Altered desensitization and internalization patterns of rodent versus human glucose-dependent insulinotropic polypeptide (GIP) receptors. An important drug discovery challenge

Abstract

Background and Purpose

The gut hormone glucose-dependent insulinotropic polypeptide (GIP) signals via the GIP receptor (GIPR), resulting in postprandial potentiation of glucose-stimulated insulin secretion. The translation of results from rodent studies to human studies has been challenged by the unexpected effects of GIPR-targeting compounds. We, therefore, investigated the variation between species, focusing on GIPR desensitization and the role of the receptor C-terminus.

Experimental Approach

The GIPR from humans, mice, rats, pigs, dogs and cats was studied in vitro for cognate ligand affinity, G protein activation (cAMP accumulation), recruitment of beta-arrestin and internalization. Variants of the mouse, rat and human GIPRs with swapped C-terminal tails were studied in parallel.

Key Results

The human GIPR is more prone to internalization than rodent GIPRs. Despite similar agonist affinities and potencies for G_αs activation, especially, the mouse GIPR shows reduced receptor desensitization, internalization and beta-arrestin recruitment. Using an enzyme-stabilized, long-acting GIP analogue, the species differences were even more pronounced. ‘Tail-swapped’ human, rat and mouse GIPRs were all fully functional in their G_αs coupling, and the mouse GIPR regained internalization and beta-arrestin 2 recruitment properties with the human tail. The human GIPR lost the ability to recruit beta-arrestin 2 when its own C-terminus was replaced by the rat or mouse tail.

Conclusions and Implications

Desensitization of the human GIPR is dependent on the C-terminal tail. The species-dependent functionality of the C-terminal tail and the different species-dependent internalization patterns, especially between human and mouse GIPRs, are important factors influencing the preclinical evaluation of GIPR-targeting therapeutic compounds.

Graphical Abstract

Abbreviations

GIPR

GIP receptor

PEI

polyethyleneimine

What is already known

• Glucose-dependent insulinotropic polypeptide receptor (GIPR) ligands seem to have different species-specific actions.
• Studies of GIPR-targeting therapeutics show diverging results in rodents and humans.

What does this study add

• The mouse GIPR, in contrast to other species, hardly recruits beta-arrestins or undergoes internalization.
• GIPR internalization and beta-arrestin recruitment are dependent on the C-terminal tail of the receptor.

What is the clinical significance

• Differential receptor internalization properties importantly affect the preclinical evaluation of GIPR-targeting compounds in mice.

1 INTRODUCTION

Glucose-dependent insulinotropic polypeptide (gastric inhibitory polypeptide; GIP) is a gut hormone secreted from enteroendocrine K cells in response to nutrient absorption (Lauritsen et al., 1980). Together with glucagon-like peptide 1 (GLP-1), GIP potentiates glucose-stimulated insulin secretion (Nauck, Bartels, et al., 1993). The actions of the two incretin hormones account for most of the postprandial rise in insulin secretion in healthy individuals (Gasbjerg, Bergmann, et al., 2019), which has led to the development of several incretin-based agents for the treatment of metabolic diseases (Andersen et al., 2018; Iqbal et al., 2022; PMID: 38062121).

The actions of GIP are mediated through the GIP receptor (GIPR), a class B1 G-protein coupled receptor (GPCR) with intracellular signalling involving cAMP formation and elevated Ca²⁺ (Baggio & Drucker, 2007; Fredriksson et al., 2003). The GIPR is expressed in many different organs, that is, the pancreas, adipose tissue, bones, vasculature and several regions in the brain (Usdin et al., 1993). However, it is mainly the abilities of both exogenous and endogenous GIPs to increase insulin secretion and reduce bone resorption that have attracted interest in humans (Gasbjerg, Helsted, et al., 2019; Helsted et al., 2020; Nauck, Bartels, et al., 1993; Nissen et al., 2014). Moreover, GIP administration to healthy individuals may induce glucagon secretion during euglycaemia and hypoglycaemia (Christensen et al., 2011) and stimulate the deposition of triglycerides in subcutaneous adipose tissue (Asmar et al., 2017).

The other incretin hormone, GLP-1, signals via the GLP-1 receptor, which also belongs to class B1 of GPCRs (Baggio & Drucker, 2007; Fredriksson et al., 2003). Several pharmaceutical compounds target the GLP-1 receptor and agonists of this are an established drug class for the treatment of type 2 diabetes and obesity (Andersen et al., 2018; Iqbal et al., 2022). Interestingly, in recent years, compounds targeting the GIPR have also been developed as anti-diabetes treatments, for example, GLP-1–GIP receptor co-agonists (Frías et al., 2021). Surprisingly, however, both GIP antagonists and agonists have been reported to cause weight loss and lower glycated haemoglobin (HbA1c) in rodents and non-human primates, alone or (especially) combined with GLP-1 agonists; (Rosenkilde et al., 2024). These paradoxical findings have spurred interest in an improved understanding of the optimal pharmacodynamic profile of GIPR therapeutics (Campbell, 2021; Killion, Lu, et al., 2020b). The insulinotropic effect of GIP in most patients with type 2 diabetes is much reduced compared with that observed in healthy individuals (Vilsbøll et al., 2003). Despite this, targeting the GIPR is still suggested to be therapeutically interesting in metabolic-related diseases (Campbell, 2021; Finan et al., 2016; Smit et al., 2021) and bone disorders (Gabe et al., 2022), and the incorporation of GIP activity has been suggested to contribute to the impressive glucose-lowering and body weight-reducing effects of the GLP-1–GIP receptor co-agonist tirzepatide (Frías et al., 2021; Jastreboff et al., 2022). Thus, a deeper understanding of the GIP system is needed to support further drug development.

Preclinical testing of drug candidates often involves animal models for proof of concept. Traditionally, mice and rats are used for endocrine and metabolic drug evaluation, but the translational value of the studies may be compromised by differences between species. For GPCRs, marked differences have been described, for instance regarding the G-protein coupled receptor 119 (GPR119) (Scott et al., 2013) and the cannabinoid receptors (Carruthers & Grimsey, 2023), but there are also receptors showing little inter-species difference, for examplein the GLP-1 system (Knudsen et al., 2012). Importantly, the GIP system is less conserved across species than the GLP-1 receptor (Sparre-Ulrich et al., 2016), but the consequences of the differences regarding receptor properties have not been thoroughly investigated. Here, we investigated species differences in the GIP system, with a focus on the mouse, rat and human GIPRs but also including pig, dog and cat GIPRs. Our studies comprise cAMP formation as a measure of G-protein coupling, beta-arrestin recruitment and receptor internalization profiles after receptor activation to elucidate the apparently incompatible results of the rodent studies underlying GIPR drug development.

2 METHODS

2.1 Cell culture and transfection

COS-7 cells (RRID:CVCL_0224) were cultured at 10% CO₂ and 37°C in Dulbecco's modified Eagle's medium (DMEM) 1885 supplemented with 10% fetal bovine serum (FBS), 2 mol·L⁻¹ glutamine, 180 units·ml⁻¹ penicillin and 45 g·ml⁻¹ streptomycin. HEK293 (RRID:CVCL_0045) and HEK293A (RRID:CVCL_6910) cells were cultured at 5% or 10% CO₂ and 37°C in DMEM-GlutaMAX™-I supplemented with 10% FBS, 180 units·ml⁻¹ penicillin and 45 g·ml⁻¹ streptomycin. MIN6 cells (mouse pancreatic cell line, RRID:CVCL_0431) were cultured at 5% CO₂ and 37°C in DMEM-GlutaMAX™-I supplemented with 10% FBS, 180 units·ml⁻¹ penicillin, 45 g·ml⁻¹ streptomycin, 20 mmol·L⁻¹ HEPES and 50 μmol·L⁻¹ beta-mercaptoethanol. HEK293 and COS-7 cells were transiently transfected using the calcium phosphate precipitation method using the plasmid pcDNA3.1(+) (Steen et al., 2013). HEK293A cells were transiently transfected using the polyethyleneimine (PEI) transfection method (Donthamsetti et al., 2015).

2.2 Receptor constructs

Human and rat GIP(1-42), as well as a long-acting GIP analogue, were purchased from Wuxi AppTec (Shanghai, China; custom-manufactured sequences in Figures 1c and 2a). Mouse GIP(1-42) was purchased from Phoenix Pharmaceuticals (Karlsruhe, Germany, Catalogue No. 027-27). The long-acting GIP analogue, which is a full agonist of both human and rat GIPRs with a half-life suitable for once-daily treatment, was provided by Bainan Biotech. All peptides had a purity of more than 95% by high-performance liquid chromatography (HPLC) analysis and had the correct molecular mass as controlled by mass spectrometry. ¹²⁵I-labelled human GIP(1-42) was purchased from PerkinElmer Life Sciences (Skovlunde, Denmark, CAS No. NEX402). cDNAs of the human, rat and mouse GIPRs were purchased from Origene (Rockville, MD, USA), and human and rat GIPRs (SC110906 and RN212314, respectively) were inserted into the pcDNA 3.1 vector (Invitrogen, Thermo Fischer Scientific). The mouse GIPR (MC216211) was cloned into the pCMV-Script vector (Agilent Technologies Denmark). For the tail-swapped human GIPRs, the rat C-terminus (54 C-terminal amino acids) or the mouse C-terminus (60 C-terminal amino acids) replaced the human GIPR from position 405. For the rat GIPR, the human C-terminus (62 C-terminal amino acids) was inserted at position 402 and for the mouse GIPR, the human C-terminus was inserted at position 401. For the SNAP-tagged receptors, the tag was placed N-terminally. All SNAP-tagged and tail-swapped GIPR constructs were synthesized by and purchased from GenScript (Piscataway, NJ, USA) and inserted into the pcDNA 3.1 vector. Full ligand and receptor sequences are provided in Table S1.

FIGURE 1

Mouse and cat glucose-dependent insulinotropic polypeptide (GIP) receptors (GIPRs) do not desensitize following agonist preincubation. The binding of ¹²⁵I-human GIP(1-42) to transiently transfected COS-7 cells expressing human, rat, mouse, pig, dog or cat GIPR was tested in (a) following 90 min of preincubation with 1, 10 or 100 nmol·L⁻¹ (nM) of GIPR agonist and in (b) in the presence of increasing amounts of GIPR agonist (without preincubation). (c) Endogenous GIPR agonists of the six species. (d) B_max values from homologous binding of GIP(1-42) displacing ¹²⁵I-human GIP(1-42) in transiently transfected COS-7 cells expressing the six GIPRs shown as percentages of human GIPR. Human GIPR n = 11, rat n = 10, mouse n = 11, pig n = 6, dog n = 6 and cat n = 6. Data are shown as means ± SEM.

FIGURE 2

Long-acting glucose-dependent insulinotropic polypeptide (GIP) analogue augments species-specific desensitization patterns. (a) Amino acid sequence of a GIP analogue. (b, c) Ligand dose–response of human, rat or mouse GIP (closed symbols) or GIP analogue (open symbols) stimulated cAMP accumulation of six GIP receptors (GIPRs) (human n = 7, rat n = 7, mouse n = 4, pig n = 3, dog n = 3 and cat n = 3) transiently expressed in HEK293 cells. (d) The binding of ¹²⁵I-human GIP(1-42) to transiently transfected COS-7 cells expressing human (n = 6), rat (n = 5), mouse (n = 5), pig (n = 3), dog (n = 3) or cat (n = 3) GIPR was tested following 90 min of preincubation with 1, 10 or 100 nmol·L⁻¹ (nM) of the GIP analogue. Data are shown as means ± SEM.

2.3 cAMP assay

Transiently transfected COS-7 cells were seeded in 96-well plates with a density of 35,000 per well and incubated overnight. The following day, the cells were washed twice with HEPES-buffered saline (HBS) buffer and incubated with HBS together with 1 mmol·L⁻¹ 3-isobutyl-1-methylxanthine (IBMX) for 30 min at 37°C. The respective ligands were added and the cells were incubated for 30 min at 37°C. The HitHunter cAMP assay (DiscoverX, Herlev, Denmark) was conducted according to the manufacturer's instructions.

2.4 Internalization assay

The time-resolved fluorescence resonance energy transfer (TR-FRET)-based internalization assay (Foster & Bräuner-Osborne, 2018) was performed with HEK293A expressing the GIPR from various species with an N-terminal SNAP tag. We previously described the use of the SNAP-tagged human GIPR (Gabe et al., 2018). The cells were seeded in white 384-well plates the day after transfection, with 15,000–20,000 cells per well. The media were removed the following day and the receptors were labelled with Tag-lite snap-lumi4-tb (0.1 pmol·μl⁻¹) (donor; Cisbio.eu/Triolab AS, Brøndby, Denmark; Catalogue No. SSNPTBD) in Opti-MEM for 60 min at 37°C. After labelling, the cells were washed four times with internalization buffer (Hanks' balanced salt solution [HBSS] supplemented with 1 mmol·L⁻¹ CaCl₂, 1 mmol·L⁻¹ MgCl₂, 20 mmol·L⁻¹ HEPES and 0.1% bovine serum albumin [BSA; pH 7.4]). Subsequently, 10 μl of 50 μmol·L⁻¹ fluorescein-O′-acetic acid (acceptor; Sigma-Aldrich, Catalogue No. 88596) was added to each well except for the wells used to record the donor signal (as a measure of receptor expression). We then added 10 μl of GIPR agonist (37°C), required for the indicated concentrations in the internalization buffer, to the plates to monitor agonist-induced internalization. The internalization was measured every 3 min at 37°C in a Perkin Elmer™ Envision 2014 multilabel reader and plotted as donor/acceptor. Background signals obtained in the absence of ligands were subtracted from the results.

To measure the impact of agonist preincubation on the surface expression of the SNAP-tagged receptors, GIP(1-42) or GIP analogue (both at 1 nmol·L⁻¹, 10 nmol·L⁻¹, 100 nmol·L⁻¹ and 1 μmol·L⁻¹) was added to the cells in serum-depleted growth media without antibiotics but supplemented with the dipeptidyl peptidase 4 (DDP-4) inhibitor Valine-Pyrrolidide (DMEM-GlutaMAX™-I, 1% FBS, 0.02 mmol·L⁻¹ Val-Pyr) and incubated for 90 min at 37°C. After this, the cells were washed four times in the internalization buffer and the donor was added for labelling. The donor signal was determined as described above.

2.5 Beta-arrestin recruitment assays

One day prior to transfection, 500,000 HEK293A cells per well were seeded in a clear 6-well plate. The cells were transiently transfected using the polyethylenimine (PEI) method with the GIPR from respective species (0.33 μg), arrestin-1-Sp1 (donor, beta-arrestin 1) (0.042 μg), arrestin-3-Sp1 (donor, beta-arrestin 2) (0.042 μg), mem-linker-citrine-SH3 (acceptor) (0.8 μg), G-protein coupled receptor kinase 2 (beta adrenergic receptor kinase 1/ GRK2; 0.8 μg) and PEI (3.94 μg). Two days following transfection, cells were washed with phosphate-buffered saline (PBS) and resuspended in 3 ml of PBS + 1% glucose (0.5 M). Afterwards, 85 μl of cell suspension was aliquoted into each well of a white 96-well plate, followed by the addition of 10 μl of coelenterazine-h (50 μmol·L⁻¹ in PBS). After 10 min of incubation, 5 μl of increasing species-specific GIP(1-42) concentrations (100 pmol·L⁻¹ to 1 μmol·L⁻¹) was added. Following 30 min of incubation at room temperature, the luminescence (emission intensity at 535 nm divided by emission intensity at 480 nm) was measured by the PerkinElmer EnVision 2104 Multilabel Reader.

2.6 Homologous competition binding

Transiently transfected COS-7 cells were seeded in white 96-well plates 1 day after the transfection. The number of cells was adjusted to result in 5%–10% specific binding of the added radioactive ligand. The MIN6 cells (a mouse pancreatic endocrine cell line) were seeded 1 day before the binding experiment at 200,000 cells per well to obtain sufficient specific binding (aiming for 5%–10%) of the added radioligand. The following day, the cells were washed twice in binding buffer at room temperature, and 15–40 pmol·L⁻¹ of radioligand (¹²⁵I-labelled human GIP(1-42)) and unlabelled ligand in binding buffer (50 mmol·L⁻¹ HEPES buffer [pH 7.2] supplemented with 0.1% casein) were added in a total volume of 100 μl (96-well plates) or 300 μl (12-well plates) and incubated for 3 h at 4°C. After incubation with radioligands and unlabelled agonists, the cells were washed twice in ice-cold binding buffer and lysed using 200 mmol·L⁻¹ NaOH with 1% sodium dodecyl sulfate (SDS) for 30 min. Nonspecific binding was determined in the presence of 100 nmol·L⁻¹ unlabelled ligand. The samples were analysed using a Wallac Wizard 1470 Gamma Counter.

To measure the impact of agonist preincubation on GIPR surface expression (receptor desensitization) using competition binding as readout, GIP(1-42) or GIP analogue (both at 1 nmol·L⁻¹, 10 nmol·L⁻¹, 100 nmol·L⁻¹ and 1 μmol·L⁻¹) was added to the cells after the two washes in a total volume of 100 μl (96-well plates, transfected HEK293 cells) or 300 μl (12-well plates, MIN6 cells) serum-depleted growth media without antibiotics but supplemented with Val-Pyr (DMEM-1885, 1% FBS, 0.02 mmol·L⁻¹ Val-Pyr). After ligand preincubation for 0, 5, 20, 45, 60 or 90 min at 37°C, the cells were washed twice in binding buffer at room temperature before the addition of radioligand and unlabelled ligand, and the competition binding experiments were hereafter conducted as described above.

2.7 Confocal microscopy

To visualize receptor localization and monitor receptor internalization by imaging, HEK293 cells (300,000 in 1 ml) were transfected in suspension with SNAP-tagged hGIPR, mGIPR or rGIPR and rGFP-CAAX (1.5 μg of plasmid DNA complexed with linear PEI; molecular weight [MW] 25,000, 3:1 PEI:DNA ratio) and seeded (3.5 × 10⁴ cells per well) in μClear white 96-well plates. SNAP-tagged receptors were imaged in living cells using the Opera Phenix Plus high-content screening system (PerkinElmer) equipped with a 40×, 1.1 NA water objective and labelled with either cell-permeable SNAP-Cell 647-SiR or cell-impermeable SNAP-Surface Alexa Fluor 647 (New England Biolabs) (1:200, 15 min) and washed twice in HBSS prior to imaging. Baseline measurements were obtained in eight image fields per condition. Cells were subsequently stimulated with hGIP(1-42) (1 μM) for 120 min, with images being acquired every 5 min (rGFP, Ex/Em: 488/500–550 and Alexa 647, Ex/Em: 640/650–760).

2.8 Data analyses and calculations

IC₅₀ and EC₅₀ values were determined by nonlinear regression carried out with the GraphPad Prism Software 9.5.1 (GraphPad, San Diego, CA, USA). K_d was determined by homologous receptor binding using the Cheng–Prusoff equation (Yung-Chi & Prusoff, 1973). Maximal binding capacity (B_max) was based on the formula for one class of binding sites in homologous competition binding studies using Microsoft Excel™ (DeBlasi et al., 1989). Baseline subtraction was performed for each individual experiment before further calculations. Area under the curve (AUC) calculations are based on the trapezoid rule and together with statistical analyses of multiple comparisons (one-way analysis of variance) and internalization rate (rate constant), they are also performed with GraphPad Prism 9.5.1. A pipeline was developed for image analysis by Python 3.12 (Python Software Foundation), consisting of Cellpose (Stringer et al., 2021; Stringer & Pachitariu, 2024) and Scikit-image (Van Der Walt et al., 2014). Images were automatically pre-processed by the following steps: eliminating exposure bias using adaptive equalization, enhancing contrast using sigmoid correction and filtering background noise using adaptive threshold. Masks of plasma membrane were segmented by Cellpose and outlines with a 5-pixel width were extracted. Connectivity domain analysis was then applied to quantify the receptors on the edges of membranes and in vesicles. All experiments were repeated at least three times, in duplicate, and if a large variation between experiments was observed, additional experiments were included. Outliers were, in general, included in data analyses and presentations. The data and statistical analysis comply with the recommendations of the British Journal of Pharmacology on experimental design and analysis in pharmacology (Curtis et al., 2022). Sample sizes of at least n=5 independent values were subjected to statistical analysis. P<0.05 is set as the threshold for statistical significance, and post hoc tests were conducted if F in the one-way analysis of variance achieved P<0.05.

2.9 Materials

The following were purchase from Sigma-Aldrich (Søborg, Denmark), beta-mercaptoetanol (M620), DMEM (D5030), FBS (F7524-500 ml), glucose (G7021), HEPES (H3784), MgCl2 (13152), NaOH (S881), and SDS (74225) while HBSS (14175-05) was purchase from ThermoFisher (Lilleroed, Denmark), CaCl2 (CA01931000) from Scarlau (Scharlab S.L., Barcelona, Spain) and PEI (cat. 23966) from Polysciences (Hirschberg an der Bergstrasse, Germany). BSA, penicillin, and streptomycin are centrally supplied from University of Copenhagen. The plasmids for arrestin recruitment (donors and acceptors) were kindly provided by Jonathan A. Javitch, Columbia University, and valine pyrrolidide was a gift from Novo Nordisk, Måløv, Denmark. Details of other materials and suppliers were provided in the specific sections.

Details of other materials and suppliers were provided in the specific sections.

2.10 Nomenclature of targets and ligands

Key protein targets and ligands in this article are hyperlinked to corresponding entries in the IUPHAR/BPS Guide to PHARMACOLOGY http://www.guidetopharmacology.org and are permanently archived in the Concise Guide to PHARMACOLOGY 2023/24 (Alexander et al., 2023).

3 RESULTS

3.1 Species-specific differences in GIPR desensitization

We have previously reported that the GIPRs from mice, rats and humans signal differently in response to the partial agonist Pro3-GIP (Sparre-Ulrich et al., 2016). To address receptor desensitization of GIPR from six species (human, rat, mouse, pig, dog and cat), we preincubated transiently transfected cells with three doses of GIP agonist (1, 10 and 100 nmol·L⁻¹ of human, rat or mouse GIP [Figure 1c]) and addressed the binding capacity after 90 min using competitive radioligand binding (Figure 1a). Compared with buffer-preincubated GIPR (vehicle), preincubation with 100 nmol·L⁻¹ GIP agonist led to a dose-dependent reduction in radioligand binding for human (56 ± 6.0%), rat (54 ± 4.3%), pig (74 ± 2.1%) and dog (62 ± 2.0%) GIPRs, while mouse and cat GIPRs showed very little or no impairment of ¹²⁵I-human GIP binding following all doses of species-specific GIP agonist (both >80% reduction by 100 nmol·L⁻¹ GIPR agonist). Confirming that the receptors were affected by preincubation and not ligand-dependent properties, all ligands were able to displace ¹²⁵I-human GIP with varying but similar K_i values in agreement with previous findings (Sparre-Ulrich et al., 2016) (Figure 1b and Table 1). The initial surface expression or number of binding sites was, however, reduced for mouse, cat and dog GIPRs compared with human GIPRs, as indicated by corresponding B_max values (Figure 1d and Table 1).

TABLE 1. K_d and B_max (competitive binding) and EC₅₀ and E_max (cAMP accumulation and beta arrestin recruitment) for species of glucose-dependent insulinotropic polypeptide (GIP) receptors (GIPRs). (a) The binding of ¹²⁵I-human GIP(1-42) to transiently transfected COS-7 cells expressing human, rat, mouse, pig, dog or cat GIPR was tested in the presence of increasing amounts of GIPR agonist. (b) Ligand dose–response of human, rat or mouse GIP or GIP analogue stimulated cAMP accumulation of the six GIPRs transiently expressed in HEK293A cells. (c) Ligand dose–response of human, rat or mouse GIP stimulated beta-arrestin 1 and 2 recruitment of human, rat and mouse GIPRs transiently expressed in HEK293 cells. Data are means ± SEM.

(a)
Receptor (competition binding)	n	Kd (log.)	SEM (log.)	Kd (nmol·L^−1)	Bmax (%)	SEM (%)
Human GIPR	23	−8.86	0.098	1.38	100	0
Rat GIPR	10	−8.85	0.18	1.42	84	29
Mouse GIPR	11	−8.58	0.18	2.64	46	8.8
Pig GIPR	6	−8.40	0.10	3.96	275	60
Dog GIPR	6	−8.94	0.09	1.15	28	6.4
Cat GIPR	6	−8.87	0.11	1.35	30	7.4

(b)
Receptor: ligand (cAMP formation)	n	EC50 (log.)	SEM (log.)	EC50 (pmol·L^−1)	Emax (%)	SEM (%)
Human GIPR: hGIP	9	−10.7	0.065	20	97	1.2
Human GIPR: GIP analogue	7	−10.7	0.13	20	107	4.2
Rat GIPR: rGIP	9	−10.2	0.24	60	95	7.8
Rat GIPR: GIP analogue	7	−10.0	0.20	96	98	7.6
Mouse GIPR: mGIP	5	−10.3	0.30	49	88	9.3
Mouse GIPR: GIP analogue	4	−9.62	0.13	24	113	6.0
Pig GIPR: hGIP	3	−11.0	0.11	11	97	3.5
Pig GIPR: GIP analogue	3	−11.5	0.19	29	145	5.5
Dog GIPR: hGIP	3	−10.1	0.10	76	94	3.5
Dog GIPR: GIP analogue	3	−10.5	0.23	29	136	8.4
Cat GIPR: hGIP	3	−10.5	0.10	32	98	3.1
Cat GIPR: GIP analogue	3	−11.2	0.18	57	96	5.5

(c)
Receptor (beta-arrestin)	n	EC50 (log.)	SEM (log.)	EC50 (pmol·L^−1)	Emax (%)	SEM (%)
Human GIPR (beta-arrestin 1)	4	−8.88	0.22	1.3	88	6.2
Human GIPR (beta-arrestin 2)	10	−8.39	0.13	4.0	100	4.2
Rat GIPR (beta-arrestin 1)	4	−9.65	0.51	0.22	52	14
Rat GIPR (beta-arrestin 2)	5	−8.62	0.21	2.4	143	10
Mouse GIPR (beta-arrestin 1)	4	−10.6	0.51	0.014	59	17
Mouse GIPR (beta-arrestin 2)	5	−9.60	0.62	0.25	28	4.7

3.2 GIP analogue augments species-specific desensitization patterns

Long-acting compounds providing continuous exposure are often suitable drug candidates. To address differences between GIPRs from different species, an enzyme-stabilized lipidated GIP analogue with amino acid substitutions at positions 2 and 17 and a C16 dicarboxylic acid attached at position 17 (Figure 2a) was first confirmed as a full agonist on receptors from all six species. The potency of the GIP analogue was highest for the pig and cat GIPRs and lowest for the rat and mouse GIPRs, and overall, it followed the potency of native GIP(1-42) on these receptors (Figure 2b,c and Table 1). As with the native agonists, receptor desensitization was probed in transiently transfected cells expressing all six GIPRs with preincubation using three doses of the GIP analogue and subsequent competitive radioligand binding after 90 min (Figure 2d). In response to preincubation with three concentrations of the GIP analogue (1, 10 and 100 nmol·L⁻¹), only the mouse GIPR did not respond with a reduced binding capacity of ¹²⁵I-human GIP after 90 min, supporting its reduced ability to desensitize as seen following preincubation with mGIP (Figure 1a). The remaining GIPRs were desensitized to an even higher degree upon the addition of the long-acting GIP analogue (Figure 2d) compared with the native GIP (Figure 1a).

3.3 Mouse GIPR shows a low degree of internalization in response to agonists

During the development of drugs targeting the GIPR, many studies have been carried out in rodents. As we observed different desensitization of mouse and rat GIPRs compared with human GIPR (Figures 1 and 2), we decided to thoroughly study the receptor internalization pattern of mouse, rat and human GIPRs using N-terminally SNAP-tagged receptors (Figure 3a). All three SNAP-tagged receptors bound ¹²⁵I-human GIP(1-42), with similar high affinity assessed by homologous competition binding (Table S2). With respect to receptor surface expression, the receptor donor signals were similar (Figure 3c) and when expressed in HEK293 cells, human and rodent receptors were primarily localized to the plasma membrane and displayed similar subcellular distributions (Figure S3). In response to 1000 nmol·L⁻¹ of either the native endogenous agonist (Figure 3d,f,h) or the GIP analogue (Figure 3e,g,i), human GIPR had the highest degree of internalization induced by either ligand as assessed by AUCs (Figures 3b and S3). There were no differences between internalization induced by the endogenous agonist or the GIP analogue for any of the three receptors (Figure 3b). Likewise, the internalization rates were reduced for mouse and rat GIPRs compared with human GIPR during both exposures to the endogenous agonist and GIP analogue (Figure 3j-m). Thus, in line with the results of the binding after preincubation with agonists (Figures 1 and 2) and similar experimental design with the SNAP-tagged receptors, where the receptors were labelled after preincubation with endogenous ligands or GIP analogues (Figure S2), the mouse GIPR had a very low degree of internalization, whereas both the rat GIPR and, especially, the human GIPR internalized to a higher degree (Figure 3d-m).

FIGURE 3

The mouse glucose-dependent insulinotropic polypeptide (GIP) receptor (GIPR) has lower internalization abilities than human and rat GIPRs. (a) SNAP-tagged receptors used for time-resolved fluorescence resonance energy transfer-based receptor internalization assays. (b) Area under the curves for 1000 nmol·L⁻¹-induced internalization of human, rat and mouse SNAP-tagged GIPRs transiently expressed in HEK293A cells in response to endogenous agonist (closed bars) or GIP analogue (open bars) compared with unpaired one-way analysis of variance and Tukey's multiple comparisons test. (c) Control experiments of surface expression of SNAP-tagged GIPRs and vector, n = 4. (d–i) Ligand-stimulated internalization ratios over time for SNAP-human GIPR (d, e), SNAP-rat GIPR (f, g) and SNAP-mouse GIPR (h, i) in response to 1, 10, 100 or 1000 nmol·L⁻¹ (nM) of endogenous agonist (d, f, h) or GIP analogue (e, g, i). J-M: Comparisons of ligand-induced internalization rates. Data are shown as means ± SEM, n = 6  (n = 3 for J and L). * indicates P < 0.05.

3.4 Human GIPR has a specific phosphorylation motif favourable for beta-arrestin recruitment

Due to the different internalization patterns across the three GIPRs (Figure 3), we assessed the beta-arrestin recruitment of the three GIPRs in parallel. In HEK293 cells transiently transfected with human, rat and mouse GIPRs, the beta-arrestin 2 recruitment efficacies in response to mouse GIP at the mouse GIPR were 28 ± 4.7% of human GIPR, whereas the rat receptor showed the opposite pattern with 143 ± 10% of human GIPR in response to rat GIP (Figure 4 and Table 1). For beta-arrestin 1 recruitment, the efficacies of both mouse and rat GIPRs were below that of human GIPR, while the potency was the highest for mouse GIPR compared with rat and human GIPRs (Table 1).

FIGURE 4

Only the human glucose-dependent insulinotropic polypeptide (GIP) receptor (GIPR) has a phosphorylation pattern that could promote beta-arrestin recruitment. (a) Ligand dose–response stimulated beta-arrestin 2 recruitment of human (n = 10), rat (n = 5) and mouse (n = 5) GIPR baseline-subtracted values shown as per cent of maximal recruitment to the human GIPR in transiently expressed HEK293 cells. Data are shown as means ± SEM. (b) A structural model of the human GIPR/beta-arrestin 2 complex indicating sites 1, 2 and 3. The model was obtained based on the rhodopsin/visual arrestin complex (PDB code: 5W0P). (c) Sequence alignment of human, rat and mouse GIPRs, including the sequence of the rhodopsin C-terminal tail as a reference. Phosphorylatable (S, T) and negatively charged (E, D) residues are highlighted in grey. The PxP(E)xxP motif, including sites 1, 2 and 3, is highlighted in red. (d) Schematic representation of how the PxPxxP motif engages the three negatively charged sites 1, 2 and 3 at beta-arrestin 2.

To understand the receptor–arrestin complex, we compared the GIPRs with the more well-characterized rhodopsin receptor, focusing on possible phosphorylation sites involved in arrestin binding. Previous research (Zhou et al., 2017) has demonstrated that specific motifs of phosphorylation sites or negatively charged residues (e.g., PxPxxP/E or PxxPxxP) promote arrestin recruitment, where P refers to a phospho-Ser (pS) or phospho-Thr (pT) and x refers to any other amino acid except proline. Interestingly, in the C-terminal tail of the human GIPR, we find only one region, ‘459–464: SRELES’, that resembles a correct spacing with a PxExxP motif (Figure 4b,c). Such specific spacing of negative charges allows strong electrostatic interactions with conserved positive residues in the N-terminal domain of beta-arrestin 2 at sites 1 (R166), 2 (K12) and 3 (K11) (Shukla et al., 2013), as marked on Figure 4b. Of note, in phosphorylation site 2 of this motif, we observe a glutamate (E) instead of a phosphorylatable residue (pS or pT). To evaluate the ability of such a C-terminal tail to engage beta-arrestin 2, we used an experimentally solved structure of a PxPxxP/E motif, namely the rhodopsin C-tail bound to arrestin (Zhou et al., 2017), as a template (see Figure 4c,d). Intriguingly, we find that the modelled C-terminal tail is able to replicate the electrostatic interaction pattern of the rhodopsin C-terminal tail (i.e. interactions with K295 in the lariat loop region), suggesting that the E residue can substitute a pS or pT residue in position 2. This finding is in line with our observation that human GIPR is able to recruit beta-arrestin. In contrast, the C-terminal tails of both the rat and murine variants of GIPR lack a complete PxPxxP or PxxPxxP motif (Figure 4c). Instead, we find that the phosphorylation site 2 exposes a valine in both cases.

3.5 The GIPRs do not desensitize in mouse pancreatic islet cell line

To address whether the lack of desensitization and internalization of the mouse GIPR also applies to naturally expressed receptors in a relevant cell, we performed competitive binding studies in the MIN6 cell line widely used for pancreatic islet studies of, for example, insulin secretion (Iwasaki et al., 2010; Nakashima et al., 2009). Increasing amounts of human GIP were able to displace ¹²⁵I-human GIP with a similar affinity (logIC₅₀-9.0 ± 0.31; Figure 5a) as mouse GIP on mouse GIPR transiently transfected in COS-7 cells (logIC₅₀-8.9 ± 0.062; Figure 1b). Following 90 min of preincubation with three doses (1, 10 or 100 nmol·L⁻¹) of either human GIP or the GIP analogue (Figure 2a), the binding of the radioligand was similar to the binding without preincubation (Figure 5b). As seen for the transiently transfected mouse GIPRs, the mouse GIPRs of the MIN6 cell lines showed no desensitization in response to agonist preincubation.

FIGURE 5

Glucose-dependent insulinotropic polypeptide (GIP) radioligand binding to the pancreatic mouse cell line is not affected by preincubation. The binding of ¹²⁵I-human GIP(1-42) to MIN6 cells (pancreatic mouse cell line) was tested in the presence of increasing amounts of (a) human GIP (without preincubation) and (b) following 90 min of preincubation with 1, 10 or 100 nmol·L⁻¹ (nM) of either human GIP (closed symbols) or the GIP analogue (open symbols). Data are shown as means ± SEM, n = 3 for all experiments.

3.6 The C-terminal tail of the GIPR is essential for beta-arrestin recruitment and internalization

As the agonist binding and G protein-dependent receptor activation properties are similar between human and rodent GIPRs, whereas their beta-arrestin recruitment and internalization patterns differ, we hypothesized that substitution of the C-terminal tails between human, rat and mouse GIPRs would result in an internalization pattern reflecting the species of the C-terminal tail. All four GIPR variants, human GIPR–rat tail (Figure 6a,b), human GIPR–mouse tail (Figure 6c,d), mouse GIPR–human tail (Figure 6e,f) and rat GIPR–human tail (Figure 6g,h), were activated by the endogenous ligand and the GIP analogue (Figure 6a,c,e,g). The species-specific ligands were also able to bind (Figure 6b,d,f,h) and activate each of the four receptors with similar E_max values as the wild-type receptors (small panels in Figure 6a,c,e,f). With respect to receptor surface expression (Table S3), B_max values were, although varying, in the same range as the untagged receptors (Table 1).

FIGURE 6

Substitution of the C-terminal tails between human, rat and mouse glucose-dependent insulinotropic polypeptide (GIP) receptors (GIPRs) results in preserved agonist stimulation and binding. Ligand dose–response (species-specific GIP [closed symbols] and GIP analogue [open symbols]) stimulated cAMP accumulation of four GIPRs transiently expressed in HEK293 cells and heterologous radioligand binding using ¹²⁵I-labelled human GIP and species-specific GIPR agonist of the GIPRs transiently expressed in COS-7 cells [human GIPR–rat tail (a, b), human GIPR–mouse tail (c, d), mouse GIPR–human tail (e, f) and rat GIPR–human tail (g, h)]. E_max values are shown as a percentage of the wild-type receptor with an endogenous ligand. Dashed lines are corresponding full-length wild-type receptors. Data are shown as means ± SEM, n = 3 for all experiments.

Consistent with the stronger beta-arrestin 2 recruitment of human GIPR, transfer of the tail of the human GIPR to the mouse or rat GIPR resulted in increased beta-arrestin 2 recruitment relative to the wild-type mouse and rat GIPRs (Figure 7c and b, respectively). In both cases, the efficacy even increased to more than that of the human GIPR (mouse GIPR–human tail to 196 ± 14% and rat GIPR–human tail to 320 ± 39% of human GIPR). Supporting the weaker arrestin recruitment of mouse and rat GIPRs, the human GIPR with the tails of either of these two was heavily impaired in their beta-arrestin recruitment (Figure 7a). The beta-arrestin recruitment pattern is therefore highly dependent on the C-terminal tail and the degree of beta-arrestin recruitment can be transferred by exchange of the C-termini.

FIGURE 7

Human C-terminal tail is essential for beta-arrestin recruitment and restores beta-arrestin recruitment of the mouse glucose-dependent insulinotropic polypeptide (GIP) receptor (GIPR). Ligand dose–response stimulated beta-arrestin 2 recruitment of human GIPR–rat tail and human GIPR–mouse tail (a), rat GIPR–human tail (b) and mouse GIPR–human tail (c) transiently expressed in HEK293 cells. Baseline-subtracted values are normalized to human wild-type GIPR stimulated with hGIP(1-42). Data are shown as means ± SEM, n = 5 for all experiments.

All four tail-swapped receptors were, however, desensitized following agonist incubation, as illustrated at 90 min (Figure 8a) and over time (Figure 8c–i). Notably, consistent with its stronger arrestin recruitment, the mouse GIPR–human tail was desensitized to a higher extent than the wild-type mouse GIPR (Figure 8a) and had reduced surface expression (Figure 8b). This was particularly visible at early time points (Figure 8h,i). The three other chimeric receptors were all desensitized to the same extent as their wild-type receptors (Figure 8c–g), reflecting the stronger beta-arrestin 2 recruitment of rat and human GIPRs and perhaps also reflecting contributions from beta-arrestin 1-dependent desensitization, as previously reported for the human GIPR (Gabe et al., 2018).

FIGURE 8

Human C-terminal tail is essential for receptor desensitization and restores desensitization of the mouse glucose-dependent insulinotropic polypeptide (GIP) receptor (GIPR). (a) The binding of ¹²⁵I-human GIP(1-42) to transiently transfected COS-7 cells expressing the four tail-swapped GIPRs (human GIPR–rat tail n = 7, human GIPR–mouse tail n = 7, rat GIPR–human tail n = 5 and mouse GIPR–human tail n = 8) was tested following 90 min of preincubation with 1, 10 or 100 nmol·L⁻¹ (nM) of species-specific GIP(1-42). Dashed lines marked the corresponding wild-type receptor results (as depicted in Figure 1a). (b) B_max values from homologous binding of GIP(1-42) displacing ¹²⁵I-human GIP(1-42) in transiently transfected COS-7 cells expressing the four tail-swapped GIPRs (human GIPR–rat tail n = 8, human GIPR–mouse tail n = 7, rat GIPR–human tail n = 6 and mouse GIPR–human tail n = 13) shown as percentage of human GIPR. (c–i) The binding of ¹²⁵I-human GIP(1-42) to transiently transfected COS-7 cells expressing wild type or one of the four tail-swapped GIPRs for 90 min with 1, 10 or 100 nmol·L⁻¹ (nM) of species-specific GIP(1-42) (closed symbols) or GIP analogue (open symbols), n = 3. Data are shown as means ± SEM.

4 DISCUSSION

In our molecular pharmacological investigations of the internalization profiles of several species of GIPRs, we found important differences between species. Most strikingly, we found that the human GIPR is more prone to internalization than rodent GIPRs and that the GIPRs from the larger species (dog and pig) resemble the human GIPR in this aspect. Despite similar agonist affinities and potencies for cAMP signalling, especially, the mouse GIPR shows reduced receptor desensitization and internalization, although the results could be influenced by reduced receptor expression. Using an enzyme-stabilized GIPR analogue designed for optimized drug properties (long plasma half-life with maintained high affinity and efficacy), the species differences were even more pronounced. Relevant for the desensitization mechanism, the mouse GIPR did not internalize to the same extent and did not recruit arrestins as the human and rat GIPRs. This altered property for the mouse GIPR was confirmed in a mouse pancreatic cell line.

The species-specific role of the C-terminal tail is poorly understood. To address the functionality of the tail, we used ‘tail-swapped’ human, rat and mouse receptors that were all fully functional in their G_αs coupling. We confirmed the importance of the C-terminus of the human GIPR for arrestin recruitment as the mouse GIPR regained internalization and beta-arrestin 2 recruitment properties with the human tail, while the human GIPR lost the ability to recruit beta-arrestin 2 when its own C-terminus was replaced by the rat or mouse tail. This finding is consistent with the analysis of the potential phosphorylation motifs associated with beta-arrestin recruitment. Although we cannot rule out that variations in receptor expression between the implemented assays affect the results, these species differences and the functional consequences are important for the translation of animal studies into human GIPR physiology.

4.1 Species differences in GIPR pharmacology

For decades, there has been an interest in GIPR as a therapeutic target in the treatment of type 2 diabetes and obesity (Gasbjerg et al., 2018; Holst & Rosenkilde, 2020; Miyawaki et al., 2002; Nauck, Bartels, et al., 1993). The insulinotropic actions of GIP, acting via GIPR on the pancreatic beta cells, are preserved across the studied species (Baggio et al., 2000; Lewis et al., 2000) but are, importantly, severely reduced in patients with type 2 diabetes (Nauck, Heimesaat, et al., 1993; Vilsbøll et al., 2003). The glucagonotropic actions of GIP, acting via the GIPR on the pancreatic alpha cells, also seem to be preserved (El et al., 2021). For other tissues, GIP actions have been difficult to translate from, especially rodent to human settings: GIPR activation (Mroz et al., 2019) or inhibition (Ravn et al., 2013) has been found to lead to weight loss in mice, whereas GIPR antagonism reduces the weight gain during a high-fat diet in non-human primates (Killion, Chen, et al., 2020a). GIPR knockout mice seem to have affected energy expenditure with a reduced respiratory coefficient and are protected against diet-induced weight gain (Miyawaki et al., 2002). Similarly, carriers of loss-of-function variants of the human GIPR display lower weight (Kizilkaya et al., 2021; Turcot et al., 2018). Recently, a long-acting GIPR agonist was reported to induce a minor weight loss in humans with type 2 diabetes, but the specific properties and molecular structure of the pharmaceutical compound were not revealed (Knop et al., 2023). In contrast, treatment with another long-acting GIP analogue (also with unrevealed pharmacological properties) did not affect body weight or glycaemic control in patients with type 2 diabetes (NovoNordisk, 2023). Paradoxically, GIPR antagonism, combined with GLP-1 agonism, results in beneficial metabolic effects and weight loss in both animals and humans (Killion, Lu, et al., 2020d; Mayendraraj et al., 2022). Whether the difference in these results lies in the pattern of receptor signalling and/or internalization properties of each ligand is still unknown, but it has been proposed that chronic GIPR stimulation leads to receptor internalization and down-regulation and thereby functional antagonism induced by agonists (Gasbjerg et al., 2023; Killion, Chen, et al., 2020c). Likely, the pharmacological mode of action for the molecules discussed here may not be the same and each molecule will need a thorough evaluation. The present data suggest that pigs would be more suitable for studies of GIPR targeting compounds than mice (Deacon et al., 2006) because the pig and human GIPRs respond more similarly to agonist activation and subsequent desensitization (Figure 2). Except for the use of the MIN6 cell line, our results are limited by the use of non-species-specific cell lines but are, however, based on experiments made in parallel and in well-established cell lines (Volz et al., 1995; Yang et al., 2022).

4.2 Species differences in receptor beta-arrestin coupling and internalization profiles

We have previously reported species differences with respect to activation of the GIPR by partial agonists and antagonists, whereas the species-specific GIP(1-42) agonists are quite similar across human, rat and mouse species (Hansen et al., 2016; Sparre-Ulrich et al., 2016, 2017). A modified GIPR ligand will, therefore, not necessarily affect the human and non-human GIPRs similarly and the findings presented here of species differences in GIPR desensitization and internalization profiles are important for the translation of murine results to the human therapeutic potential.

The human GIPR readily and rapidly internalizes, but it is unclear how important this mechanism is for the clinical effects of the GLP-1–GIP receptor co-agonist tirzepatide (Mayendraraj et al., 2022; Willard et al., 2020), which is now marketed as the treatment of type 2 diabetes and approved for the treatment of obesity (Jastreboff et al., 2022). In vitro pharmacological characterization revealed that tirzepatide is prone towards cAMP accumulation and not beta-arrestin recruitment (and receptor internalization) when interacting with the GLP-1 receptor, whereas the GIPR is both activated and internalized upon tirzepatide binding (Willard et al., 2020). A recent investigation of species revealed that tirzepatide acts primarily through the GLP-1 receptor in murine pancreatic islets, whereas both the GLP-1 and GIP receptor were responsible for the insulin secretion of human pancreatic islets (as expected for islets from non-diabetic humans) (El et al., 2023). Based on the results presented here, the maintained arrestin recruitment (and receptor internalization) could be an important factor for the GIPR-dependent effects of tirzepatide in human pancreatic islets. The compound has potent but otherwise similar therapeutic effects and adverse effects as other GLP-1 agonists (Frías et al., 2021). Moreover, for the GLP-1 receptor, GIPR and the related glucagon receptor biased agonists with reduced beta-arrestin recruitment and, therefore, reduced receptor desensitization relative to the native hormones are suggested to be beneficial for human therapies (Jones et al., 2021). We previously showed that the internalization of the human GIPR markedly depends on beta-arrestins (Gabe et al., 2018), whereas the GLP-1 receptor and the GLP-2 receptor show a high degree of arrestin-independent internalization (Gabe et al., 2023; Jones et al., 2021), thereby illustrating that the internalization of even very similar receptors may vary in their arrestin dependency (von Moo et al., 2021). For preclinical screening of compounds, the lack of receptor internalization and poor beta-arrestin recruitment for the mouse GIPR must therefore be considered during the preclinical development of new compounds for human diseases.

4.3 The role of the C-terminus for GIP receptors

For family B1 GPCRs, the structure and function of the receptor N-terminus and transmembrane core, as well as the ligand binding pocket, have now been addressed by X-ray and cryo-electron microscopy studies (Tikhele et al., 2010; Zhang et al., 2017; Zhao et al., 2021, 2022). This also includes the structures of the human GIPR and models for its activation (Smit et al., 2021; Zhao et al., 2021). We find that GIP binding and GIPR activation via cAMP accumulation are widely preserved across species, irrespective of the origin of the C-terminus, but that the role of the C-terminal tail for arrestin recruitment is species specific. It has been established that the most important part of the receptor for arrestin recruitment is the C-terminus, although intracellular loops may contribute (Maharana et al., 2022). Consistent with this, we found that replacement of the C-terminal tail of the human GIPR with that of the mouse or rat GIPR leads to a loss of the ability to recruit beta-arrestin. Comparing mouse, rat and human GIPRs, we found a phosphorylation motif in human GIPR identical to the phosphorylation code shown to be relevant for beta-arrestin 2 recruitment to the rhodopsin receptor (Zhou et al., 2017). This motif is present in neither the mouse nor rat C-terminal tails, and its absence may explain the reduction in beta-arrestin 2 recruitment when the human GIPR was fused to the mouse or rat C-terminal tail. Nevertheless, it is of interest that the rat GIPR shows robust beta-arrestin 2 recruitment despite lacking a complete phosphorylation code. This might point to the existence of an additional structural mechanism for this receptor, which can compensate for the lack of the PxPxxP/PxxPxxP motif in the C-terminal tail. Together, these findings highlight the C-terminal tail as an important functional unit for the GIPRs in a species-dependent manner. In future studies, thorough site-specific mutational studies may reveal the areas of the tail involved in beta-arrestin recruitment and/or the initiation of receptor internalization.

5 CONCLUSION

The human GIPR depends entirely upon arrestin recruitment for its internalization, whereby it differs from the GLP-1 receptor. The human GIPR is markedly desensitized upon agonist binding, whereas, especially, the mouse GIPR is not. The receptor desensitization is dependent on the human GIPR C-terminal tail, which can restore beta-arrestin recruitment and receptor internalization when substituted in the mouse GIPR. The species-specific, different functionalities of the C-terminal tail and receptor internalization patterns are crucial factors for the results of rodent studies of GIPR-targeting compounds.

AUTHOR CONTRIBUTIONS

L. S. Gasbjerg: Conceptualization (equal); data curation (equal); formal analysis (equal); funding acquisition (supporting); investigation (supporting); methodology (supporting); project administration (equal); resources (equal); software (lead); supervision (equal); validation (equal); visualization (lead); writing—original draft (lead); writing—review and editing (lead). R. S. Rasmussen: Data curation (equal); formal analysis (equal); visualization (equal); writing—review and editing (equal). A. Dragan: Data curation (equal); investigation (equal); writing—review and editing (equal). P. Lindquist: Data curation (equal); investigation (equal); writing—review and editing (equal). J. U. Melchiorsen: Formal analysis (equal); software (equal); writing—review and editing (equal). TM Stepniewski: Methodology (supporting); software (equal); formal analysis (supporting); writing—original draft (supporting); visualization (supporting). S. Schiellerup: Data curation (equal); investigation (equal); writing—review and editing (equal). E. K. Tordrup: Data curation (equal); investigation (equal); writing—review and editing (equal). S. Gadgaard: Data curation (equal); investigation (equal); writing—review and editing (equal). H. S. Kizilkaya: Data curation (equal); investigation (equal); writing—review and editing (equal). S Willems: Investigation (supporting); visualization (supporting). Y. Zhong: Investigation (supporting); visualization (supporting). Y. Wang: Investigation (supporting); visualization (supporting). SC Wright: Formal analysis (supporting); writing—review and editing (supporting); visualization (equal); supervision (supporting). VM Lauschke: Resources (supporting); formal analysis (supporting); visualization (equal); supervision (supporting). B. Hartmann: Project administration (equal); supervision (equal); writing—review and editing (equal). J. J. Holst: Project administration (equal); supervision (equal); validation (equal); writing—review and editing (equal). J Selent: Methodology (supporting); formal analysis (supporting); writing—original draft (supporting); visualization (supporting); supervision (supporting). M. M. Rosenkilde: Conceptualization (lead); data curation (lead); formal analysis (lead); funding acquisition (lead); investigation (equal); methodology (lead); project administration (lead); resources (equal); software (equal); supervision (lead); validation (supporting); visualization (supporting); writing—original draft (supporting); writing—review and editing (equal).

ACKNOWLEDGEMENTS

We would like to thank Søren Petersen and Maibritt Sigvardt Baggesen for their excellent technical assistance. We also thank Michel Bouvier (Université de Montréal) for rGFP-CAAX. This work was supported by the Department of Biomedical Sciences, University of Copenhagen, by a grant from Novo Nordisk Foundation (NNF18SA0034956) to LSG and grants to MMR from Novo Nordisk Foundation (NNF21OC00671 and NNF21OC0070347) and by a donation from deceased Valter Alex Torbjørn Eichmuller (VAT Eichmuller) (2020-117043) and from Kirsten and Freddy Johansens Foundation (KFJ) (2017-112697). SW was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) (533772148). SCW is supported by a fellowship from the Swedish Society for Medical Research (PD20-0153). VML is supported by the Swedish Research Council (Grant Agreement Numbers 2021-02801 and 2023-03015), by the EU/EFPIA/OICR/McGill/KTH/Diamond Innovative Medicines Initiative 2 Joint Undertaking (EUbOPEN Grant Number 875510) and by the Robert Bosch Foundation, Stuttgart, Germany.

CONFLICT OF INTEREST STATEMENT

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. VML is co-founder, CEO and shareholder of HepaPredict AB, as well as co-founder and shareholder of PersoMedix AB. BH, JJH and MMR are co-founders of Bainan Biotech, and AD and SG were, during the presented work, employed at Bainan Biotech. LSG, JJH and MMR are co-founders of Antag Therapeutics.

DECLARATION OF TRANSPARENCY AND SCIENTIFIC RIGOUR

This Declaration acknowledges that this paper adheres to the principles for transparent reporting and scientific rigour of preclinical research as stated in the BJP guidelines for Design and Analysis and as recommended by funding agencies, publishers and other organizations engaged with supporting research.

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DATA AVAILABILITY STATEMENT

All data are available upon request to the corresponding author.