Characterization of genetic variants of GIPR reveals a contribution of ß-arrestin to metabolic phenotypes.

Incretin-based therapies are highly successful in combatting obesity and type 2 diabetes1. Yet both activation and inhibition of the glucose-dependent insulinotropic polypeptide (GIP) receptor (GIPR) in combination with glucagon-like peptide-1 (GLP-1) receptor (GLP-1R) activation have resulted in similar clinical outcomes, as demonstrated by the GIPR-GLP-1R co-agonist tirzepatide2 and AMG-133 (ref. 3) combining GIPR antagonism with GLP-1R agonism. This underlines the importance of a better understanding of the GIP system. Here we show the necessity of ß-arrestin recruitment for GIPR function, by combining in vitro pharmacological characterization of 47 GIPR variants with burden testing of clinical phenotypes and in vivo studies. Burden testing of variants with distinct ligand-binding capacity, Gs activation (cyclic adenosine monophosphate production) and ß-arrestin 2 recruitment and internalization shows that unlike variants solely impaired in Gs signalling, variants impaired in both Gs and ß-arrestin 2 recruitment contribute to lower adiposity-related traits. Endosomal Gs-mediated signalling of the variants shows a ß-arrestin dependency and genetic ablation of ß-arrestin 2 impairs cyclic adenosine monophosphate production and decreases GIP efficacy on glucose control in male mice. This study highlights a crucial impact of ß-arrestins in regulating GIPR signalling and overall preservation of biological activity that may facilitate new developments in therapeutic targeting of the GIPR system.

Effects of Exogenous GIP and GLP-2 on Bone Turnover in Individuals With Type 2 Diabetes.

CONTEXT: Individuals with type 2 diabetes (T2D) have an increased risk of bone fractures despite normal or increased bone mineral density. The underlying causes are not well understood but may include disturbances in the gut-bone axis, in which both glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-2 (GLP-2) are regulators of bone turnover. Thus, in healthy fasting participants, both exogenous GIP and GLP-2 acutely reduce bone resorption. OBJECTIVE: The objective of this study was to investigate the acute effects of subcutaneously administered GIP and GLP-2 on bone turnover in individuals with T2D. METHODS: We included 10 men with T2D. Participants met fasting in the morning on 3 separate test days and were injected subcutaneously with GIP, GLP-2, or placebo in a randomized crossover design. Blood samples were drawn at baseline and regularly after injections. Bone turnover was estimated by circulating levels of collagen type 1 C-terminal telopeptide (CTX), procollagen type 1 N-terminal propeptide (P1NP), sclerostin, and PTH. RESULTS: GIP and GLP-2 significantly reduced CTX to (mean ± SEM) 66 ± 7.8% and 74 ± 5.9% of baseline, respectively, compared with after placebo (P = .001). In addition, P1NP and sclerostin increased acutely after GIP whereas a decrease in P1NP was seen after GLP-2. PTH levels decreased to 67 ± 2.5% of baseline after GLP-2 and to only 86 ± 3.4% after GIP. CONCLUSION: Subcutaneous GIP and GLP-2 affect CTX and P1NP in individuals with T2D to the same extent as previously demonstrated in healthy individuals.

Re-routing GPR56 signalling using Gα12/13 G protein chimeras.

Adhesion G protein-coupled receptors (aGPCRs) constitute the second largest subclass of the GPCR superfamily. Although canonical GPCRs are explored pharmacologically as drug targets, no clinically approved drugs target the aGPCR family so far. The aGPCR GPR56/ADGRG1 stands out as an especially promising target, given its direct link to the monogenetic disease bilateral frontoparietal polymicrogyria and implications in cancers. Key to understanding GPCR pharmacology has been mapping out intracellular signalling activity. Detection of GPCR signalling in the Gαs /Gαi /Gαq G protein pathways is feasible with second messenger detection systems. However, in the case of Gα12/13 -coupled receptors, like GPR56, signalling detection is more challenging due to the lack of direct second messenger generation. To overcome this challenge, we engineered a Gαq chimera to translate Gα12/13 signalling. We show the ability of the chimeric GαΔ6q12myr and GαΔ6q13myr to translate basal Gα12/13 signalling of GPR56 to a Gαq readout in transcription factor luciferase reporter systems and show that the established peptide ligands (P7 and P19) function to enhance this signal. We further demonstrate the ability to directly influence the generation of second messengers in inositol-3-phosphate assays. In the future, these chimeric G proteins could facilitate basic functional studies, drug screenings and deorphanization of other aGPCRs.

The importance of glucose-dependent insulinotropic polypeptide receptor activation for the effects of tirzepatide.

Tirzepatide is a unimolecular co-agonist of the glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) receptors recently approved for the treatment of type 2 diabetes by the US Food and Drug Administration and the European Medicine Agency. Tirzepatide treatment results in an unprecedented improvement of glycaemic control and lowering of body weight, but the contribution of the GIP receptor-activating component of tirzepatide to these effects is uncertain. In this review, we present the current knowledge about the physiological roles of the incretin hormones GLP-1 and GIP, their receptors, and previous results of co-targeting the two incretin hormone receptors in humans. We also analyse the molecular pharmacological, preclinical and clinical effects of tirzepatide to discuss the role of GIP receptor activation for the clinical effects of tirzepatide. Based on the available literature on the combination of GLP-1 and GIP receptor activation, tirzepatide does not seem to have a classical co-activating mode of action in humans. Rather, in vitro studies of the human GLP-1 and GIP receptors reveal a biased GLP-1 receptor activation profile and GIP receptor downregulation. Therefore, we propose three hypotheses for the mode of action of tirzepatide, which can be addressed in future, elaborate clinical trials.

The Secretome of Parental and Bone Metastatic Breast Cancer Elicits Distinct Effects in Human Osteoclast Activity after Activation of ß2 Adrenergic Signaling.

The sympathetic nervous system (SNS), particularly through the ß2 adrenergic receptor (ß2-AR), has been linked with breast cancer (BC) and the development of metastatic BC, specifically in the bone. Nevertheless, the potential clinical benefits of exploiting ß2-AR antagonists as a treatment for BC and bone loss-associated symptoms remain controversial. In this work, we show that, when compared to control individuals, the epinephrine levels in a cohort of BC patients are augmented in both earlier and late stages of the disease. Furthermore, through a combination of proteomic profiling and functional in vitro studies with human osteoclasts and osteoblasts, we demonstrate that paracrine signaling from parental BC under ß2-AR activation causes a robust decrease in human osteoclast differentiation and resorption activity, which is rescued in the presence of human osteoblasts. Conversely, metastatic bone tropic BC does not display this anti-osteoclastogenic effect. In conclusion, the observed changes in the proteomic profile of BC cells under ß-AR activation that take place after metastatic dissemination, together with clinical data on epinephrine levels in BC patients, provided new insights on the sympathetic control of breast cancer and its implications on osteoclastic bone resorption.

Rare Heterozygous Loss-of-Function Variants in the Human GLP-1 Receptor Are Not Associated With Cardiometabolic Phenotypes.

CONTEXT: Lost glucagon-like peptide 1 receptor (GLP-1R) function affects human physiology. OBJECTIVE: This work aimed to identify coding nonsynonymous GLP1R variants in Danish individuals to link their in vitro phenotypes and clinical phenotypic associations. METHODS: We sequenced GLP1R in 8642 Danish individuals with type 2 diabetes or normal glucose tolerance and examined the ability of nonsynonymous variants to bind GLP-1 and to signal in transfected cells via cyclic adenosine monophosphate (cAMP) formation and ß-arrestin recruitment. We performed a cross-sectional study between the burden of loss-of-signaling (LoS) variants and cardiometabolic phenotypes in 2930 patients with type 2 diabetes and 5712 participants in a population-based cohort. Furthermore, we studied the association between cardiometabolic phenotypes and the burden of the LoS variants and 60 partly overlapping predicted loss-of-function (pLoF) GLP1R variants found in 330 566 unrelated White exome-sequenced participants in the UK Biobank cohort. RESULTS: We identified 36 nonsynonymous variants in GLP1R, of which 10 had a statistically significant loss in GLP-1-induced cAMP signaling compared to wild-type. However, no association was observed between the LoS variants and type 2 diabetes, although LoS variant carriers had a minor increased fasting plasma glucose level. Moreover, pLoF variants from the UK Biobank also did not reveal substantial cardiometabolic associations, despite a small effect on glycated hemoglobin A1c. CONCLUSION: Since no homozygous LoS nor pLoF variants were identified and heterozygous carriers had similar cardiometabolic phenotype as noncarriers, we conclude that GLP-1R may be of particular importance in human physiology, due to a potential evolutionary intolerance of harmful homozygous GLP1R variants.

GIP reduces osteoclast activity and improves osteoblast survival in primary human bone cells.

OBJECTIVE: Drugs targeting the glucose-dependent insulinotropic polypeptide (GIP) receptor (GIPR) are emerging as treatments for type-2 diabetes and obesity. GIP acutely decreases serum markers of bone resorption and transiently increases bone formation markers in short-term clinical investigations. However, it is unknown whether GIP acts directly on bone cells to mediate these effects. Using a GIPR-specific antagonist, we aimed to assess whether GIP acts directly on primary human osteoclasts and osteoblasts. METHODS: Osteoclasts were differentiated from human CD14+ monocytes and osteoblasts from human bone. GIPR expression was determined using RNA-seq in primary human osteoclasts and in situ hybridization in human femoral bone. Osteoclastic resorptive activity was assessed using microscopy. GIPR signaling pathways in osteoclasts and osteoblasts were assessed using LANCE cAMP and AlphaLISA phosphorylation assays, intracellular calcium imaging and confocal microscopy. The bioenergetic profile of osteoclasts was evaluated using Seahorse XF-96. RESULTS: GIPR is robustly expressed in mature human osteoclasts. GIP inhibits osteoclastogenesis, delays bone resorption, and increases osteoclast apoptosis by acting upon multiple signaling pathways (Src, cAMP, Akt, p38, Akt, NFκB) to impair nuclear translocation of nuclear factor of activated T cells-1 (NFATc1) and nuclear factor-κB (NFκB). Osteoblasts also expressed GIPR, and GIP improved osteoblast survival. Decreased bone resorption and improved osteoblast survival were also observed after GIP treatment of osteoclast-osteoblast co-cultures. Antagonizing GIPR with GIP(3-30)NH2 abolished the effects of GIP on osteoclasts and osteoblasts. CONCLUSIONS: GIP inhibits bone resorption and improves survival of human osteoblasts, indicating that drugs targeting GIPR may impair bone resorption, whilst preserving bone formation.

Patterns of human and porcine gammaherpesvirus-encoded BILF1 receptor endocytosis.

BACKGROUND: The viral G-protein-coupled receptor (vGPCR) BILF1 encoded by the Epstein-Barr virus (EBV) is an oncogene and immunoevasin and can downregulate MHC-I molecules at the surface of infected cells. MHC-I downregulation, which presumably occurs through co-internalization with EBV-BILF1, is preserved among BILF1 receptors, including the three BILF1 orthologs encoded by porcine lymphotropic herpesviruses (PLHV BILFs). This study aimed to understand the detailed mechanisms of BILF1 receptor constitutive internalization, to explore the translational potential of PLHV BILFs compared with EBV-BILF1. METHODS: A novel real-time fluorescence resonance energy transfer (FRET)-based internalization assay combined with dominant-negative variants of dynamin-1 (Dyn K44A) and the chemical clathrin inhibitor Pitstop2 in HEK-293A cells was used to study the effect of specific endocytic proteins on BILF1 internalization. Bioluminescence resonance energy transfer (BRET)-saturation analysis was used to study BILF1 receptor interaction with ß-arrestin2 and Rab7. In addition, a bioinformatics approach informational spectrum method (ISM) was used to investigate the interaction affinity of BILF1 receptors with ß-arrestin2, AP-2, and caveolin-1. RESULTS: We identified dynamin-dependent, clathrin-mediated constitutive endocytosis for all BILF1 receptors. The observed interaction affinity between BILF1 receptors and caveolin-1 and the decreased internalization in the presence of a dominant-negative variant of caveolin-1 (Cav S80E) indicated the involvement of caveolin-1 in BILF1 trafficking. Furthermore, after BILF1 internalization from the plasma membrane, both the recycling and degradation pathways are proposed for BILF1 receptors. CONCLUSIONS: The similarity in the internalization mechanisms observed for EBV-BILF1 and PLHV1-2 BILF1 provide a foundation for further studies exploring a possible translational potential for PLHVs, as proposed previously, and provides new information about receptor trafficking.

The naturally occurring GIP(1-30)NH2 is a GIP receptor agonist in humans.

OBJECTIVE: The gut hormone glucose-dependent insulinotropic polypeptide (GIP) is an important regulator of glucose and bone metabolism. In rodents, the naturally occurring GIP variant, GIP(1-30)NH2, has shown similar effects as full-length GIP (GIP(1-42)), but its effects in humans are unsettled. Here, we investigated the actions of GIP(1-30)NH2 compared to GIP(1-42) on glucose and bone metabolism in healthy men and in isolated human pancreatic islets. METHODS: Nine healthy men completed three separate three-step glucose clamps (0-60 minutes at fasting plasma glucose (FPG) level, 60-120 minutes at 1.5× FPG, and 120-180 minutes at 2× FPG) with infusion of GIP(1-42) (4 pmol/kg/min), GIP(1-30)NH2 (4 pmol/kg/min), and saline (9 mg/mL) in randomised order. Blood was sampled for measurement of relevant hormones and bone turnover markers. Human islets were incubated with low (2 mmol/L) or high (20 mmol/L) d-glucose with or without GIP(1-42) or GIP(1-30)NH2 in three different concentrations for 30 minutes, and secreted insulin and glucagon were measured. RESULTS: Plasma glucose (PG) levels at FPG, 1.5× FPG, and 2× FPG were obtained by infusion of 1.45 g/kg, 0.97 g/kg, and 0.6 g/kg of glucose during GIP(1-42), GIP(1-30)NH2, and saline, respectively (P = .18), and were similar on the three experimental days. Compared to placebo, GIP(1-30)NH2 resulted in similar glucagonotropic, insulinotropic, and carboxy-terminal type 1 collagen crosslinks-suppressing effects as GIP(1-42). In vitro experiments on human islets showed similar insulinotropic and glucagonotropic effects of the two GIP variants. CONCLUSIONS: GIP(1-30)NH2 has similar effects on glucose and bone metabolism in healthy individuals and in human islets in vitro as GIP(1-42).

Loss of Adgra3 causes obstructive azoospermia with high penetrance in male mice.

The adhesion receptor ADGRA3 (GPR125) is a known spermatogonial stem cell marker, but its impact on male reproduction and fertility has not been examined. Using a mouse model lacking Adgra3 (Adgra3-/- ), we show that 55% of the male mice are infertile from puberty despite having normal spermatogenesis and epididymal sperm count. Instead, male mice lacking Adgra3 exhibited decreased estrogen receptor alpha expression and transient dilation of the epididymis. Combined with an increased estradiol production, this indicates a post-pubertal hormonal imbalance and fluid retention. Dye injection revealed a blockage between the ejaculatory duct and the urethra, which is rare in mice suffering from infertility, thereby mimicking the etiologies of obstructive azoospermia found in human male infertility. To summarize, male reproductive tract development is dependent on ADGRA3 function that in concert with estrogen signaling may influence fluid handling during sperm maturation and storage.

The Importance of Endogenously Secreted GLP-1 and GIP for Postprandial Glucose Tolerance and ß-Cell Function After Roux-en-Y Gastric Bypass and Sleeve Gastrectomy Surgery.

Enhanced secretion of glucagon-like peptide 1 (GLP-1) seems to be essential for improved postprandial ß-cell function after Roux-en-Y gastric bypass (RYGB) but is less studied after sleeve gastrectomy (SG). Moreover, the role of the other major incretin hormone, glucose-dependent insulinotropic polypeptide (GIP), is relatively unexplored after bariatric surgery. We studied the effects of separate and combined GLP-1 receptor (GLP-1R) and GIP receptor (GIPR) blockade during mixed-meal tests in unoperated (CON), SG-operated, and RYGB-operated people with no history of diabetes. Postprandial GLP-1 concentrations were highest after RYGB but also higher after SG compared with CON. In contrast, postprandial GIP concentrations were lowest after RYGB. The effect of GLP-1R versus GIPR blockade differed between groups. GLP-1R blockade reduced ß-cell glucose sensitivity and increased or tended to increase postprandial glucose responses in the surgical groups but had no effect in CON. GIPR blockade reduced ß-cell glucose sensitivity and increased or tended to increase postprandial glucose responses in the CON and SG groups but had no effect in the RYGB group. Our results support that GIP is the most important incretin hormone in unoperated people, whereas GLP-1 and GIP are equally important after SG, and GLP-1 is the most important incretin hormone after RYGB.

Endogenous Glucose-Dependent Insulinotropic Polypeptide Contributes to Sitagliptin-Mediated Improvement in ß-Cell Function in Patients With Type 2 Diabetes.

Dipeptidyl peptidase 4 (DPP-4) degrades the incretin hormones glucagon-like peptide 1 and glucose-dependent insulinotropic polypeptide (GIP). DPP-4 inhibitors improve glycemic control in type 2 diabetes, but the importance of protecting GIP from degradation for their clinical effects is unknown. We included 12 patients with type 2 diabetes (mean ± SD BMI 27 ± 2.6 kg/m2, HbA1c 7.1 ± 1.4% [54 ± 15 mmol/mol]) in this double-blind, placebo-controlled, crossover study to investigate the contribution of endogenous GIP to the effects of the DPP-4 inhibitor sitagliptin. Participants underwent two randomized, 13-day treatment courses of sitagliptin (100 mg/day) and placebo, respectively. At the end of each treatment period, we performed two mixed-meal tests with infusion of the GIP receptor antagonist GIP(3-30)NH2 (1,200 pmol/kg/min) or saline placebo. Sitagliptin lowered mean fasting plasma glucose by 1.1 mmol/L compared with placebo treatment. During placebo treatment, postprandial glucose excursions were increased during GIP(3-30)NH2 compared with saline (difference in area under the curve ± SEM 7.3 ± 2.8%) but were unchanged during sitagliptin treatment. Endogenous GIP improved ß-cell function by 37 ± 12% during DPP-4 inhibition by sitagliptin. This was determined by the insulin secretion rate/plasma glucose ratio. We calculated an estimate of the absolute sitagliptin-mediated impact of GIP on ß-cell function as the insulinogenic index during sitagliptin treatment plus saline infusion minus the insulinogenic index during sitagliptin plus GIP(3-30)NH2. This estimate was expressed relative to the maximal potential contribution of GIP to the effect of sitagliptin (100%), defined as the difference between the full sitagliptin treatment effect, including actions mediated by GIP (sitagliptin + saline), and the physiological response minus any contribution by GIP [placebo treatment + GIP(3-30)NH2]. We demonstrate insulinotropic and glucose-lowering effects of endogenous GIP in patients with type 2 diabetes and that endogenous GIP contributes to the improved ß-cell function observed during DPP-4 inhibition.

Viral G Protein-Coupled Receptors Encoded by ß- and γ-Herpesviruses.

Herpesviruses are ancient large DNA viruses that have exploited gene capture as part of their strategy to escape immune surveillance, promote virus spreading, or reprogram host cells to benefit their survival. Most acquired genes are transmembrane proteins and cytokines, such as viral G protein-coupled receptors (vGPCRs), chemokines, and chemokine-binding proteins. This review focuses on the vGPCRs encoded by the human ß- and γ-herpesviruses. These include receptors from human cytomegalovirus, which encodes four vGPCRs: US27, US28, UL33, and UL78; human herpesvirus 6 and 7 with two receptors: U12 and U51; Epstein-Barr virus with one: BILF1; and Kaposi's sarcoma-associated herpesvirus with one: open reading frame 74, ORF74. We discuss ligand binding, signaling, and structures of the vGPCRs in light of robust differences from endogenous receptors. Finally, we briefly discuss the therapeutic targeting of vGPCRs as future treatment of acute and chronic herpesvirus infections.

Epstein-Barr Virus-Encoded BILF1 Orthologues From Porcine Lymphotropic Herpesviruses Display Common Molecular Functionality.

Infection of immunosuppressed transplant patients with the human γ-herpesvirus Epstein-Barr virus (EBV) is associated with post-transplant lymphoproliferative disease (PTLD), an often fatal complication. Immunosuppressed miniature pigs infected with γ-herpesvirus porcine lymphotropic herpesvirus 1 (PLHV1) develop a similar disease, identifying pigs as a potential preclinical model for PTLD in humans. BILF1 is a G protein-coupled receptor (GPCR) encoded by EBV with constitutive activity linked to tumorigenesis and immunoevasive function downregulating MHC-I. In the present study, we compared BILF1-orthologues encoded by the three known PLHVs (PLHV1-3) with EBV-BILF1 to determine pharmacological suitability of BILF1 orthologues as model system to study EBV-BILF1 druggability. Cell surface localization, constitutive internalization, and MHC-I downregulation as well as membrane proximal constitutive Gαi signaling patterns were conserved across all BILFs. Only subtle differences between the individual BILFs were observed in downstream transcription factor activation. Using Illumina sequencing, PLHV1 was observed in lymphatic tissue from PTLD-diseased, but not non-diseased pigs. Importantly, these tissues showed enhanced expression of PLHV1-BILF1 supporting its involvement in PTLD infection.

GLP-1 and GIP receptor signaling in beta cells - A review of receptor interactions and co-stimulation.

Glucagon-like peptide 1 receptor (GLP-1R) and glucose-dependent insulinotropic polypeptide receptor (GIPR) are two class B1 G protein-coupled receptors, which are stimulated by the gastrointestinal hormones GLP-1 and GIP, respectively. In the pancreatic beta cells, activation of both receptors lead to increased cyclic adenosine monophosphate (cAMP) and glucose-dependent insulin secretion. Marketed GLP-1R agonists such as dulaglutide, liraglutide, exenatide and semaglutide constitute an expanding drug class with beneficial effects for persons suffering from type 2 diabetes and/or obesity. In recent years another drug class, the GLP-1R-GIPR co-agonists, has emerged. Especially the peptide-based, co-agonist tirzepatide is a promising candidate for a better treatment of type 2 diabetes by improving glycemic control and weight reduction. The mechanism of action for tirzepatide include biased signaling of the GLP-1R as well as potent GIPR signaling. Since the implications of co-targeting these closely related receptors concomitantly are challenging to study in vivo, the pharmacodynamic mechanisms and downstream signaling pathways of the GLP-1R-GIPR co-agonists in general, are not fully elucidated. In this review, we present the individual signaling pathways for GLP-1R and GIPR in the pancreatic beta cell with a focus on the shared signaling pathways of the two receptors and interpret the implications of GLP-1R-GIPR co-activation in the light of recent co-activating therapeutic compounds.

The glucagon receptor antagonist LY2409021 has no effect on postprandial glucose in type 2 diabetes.

OBJECTIVE: Type 2 diabetes (T2D) pathophysiology includes fasting and postprandial hyperglucagonemia, which has been linked to hyperglycemia via increased endogenous glucose production (EGP). We used a glucagon receptor antagonist (LY2409021) and stable isotope tracer infusions to investigate the consequences of hyperglucagonemia in T2D. DESIGN: A double-blinded, randomized, placebo-controlled crossover study was conducted. METHODS: Ten patients with T2D and ten matched non-diabetic controls underwent two liquid mixed meal tests preceded by single-dose administration of LY2409021 (100 mg) or placebo. Double-tracer technique was used to quantify EGP. Antagonist selectivity toward related incretin receptors was determined in vitro. RESULTS: Compared to placebo, LY2409021 lowered the fasting plasma glucose (FPG) from 9.1 to 7.1 mmol/L in patients and from 5.6 to 5.0 mmol/L in controls (both P < 0.001) by mechanisms involving reduction of EGP. Postprandial plasma glucose excursions (baseline-subtracted area under the curve) were unaffected by LY2409021 in patients and increased in controls compared to placebo. Glucagon concentrations more than doubled during glucagon receptor antagonism. The antagonist interfered with both glucagon-like peptide 1 and glucose-dependent insulinotropic polypeptide receptors, complicating the interpretation of the postprandial data. CONCLUSIONS: LY2409021 lowered FPG concentrations but did not improve postprandial glucose tolerance after a meal in patients with T2D and controls. The metabolic consequences of postprandial hyperglucagonemia are difficult to evaluate using LY2409021 because of its antagonizing effects on the incretin receptors.

Novel agonist and antagonist radioligands for the GLP-2 receptor. Useful tools for studies of basic GLP-2 receptor pharmacology.

BACKGROUND: Glucagon-like peptide-2 (GLP-2) is a pro-glucagon-derived hormone secreted from intestinal enteroendocrine L cells with actions on gut and bones. GLP-2(1-33) is cleaved by DPP-4, forming GLP-2(3-33), having low intrinsic activity and competitive antagonism properties at GLP-2 receptors. We created radioligands based on these two molecules. EXPERIMENTAL APPROACH: The methionine in position 10 of GLP-2(1-33) and GLP-2(3-33) was substituted with tyrosine (M10Y) enabling oxidative iodination, creating [125 I]-hGLP-2(1-33,M10Y) and [125 I]-hGLP-2(3-33,M10Y). Both were characterized by competition binding, on-and-off-rate determination and receptor activation. Receptor expression was determined by target-tissue autoradiography and immunohistochemistry. KEY RESULTS: Both M10Y-substituted peptides induced cAMP production via the GLP-2 receptor comparable to the wildtype peptides. GLP-2(3-33,M10Y) maintained the antagonistic properties of GLP-2(3-33). However, hGLP-2(1-33,M10Y) had lower arrestin recruitment than hGLP-2(1-33). High affinities for the hGLP-2 receptor were observed using [125 I]-hGLP-2(1-33,M10Y) and [125 I]-hGLP-2(3-33,M10Y) with KD values of 59.3 and 40.6 nM. The latter (with antagonistic properties) had higher Bmax and faster on and off rates compared to the former (full agonist). Both bound the hGLP-1 receptor with low affinity (Ki of 130 and 330 nM, respectively). Autoradiography in wildtype mice revealed strong labelling of subepithelial myofibroblasts, confirmed by immunohistochemistry using a GLP-2 receptor specific antibody that in turn was confirmed in GLP-2 receptor knock-out mice. CONCLUSION AND IMPLICATIONS: Two new radioligands with different binding kinetics, one a full agonist and the other a weak partial agonist with antagonistic properties were developed and subepithelial myofibroblasts identified as a major site for GLP-2 receptor expression.

Loss of Function Glucose-Dependent Insulinotropic Polypeptide Receptor Variants Are Associated With Alterations in BMI, Bone Strength and Cardiovascular Outcomes.

Glucose-dependent insulinotropic polypeptide (GIP) and its receptor (GIPR) are involved in multiple physiological systems related to glucose metabolism, bone homeostasis and fat deposition. Recent research has surprisingly indicated that both agonists and antagonists of GIPR may be useful in the treatment of obesity and type 2 diabetes, as both result in weight loss when combined with GLP-1 receptor activation. To understand the receptor signaling related with weight loss, we examined the pharmacological properties of two rare missense GIPR variants, R190Q (rs139215588) and E288G (rs143430880) linked to lower body mass index (BMI) in carriers. At the molecular and cellular level, both variants displayed reduced G protein coupling, impaired arrestin recruitment and internalization, despite maintained high GIP affinity. The physiological phenotyping revealed an overall impaired bone strength, increased systolic blood pressure, altered lipid profile, altered fat distribution combined with increased body impedance in human carriers, thereby substantiating the role of GIP in these physiological processes.

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.