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.

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.

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.

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.

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).

Association of gastric inhibitory polypeptide receptor (GIPR) gene polymorphism with type 2 diabetes mellitus in iranian patients.

INTRODUCTION: Gastric inhibitory polypeptide receptor (GIPR) encodes a G-protein coupled receptor for gastric inhibitory polypeptide (GIP), which was demonstrated to stimulate insulin secretion. Relation of GIPR gene variation to impaired insulin response has been suggested in previous studies. However, little information is available regarding GIPR polymorphisms and type 2 diabetes mellitus (T2DM). Hence, the aim of the study was to investigate single nucleotide polymorphisms (SNPs) in the promoter and coding regions of GIPR in Iranian T2DM patients. MATERIALS AND METHODS: Two hundred subjects including 100 healthy and 100 T2DM patients were recruited in the study. Genotypes and allele frequency of rs34125392, rs4380143 and rs1800437 in the promoter, 5' UTR and coding region of GIPR were investigated by RFLP-PCR and Nested-PCR. RESULTS: Our finding indicated that rs34125392 genotype distribution was statistically different between T2DM and healthy groups (P = 0.043). In addition, distribution of T/- + -/- versus TT was significantly different between the both groups (P = 0.021). Moreover, rs34125392 T/- genotype increased the risk of T2DM (OR = 2.68, 95%CI = 1.203-5.653, P = 0.015). However, allele frequency and genotype distributions of rs4380143 and rs1800437 were not statistically different between the groups (P > 0.05). Multivariate analysis showed that the tested polymorphisms had no effect on biochemical variables. CONCLUSION: We concluded that GIPR gene polymorphism is associated with T2DM. In addition; rs34125392 heterozygote genotype may increase the risk of T2DM. More studies with large sample size in other populations are recommended to show the ethnical relation of these polymorphisms to T2DM.

Enhanced Endosomal Signaling and Desensitization of GLP-1R vs GIPR in Pancreatic Beta Cells.

The incretin receptors, glucagon-like peptide-1 receptor (GLP-1R) and glucose-dependent insulinotropic polypeptide receptor (GIPR), are prime therapeutic targets for the treatment of type 2 diabetes (T2D) and obesity. They are expressed in pancreatic beta cells where they potentiate insulin release in response to food intake. Despite GIP being the main incretin in healthy individuals, GLP-1R has been favored as a therapeutic target due to blunted GIPR responses in T2D patients and conflicting effects of GIPR agonists and antagonists in improving glucose tolerance and preventing weight gain. There is, however, a recently renewed interest in GIPR biology, following the realization that GIPR responses can be restored after an initial period of blood glucose normalization and the recent development of dual GLP-1R/GIPR agonists with superior capacity for controlling blood glucose levels and weight. The importance of GLP-1R trafficking and subcellular signaling in the control of receptor outputs is well established, but little is known about the pattern of spatiotemporal signaling from the GIPR in beta cells. Here, we have directly compared surface expression, trafficking, and signaling characteristics of both incretin receptors in pancreatic beta cells to identify potential differences that might underlie distinct pharmacological responses associated with each receptor. Our results indicate increased cell surface levels, internalization, degradation, and endosomal vs plasma membrane activity for the GLP-1R, while the GIPR is instead associated with increased plasma membrane recycling, reduced desensitization, and enhanced downstream signal amplification. These differences might have potential implications for the capacity of each incretin receptor to control beta cell function.

Characterization of sporadic somatotropinomas with high GIP receptor expression.

PURPOSE: To analyze the expression of glucose-dependent insulinotropic polypeptide receptor (GIPR) in somatotropinomas specimens and compare clinical, biochemical, radiological, therapeutic, molecular, and pathological data among those who overexpressed (GIPR +) and those who did not overexpress (GIPR - ) GIPR. METHODS: Clinical, biochemical, radiological, molecular, and pathological data were collected. GNAS1 sequencing was performed with the Sanger method. Protein expression of somatostatin receptor subtypes 2 and 5 and CAM 5.2 were analyzed by immunohistochemistry. Quantitative real-time PCR was performed to analyze the mRNA expression of GIPR with the TaqMan® method. Positive expression was considered when the fold change (FC) was above 17.2 (GIPR +). RESULTS: A total of 74 patients (54% female) were included. Eighteen tumors (24%) were GIPR + . Gsp mutation was detected in 30 tumors (40%). GIPR + tumors were more frequently densely granulated adenomas (83% vs 47%, p = 0.028). There was no difference in clinical, biochemical, radiological, therapeutic (surgical cure or response to medical therapy), or other pathological features between GIPR + and GIPR -  tumors. Twenty-eight out of 56 (50%) GIPR -  tumors harbored a gsp mutation, whereas two out of 18 (11%) GIPR + tumors harbored a gsp mutation (p = 0.005). CONCLUSION: We described, for the first time, that GIPR + and gsp mutations are not mutually exclusive, but gsp mutations are less common in GIPR + tumors. GIPR + and GIPR -  tumors have similar clinical, biochemical, radiological, therapeutic, and pathological features, with the exception of a high frequency of densely granulated adenomas among GIPR + tumors.

The Link between Three Single Nucleotide Variants of the GIPR Gene and Metabolic Health.

Single nucleotide variants (SNVs) of the GIPR gene have been associated with BMI and type 2 diabetes (T2D), suggesting the role of the variation in this gene in metabolic health. To increase our understanding of this relationship, we investigated the association of three GIPR SNVs, rs11672660, rs2334255 and rs10423928, with anthropometric measurements, selected metabolic parameters, and the risk of excessive body mass and metabolic syndrome (MS) in the Polish population. Normal-weight subjects (n = 340, control group) and subjects with excessive body mass (n = 600, study group) participated in this study. For all participants, anthropometric measurements and metabolic parameters were collected, and genotyping was performed using the high-resolution melting curve analysis. We did not find a significant association between rs11672660, rs2334255 and rs10423928 variants with the risk of being overweight. Differences in metabolic and anthropometric parameters were found for investigated subgroups. An association between rs11672660 and rs10423928 with MS was identified. Heterozygous CT genotype of rs11672660 and AT genotype of rs10423928 were significantly more frequent in the group with MS (OR = 1.38, 95%CI: 1.03-1.85; p = 0.0304 and OR = 1.4, 95%CI: 1.05-1.87; p = 0.0222, respectively). Moreover, TT genotype of rs10423928 was less frequent in the MS group (OR = 0.72, 95%CI: 0.54-0.95; p = 0.0221).

Liver secretin receptor predicts portoenterostomy outcomes and liver injury in biliary atresia.

Biliary atresia (BA) is a chronic neonatal cholangiopathy characterized by fibroinflammatory bile duct damage. Reliable biomarkers for predicting native liver survival (NLS) following portoenterostomy (PE) surgery are lacking. Herein we explore the utility of 22 preidentified profibrotic molecules closely connected to ductular reaction (DR) and prevailing after successful PE (SPE), in predicting PE outcomes and liver injury. We used qPCR and immunohistochemistry in a BA cohort including liver samples obtained at PE (n = 53) and during postoperative follow-up after SPE (n = 25). Of the 13 genes over-expressed in relation to cholestatic age-matched controls at PE, only secretin receptor (SCTR) expression predicted cumulative 5-year NLS and clearance of jaundice. Patients in the highest SCTR expression tertile showed 34-55% lower NLS than other groups at 1-5 years after PE (P = 0.006-0.04 for each year). SCTR expression was also significantly lower [42 (24-63) vs 75 (39-107) fold, P = 0.015] among those who normalized their serum bilirubin after PE. Liver SCTR expression localized in cholangiocytes and correlated positively with liver fibrosis, DR, and transcriptional markers of fibrosis (ACTA2) and cholangiocytes (KRT7, KRT19) both at PE and after SPE. SCTR is a promising prognostic marker for PE outcomes and associates with liver injury in BA.

Enrichment of Motilin Receptor Loss-of-Function Variants in Gastroparesis.

INTRODUCTION: Gastroparesis is a serious medical condition characterized by delayed gastric emptying and symptoms of nausea, vomiting, bloating, fullness after meals, and abdominal pain. METHODS: To ascertain the genetic risk factors for gastroparesis, we conducted the largest thus far whole-genome sequencing study of gastroparesis. We investigated the frequency and effect of rare loss-of-function variants in patients with both idiopathic and diabetic gastroparesis enrolled in a clinical study of gastroparesis. RESULTS: Among rare loss-of-function variants, we reported an increased frequency of a frameshift mutation p.Leu202ArgfsTer105, within the motilin receptor gene, variant rs562138828 (odds ratio 4.9). We currently replicated this finding in an independent large cohort of gastroparesis samples obtained from patients participating in the ongoing phase III gastroparesis clinical study. DISCUSSION: Motilin receptor is an important therapeutic target for the treatment of hypomotility disorders. The identified genetic variants may be important risk factors for disease as well as may inform treatments, especially those targeting motilin receptor.

GIPR Is Predominantly Localized to Nonadipocyte Cell Types Within White Adipose Tissue.

The incretin hormone glucose-dependent insulinotropic polypeptide (GIP) augments glucose-dependent insulin secretion through its receptor expressed on islet ß-cells. GIP also acts on adipose tissue; yet paradoxically, both enhanced and reduced GIP receptor (GIPR) signaling reduce adipose tissue mass and attenuate weight gain in response to nutrient excess. Moreover, the precise cellular localization of GIPR expression within white adipose tissue (WAT) remains uncertain. We used mouse genetics to target Gipr expression within adipocytes. Surprisingly, targeting Cre expression to adipocytes using the adiponectin (Adipoq) promoter did not produce meaningful reduction of WAT Gipr expression in Adipoq-Cre:Giprflx/flx mice. In contrast, adenoviral expression of Cre under the control of the cytomegalovirus promoter, or transgenic expression of Cre using nonadipocyte-selective promoters (Ap2/Fabp4 and Ubc) markedly attenuated WAT Gipr expression. Analysis of single-nucleus RNA-sequencing, adipose tissue data sets localized Gipr/GIPR expression predominantly to pericytes and mesothelial cells rather than to adipocytes. Together, these observations reveal that adipocytes are not the major GIPR+ cell type within WAT-findings with mechanistic implications for understanding how GIP and GIP-based co-agonists control adipose tissue biology.

GIP and GLP-2 together improve bone turnover in humans supporting GIPR-GLP-2R co-agonists as future osteoporosis treatment.

The intestinal hormones glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-2 (GLP-2) are key regulators of postprandial bone turnover in humans. We hypothesized that GIP and GLP-2 co-administration would provide stronger effect on bone turnover than administration of the hormones separately, and tested this using subcutaneous injections of GIP and GLP-2 alone or in combination in humans. Guided by these findings, we designed series of GIPR-GLP-2R co-agonists as template for new osteoporosis treatment. The clinical experiment was a randomized cross-over design including 10 healthy men administered subcutaneous injections of GIP and GLP-2 alone or in combination. The GIPR-GLP-2R co-agonists were characterized in terms of binding and activation profiles on human and rodent GIP and GLP-2 receptors, and their pharmacokinetic (PK) profiles were improved by dipeptidyl peptidase-4 protection and site-directed lipidation. Co-administration of GIP and GLP-2 in humans resulted in an additive reduction in bone resorption superior to each hormone individually. The GIPR-GLP-2R co-agonists, designed by combining regions of importance for cognate receptor activation, obtained similar efficacies as the two native hormones and nanomolar potencies on both human receptors. The PK-improved co-agonists maintained receptor activity along with their prolonged half-lives. Finally, we found that the GIPR-GLP-2R co-agonists optimized toward the human receptors for bone remodeling are not feasible for use in rodent models. The successful development of potent and efficacious GIPR-GLP-2R co-agonists, combined with the improved effect on bone metabolism in humans by co-administration, support these co-agonists as a future osteoporosis treatment.

Secretin receptor-deficient mice exhibit robust food anticipatory activity.

In mammals, the suprachiasmatic nucleus (SCN) is a principal circadian pacemaker that optimizes the timing of behavioral rhythms and physiological events. Normally, circadian behavioral rhythms are entrained by the environmental light-dark (LD) cycle via the SCN. However, daily rhythms of other synchronizing signals, such as food availability, also emerge. When food availability is restricted to a single recurring daytime meal in nocturnal rodents, they exhibit increased activity during the hours immediately preceding feeding time; this is called food anticipatory activity (FAA). Many reports suggest that FAA is mediated by the food-entrainable oscillator (FEO) with circadian properties, but not the SCN. However, the neural locus and timekeeping mechanisms of the FEO, including its relationship with gastrointestinal hormone signaling, remain unclear. Herein, to examine whether secretin receptor signaling is necessary for the FEO, the effect of daily food restriction was studied in secretin receptor-deficient (Sctr-/-) mice. Adult wild-type (WT) and Sctr-/- mice were housed in separate cages containing a running wheel, with ad libitum food access and in a LD cycle (12 hours:12 hours) for at least 2 weeks. After acclimation to the condition, food access times were gradually restricted and 4-hour restricted feeding lasted over 10 days. Subsequently, mice had ad libitum food access for 2 days and then fasted for 2 days. Thereafter, robust FAAs were observed in both WT and Sctr-/- mice during restricted feeding and subsequent fasting. These results indicate that secretin receptor signaling is not essential for the timekeeping mechanism of FEO.

Reagents and models for detecting endogenous GLP1R and GIPR.

Glucagon-like peptide-1 receptor (GLP1R) agonists target the GLP1R, whereas dual GLP1R/ gastric inhibitory polypeptide receptor (GIPR) agonists target both the GLP1R and GIPR. Despite the importance of these drug classes for the treatment of diabetes and obesity, still very little is known about the localization of GLP1R and GIPR themselves. Complicating matters is the low abundance of GLP1R and GIPR mRNA/protein, as well as a lack of specific and validated reagents for their detection. Without knowing where GLP1R and GIPR are located, it is difficult to propose mechanisms of action in the various target organs, and whether this is indirect or direct. In the current review, we will explain the steps needed to properly validate reagents for endogenous GLP1R/GIPR detection, describe the available approaches to visualize GLP1R/GIPR, and provide an update on the state-of-art. The overall aim is to provide a reference resource for researchers interested in GLP1R and GIPR signaling.

Leveraging human genetic data to investigate the cardiometabolic effects of glucose-dependent insulinotropic polypeptide signalling.

AIMS/HYPOTHESIS: The aim of this study was to leverage human genetic data to investigate the cardiometabolic effects of glucose-dependent insulinotropic polypeptide (GIP) signalling. METHODS: Data were obtained from summary statistics of large-scale genome-wide association studies. We examined whether genetic associations for type 2 diabetes liability in the GIP and GIPR genes co-localised with genetic associations for 11 cardiometabolic outcomes. For those outcomes that showed evidence of co-localisation (posterior probability >0.8), we performed Mendelian randomisation analyses to estimate the association of genetically proxied GIP signalling with risk of cardiometabolic outcomes, and to test whether this exceeded the estimate observed when considering type 2 diabetes liability variants from other regions of the genome. RESULTS: Evidence of co-localisation with genetic associations of type 2 diabetes liability at both the GIP and GIPR genes was observed for five outcomes. Mendelian randomisation analyses provided evidence for associations of lower genetically proxied type 2 diabetes liability at the GIP and GIPR genes with lower BMI (estimate in SD units -0.16, 95% CI -0.30, -0.02), C-reactive protein (-0.13, 95% CI -0.19, -0.08) and triacylglycerol levels (-0.17, 95% CI -0.22, -0.12), and higher HDL-cholesterol levels (0.19, 95% CI 0.14, 0.25). For all of these outcomes, the estimates were greater in magnitude than those observed when considering type 2 diabetes liability variants from other regions of the genome. CONCLUSIONS/INTERPRETATION: This study provides genetic evidence to support a beneficial role of sustained GIP signalling on cardiometabolic health greater than that expected from improved glycaemic control alone. Further clinical investigation is warranted. DATA AVAILABILITY: All data used in this study are publicly available. The scripts for the analysis are available at: https://github.com/vkarhune/GeneticallyProxiedGIP .

Insights into agonist-elicited activation of the human glucose-dependent insulinotropic polypeptide receptor.

Glucose-dependent insulinotropic polypeptide (GIP) and its receptor (GIPR) are part of the incretin system that regulates glucose homeostasis. A series of GIPR residues putatively important for ligand binding and receptor activation were mutated and pharmacologically evaluated using GIPR selective agonists in cAMP accumulation, ERK1/2 phosphorylation (pERK1/2) and ß-arrestin 2 recruitment assays. The impact of mutation on ligand efficacy was determined by operational modelling of experimental data for each mutant, with results mapped onto the full-length, active-state GIPR structure. Two interaction networks, comprising transmembrane helix (TM) 7, TM1 and TM2, and extracellular loop (ECL) 2, TM5 and ECL3 were revealed, respectively. Both networks were critical for Gαs-mediated cAMP accumulation and the recruitment of ß-arrestin 2, however, cAMP response was more sensitive to alanine substitution, with most mutated residues displaying reduced signaling. Unlike the other two assays, activation of ERK1/2 was largely independent of the network involving ECL2, TM5 and ECL3, indicating that pERK1/2 is at least partially distinct from Gαs or ß-arrestin pathways and this network is also crucial for potential biased agonism at GIPR. Collectively, our work advances understanding of the structure-function relationship of GIPR and provides a framework for the design and/or interpretation of GIP analogues with unique signaling profiles.

Genetically Predicted Glucose-Dependent Insulinotropic Polypeptide (GIP) Levels and Cardiovascular Disease Risk Are Driven by Distinct Causal Variants in the GIPR Region.

There is considerable interest in GIPR agonism to enhance the insulinotropic and extrapancreatic effects of GIP, thereby improving glycemic and weight control in type 2 diabetes (T2D) and obesity. Recent genetic epidemiological evidence has implicated higher GIPR-mediated GIP levels in raising coronary artery disease (CAD) risk, a potential safety concern for GIPR agonism. We therefore aimed to quantitatively assess whether the association between higher GIPR-mediated fasting GIP levels and CAD risk is mediated via GIPR or is instead the result of linkage disequilibrium (LD) confounding between variants at the GIPR locus. Using Bayesian multitrait colocalization, we identified a GIPR missense variant, rs1800437 (G allele; E354), as the putatively causal variant shared among fasting GIP levels, glycemic traits, and adiposity-related traits (posterior probability for colocalization [PPcoloc] > 0.97; PP explained by the candidate variant [PPexplained] = 1) that was independent from a cluster of CAD and lipid traits driven by a known missense variant in APOE (rs7412; distance to E354 ∼770 Kb; R 2 with E354 = 0.004; PPcoloc > 0.99; PPexplained = 1). Further, conditioning the association between E354 and CAD on the residual LD with rs7412, we observed slight attenuation in association, but it remained significant (odds ratio [OR] per copy of E354 after adjustment 1.03; 95% CI 1.02, 1.04; P = 0.003). Instead, E354's association with CAD was completely attenuated when conditioning on an additional established CAD signal, rs1964272 (R 2 with E354 = 0.27), an intronic variant in SNRPD2 (OR for E354 after adjustment for rs1964272: 1.01; 95% CI 0.99, 1.03; P = 0.06). We demonstrate that associations with GIP and anthropometric and glycemic traits are driven by genetic signals distinct from those driving CAD and lipid traits in the GIPR region and that higher E354-mediated fasting GIP levels are not associated with CAD risk. These findings provide evidence that the inclusion of GIPR agonism in dual GIPR/GLP1R agonists could potentiate the protective effect of GLP-1 agonists on diabetes without undue CAD risk, an aspect that has yet to be assessed in clinical trials.

CD8+ T Cells Involved in Metabolic Inflammation in Visceral Adipose Tissue and Liver of Transgenic Pigs.

Anti-inflammatory therapies have the potential to become an effective treatment for obesity-related diseases. However, the huge gap of immune system between human and rodent leads to limitations of drug discovery. This work aims at constructing a transgenic pig model with higher risk of metabolic diseases and outlining the immune responses at the early stage of metaflammation by transcriptomic strategy. We used CRISPR/Cas9 techniques to targeted knock-in three humanized disease risk genes, GIPRdn , hIAPP and PNPLA3I148M . Transgenic effect increased the risk of metabolic disorders. Triple-transgenic pigs with short-term diet intervention showed early symptoms of type 2 diabetes, including glucose intolerance, pancreatic lipid infiltration, islet hypertrophy, hepatic lobular inflammation and adipose tissue inflammation. Molecular pathways related to CD8+ T cell function were significantly activated in the liver and visceral adipose samples from triple-transgenic pigs, including antigen processing and presentation, T-cell receptor signaling, co-stimulation, cytotoxicity, and cytokine and chemokine secretion. The similar pro-inflammatory signaling in liver and visceral adipose tissue indicated that there might be a potential immune crosstalk between the two tissues. Moreover, genes that functionally related to liver antioxidant activity, mitochondrial function and extracellular matrix showed distinct expression between the two groups, indicating metabolic stress in transgenic pigs' liver samples. We confirmed that triple-transgenic pigs had high coincidence with human metabolic diseases, especially in the scope of inflammatory signaling at early stage metaflammation. Taken together, this study provides a valuable large animal model for the clinical study of metaflammation and metabolic diseases.