefits of GLP-1 receptor (GLP-1R) agonism are widely acknowledged, the role of the GIP system in regulating systems metabolism remains controversial. When given in adjunct to GLP-1R agonism, both agonism and antagonism of the GIP receptor (GIPR) improves metabolic outcome in preclinical and clinical studies, and despite persistent concerns about its potential obesogenic nature, there is accumulating evidence indicating that GIP has beneficial metabolic effects via central GIPR agonism. Nonetheless, despite growing recognition of the GIP system as a valuable pharmacological target, there remains great uncertainty as to where and how GIP acts in the brain to regulate metabolism, and how GIPR agonism may differ from GIPR antagonism in control of energy metabolism. In this review we highlight current knowledge on the central action of GIP, and discuss open questions related to its multifaceted biology in the brain and the periphery.

Immunomodulation and inflammation: Role of GLP-1R and GIPR expressing cells within the gut.

Glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are peptide hormones produced by enteroendocrine cells in the small intestine. Despite being produced in the gut, the leveraging of their role in potentiating glucose-stimulated insulin secretion, also known as the incretin effect, has distracted from discernment of direct intestinal signaling circuits. Both preclinical and clinical evidence have highlighted a role for the incretins in inflammation. In this review, we highlight the discoveries of GLP-1 receptor (GLP-1R)+ natural (TCRαß and TCRγδ) and induced (TCRαß+CD4+ cells and TCRαß+CD8αß+) intraepithelial lymphocytes. Both endogenous signaling and pharmacological activation of GLP-1R impact local and systemic inflammation, the gut microbiota, whole-body metabolism, as well as the control of GLP-1 bioavailability. While GIPR signaling has been documented to impact hematopoiesis, the impact of these bone marrow-derived cells in gut immunology is not well understood. We uncover gaps in the literature of the evaluation of the impact of sex in these GLP-1R and GIP receptor (GIPR) signaling circuits and provide speculations of the maintenance roles these hormones play within the gut in the fasting-refeeding cycles. GLP-1R agonists and GLP-1R/GIPR agonists are widely used as treatments for diabetes and weight loss, respectively; however, their impact on gut homeostasis has not been fully explored. Advancing our understanding of the roles of GLP-1R and GIPR signaling within the gut at homeostasis as well as metabolic and inflammatory diseases may provide targets to improve disease management.

GLP1R and GIPR expression and signaling in pancreatic alpha cells, beta cells and delta cells.

Glucagon-like peptide-1 receptor (GLP1R) and glucose-dependent insulinotropic polypeptide receptor (GIPR) are transmembrane receptors involved in insulin, glucagon and somatostatin secretion from the pancreatic islet. Therapeutic targeting of GLP1R and GIPR restores blood glucose levels in part by influencing beta cell, alpha cell and delta cell function. Despite the importance of the incretin-mimetics for diabetes therapy, our understanding of GLP1R and GIPR expression patterns and signaling within the islet remain incomplete. Here, we present the evidence for GLP1R and GIPR expression in the major islet cell types, before addressing signaling pathway(s) engaged, as well as their influence on cell survival and function. While GLP1R is largely a beta cell-specific marker within the islet, GIPR is expressed in alpha cells, beta cells, and (possibly) delta cells. GLP1R and GIPR engage Gs-coupled pathways in most settings, although the exact outcome on hormone release depends on paracrine communication and promiscuous signaling. Biased agonism away from beta-arrestin is an emerging concept for improving therapeutic efficacy, and is also relevant for GLP1R/GIPR dual agonism. Lastly, dual agonists exert multiple effects on islet function through GIPR > GLP1R imbalance, increased GLP1R surface expression and cAMP signaling, as well as beneficial alpha cell-beta cell-delta cell crosstalk.

GIPR Agonism Enhances TZD-Induced Insulin Sensitivity in Obese IR Mice.

Recent studies have found that glucose-dependent insulinotropic polypeptide receptor (GIPR) agonism can enhance the metabolic efficacy of glucagon-like peptide-1 receptor agonist treatment by promoting both weight-dependent and -independent improvements on systemic insulin sensitivity. These findings have prompted new investigations aimed at better understanding the broad metabolic benefit of GIPR activation. Herein, we determined whether GIPR agonism favorably influenced the pharmacologic efficacy of the insulin-sensitizing thiazolidinedione (TZD) rosiglitazone in obese insulin-resistant (IR) mice. Genetic and pharmacological approaches were used to examine the role of GIPR signaling on rosiglitazone-induced weight gain, hyperphagia, and glycemic control. RNA sequencing was conducted to uncover potential mechanisms by which GIPR activation influences energy balance and insulin sensitivity. In line with previous findings, treatment with rosiglitazone induced the mRNA expression of the GIPR in white and brown fat. However, obese GIPR-null mice dosed with rosiglitazone had equivalent weight gain to that of wild-type (WT) animals. Strikingly, chronic treatment of obese IR WT animals with a long-acting GIPR agonist prevented rosiglitazone-induced weight-gain and hyperphagia, and it enhanced the insulin-sensitivity effect of this TZD. The systemic insulin sensitization was accompanied by increased glucose disposal in brown adipose tissue, which was underlined by the recruitment of metabolic and thermogenic genes. These findings suggest that GIPR agonism can counter the negative consequences of rosiglitazone treatment on body weight and adiposity, while improving its insulin-sensitizing efficacy at the same time.

Molecular basis of signal transduction mediated by the human GIPR splice variants.

Glucose-dependent insulinotropic polypeptide receptor (GIPR) is a potential drug target for metabolic disorders. It works with glucagon-like peptide-1 receptor and glucagon receptor in humans to maintain glucose homeostasis. Unlike the other two receptors, GIPR has at least 13 reported splice variants (SVs), more than half of which have sequence variations at either C or N terminus. To explore their roles in endogenous peptide-mediated GIPR signaling, we determined the cryoelectron microscopy (cryo-EM) structures of the two N terminus-altered SVs (referred as GIPR-202 and GIPR-209 in the Ensembl database, SV1 and SV2 here, respectively) and investigated the outcome of coexpressing each of them in question with GIPR in HEK293T cells with respect to ligand binding, receptor expression, cAMP (adenosine 3,5-cyclic monophosphate) accumulation, ß-arrestin recruitment, and cell surface localization. It was found that while both N terminus-altered SVs of GIPR neither bound to the hormone nor elicited signal transduction per se, they suppressed ligand binding and cAMP accumulation of GIPR. Meanwhile, SV1 reduced GIPR-mediated ß-arrestin 2 responses. The cryo-EM structures of SV1 and SV2 showed that they reorganized the extracellular halves of transmembrane helices 1, 6, and 7 and extracellular loops 2 and 3 to adopt a ligand-binding pocket-occupied conformation, thereby losing binding ability to the peptide. The results suggest a form of signal bias that is constitutive and ligand-independent, thus expanding our knowledge of biased signaling beyond pharmacological manipulation (i.e., ligand specific) as well as constitutive and ligand-independent (e.g., SV1 of the growth hormone-releasing hormone receptor).

The Methylation Analysis of the Glucose-Dependent Insulinotropic Polypeptide Receptor (GIPR) Locus in GH-Secreting Pituitary Adenomas.

The glucose-dependent insulinotropic polypeptide receptor (GIPR) is aberrantly expressed in about one-third of GH-secreting pituitary adenomas (GH-PAs) and has been associated with a paradoxical increase of GH after a glucose load. The reason for such an overexpression has not yet been clarified. In this work, we aimed to evaluate whether locus-specific changes in DNA methylation patterns could contribute to this phenomenon. By cloning bisulfite-sequencing PCR, we compared the methylation pattern of the GIPR locus in GIPR-positive (GIPR+) and GIPR-negative (GIPR-) GH-PAs. Then, to assess the correlation between Gipr expression and locus methylation, we induced global DNA methylation changes by treating the lactosomatotroph GH3 cells with 5-aza-2'-deoxycytidine. Differences in methylation levels were observed between GIPR+ and GIPR- GH-PAs, both within the promoter (31.9% vs. 68.2%, p < 0.05) and at two gene body regions (GB_1 20.7% vs. 9.1%; GB_2 51.2% vs. 65.8%, p < 0.05). GH3 cells treated with 5-aza-2'-deoxycytidine showed a ~75% reduction in Gipr steady-state level, possibly associated with the observed decrease in CpGs methylation. These results indicate that epigenetic regulation affects GIPR expression in GH-PAs, even though this possibly represents only a part of a much more complex regulatory mechanism.

Hypothalamic and brainstem glucose-dependent insulinotropic polypeptide receptor neurons employ distinct mechanisms to affect feeding.

Central glucose-dependent insulinotropic polypeptide (GIP) receptor (GIPR) signaling is critical in GIP-based therapeutics' ability to lower body weight, but pathways leveraged by GIPR pharmacology in the brain remain incompletely understood. We explored the role of Gipr neurons in the hypothalamus and dorsal vagal complex (DVC) - brain regions critical to the control of energy balance. Hypothalamic Gipr expression was not necessary for the synergistic effect of GIPR/GLP-1R coagonism on body weight. While chemogenetic stimulation of both hypothalamic and DVC Gipr neurons suppressed food intake, activation of DVC Gipr neurons reduced ambulatory activity and induced conditioned taste avoidance, while there was no effect of a short-acting GIPR agonist (GIPRA). Within the DVC, Gipr neurons of the nucleus tractus solitarius (NTS), but not the area postrema (AP), projected to distal brain regions and were transcriptomically distinct. Peripherally dosed fluorescent GIPRAs revealed that access was restricted to circumventricular organs in the CNS. These data demonstrate that Gipr neurons in the hypothalamus, AP, and NTS differ in their connectivity, transcriptomic profile, peripheral accessibility, and appetite-controlling mechanisms. These results highlight the heterogeneity of the central GIPR signaling axis and suggest that studies into the effects of GIP pharmacology on feeding behavior should consider the interplay of multiple regulatory pathways.

Structural basis for motilin and erythromycin recognition by motilin receptor.

Motilin is an endogenous peptide hormone almost exclusively expressed in the human gastrointestinal (GI) tract. It activates the motilin receptor (MTLR), a class A G protein-coupled receptor (GPCR), and stimulates GI motility. To our knowledge, MTLR is the first GPCR reported to be activated by macrolide antibiotics, such as erythromycin. It has attracted extensive attention as a potential drug target for GI disorders. We report two structures of Gq-coupled human MTLR bound to motilin and erythromycin. Our structures reveal the recognition mechanism of both ligands and explain the specificity of motilin and ghrelin, a related gut peptide hormone, for their respective receptors. These structures also provide the basis for understanding the different recognition modes of erythromycin by MTLR and ribosome. These findings provide a framework for understanding the physiological regulation of MTLR and guiding drug design targeting MTLR for the treatment of GI motility disorders.

Differential effects of glucose-dependent insulinotropic polypeptide receptor/glucagon-like peptide-1 receptor heteromerization on cell signaling when expressed in HEK-293 cells.

The incretin hormones: glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) are important regulators of many aspects of metabolism including insulin secretion. Their receptors (GIPR and GLP-1R) are closely related members of the secretin class of G-protein-coupled receptors. As both receptors are expressed on pancreatic ß-cells there is at least the hypothetical possibility that they may form heteromers. In the present study, we investigated GIPR/GLP-1R heteromerization and the impact of GIPR on GLP-1R-mediated signaling and vice versa in HEK-293 cells. Real-time fluorescence resonance energy transfer (FRET) and bioluminescence resonance energy transfer (BRET) saturation experiments confirm that GLP-1R and GIPR form heteromers. Stimulation with 1 µM GLP-1 caused an increase in both FRET and BRET ratio, whereas stimulation with 1 µM GIP caused a decrease. The only other ligand tested to cause a significant change in BRET signal was the GLP-1 metabolite, GLP-1 (9-36). GIPR expression had no significant effect on mini-Gs recruitment to GLP-1R but significantly inhibited GLP-1 stimulated mini-Gq and arrestin recruitment. In contrast, the presence of GLP-1R improved GIP stimulated mini-Gs and mini-Gq recruitment to GIPR. These data support the hypothesis that GIPR and GLP-1R form heteromers with differential consequences on cell signaling.

LY3437943, a novel triple glucagon, GIP, and GLP-1 receptor agonist for glycemic control and weight loss: From discovery to clinical proof of concept.

With an increasing prevalence of obesity, there is a need for new therapies to improve body weight management and metabolic health. Multireceptor agonists in development may provide approaches to fulfill this unmet medical need. LY3437943 is a novel triple agonist peptide at the glucagon receptor (GCGR), glucose-dependent insulinotropic polypeptide receptor (GIPR), and glucagon-like peptide-1 receptor (GLP-1R). In vitro, LY3437943 shows balanced GCGR and GLP-1R activity but more GIPR activity. In obese mice, administration of LY3437943 decreased body weight and improved glycemic control. Body weight loss was augmented by the addition of GCGR-mediated increases in energy expenditure to GIPR- and GLP-1R-driven calorie intake reduction. In a phase 1 single ascending dose study, LY3437943 showed a safety and tolerability profile similar to other incretins. Its pharmacokinetic profile supported once-weekly dosing, and a reduction in body weight persisted up to day 43 after a single dose. These findings warrant further clinical assessment of LY3437943.

Activation of GIPR Exerts Analgesic and Anxiolytic-Like Effects in the Anterior Cingulate Cortex of Mice.

Background: Chronic pain is defined as pain that persists typically for a period of over six months. Chronic pain is often accompanied by an anxiety disorder, and these two tend to exacerbate each other. This can make the treatment of these conditions more difficult. Glucose-dependent insulinotropic polypeptide (GIP) is a member of the incretin hormone family and plays a critical role in glucose metabolism. Previous research has demonstrated the multiple roles of GIP in both physiological and pathological processes. In the central nervous system (CNS), studies of GIP are mainly focused on neurodegenerative diseases; hence, little is known about the functions of GIP in chronic pain and pain-related anxiety disorders. Methods: The chronic inflammatory pain model was established by hind paw injection with complete Freund's adjuvant (CFA) in C57BL/6 mice. GIP receptor (GIPR) agonist (D-Ala2-GIP) and antagonist (Pro3-GIP) were given by intraperitoneal injection or anterior cingulate cortex (ACC) local microinjection. Von Frey filaments and radiant heat were employed to assess the mechanical and thermal hypersensitivity. Anxiety-like behaviors were detected by open field and elevated plus maze tests. The underlying mechanisms in the peripheral nervous system and CNS were explored by GIPR shRNA knockdown in the ACC, enzyme-linked immunosorbent assay, western blot analysis, whole-cell patch-clamp recording, immunofluorescence staining and quantitative real-time PCR. Results: In the present study, we found that hind paw injection with CFA induced pain sensitization and anxiety-like behaviors in mice. The expression of GIPR in the ACC was significantly higher in CFA-injected mice. D-Ala2-GIP administration by intraperitoneal or ACC local microinjection produced analgesic and anxiolytic effects; these were blocked by Pro3-GIP and GIPR shRNA knockdown in the ACC. Activation of GIPR inhibited neuroinflammation and activation of microglia, reversed the upregulation of NMDA and AMPA receptors, and suppressed the enhancement of excitatory neurotransmission in the ACC of model mice. Conclusions: GIPR activation was found to produce analgesic and anxiolytic effects, which were partially due to attenuation of neuroinflammation and inhibition of excitatory transmission in the ACC. GIPR may be a suitable target for treatment of chronic inflammatory pain and pain-related anxiety.

Structural determinants of dual incretin receptor agonism by tirzepatide.

SignificanceTirzepatide is a dual agonist of the glucose-dependent insulinotropic polypeptide receptor (GIPR) and the glucagon-like peptide-1 receptor (GLP-1R), which are incretin receptors that regulate carbohydrate metabolism. This investigational agent has proven superior to selective GLP-1R agonists in clinical trials in subjects with type 2 diabetes mellitus. Intriguingly, although tirzepatide closely resembles native GIP in how it activates the GIPR, it differs markedly from GLP-1 in its activation of the GLP-1R, resulting in less agonist-induced receptor desensitization. We report how cryogenic electron microscopy and molecular dynamics simulations inform the structural basis for the unique pharmacology of tirzepatide. These studies reveal the extent to which fatty acid modification, combined with amino acid sequence, determines the mode of action of a multireceptor agonist.