Characterization of metabolic phenotypes and distinctive genes in mice with low-weight gain.

Being overweight exacerbates various metabolic diseases, necessitating the identification of target molecules for obesity control. In the current study, we investigated common physiological features related to metabolism in mice with low weight gain: (1) G protein-coupled receptor, family C, group 5, member B-knockout; (2) gastric inhibitory polypeptide receptor-knockout; and (3) Iroquois-related homeobox 3-knockout. Moreover, we explored genes involved in metabolism by analyzing differentially expressed genes (DEGs) between low-weight gain mice and the respective wild-type control mice. The common characteristics of the low-weight gain mice were low inguinal white adipose tissue (iWAT) and liver weight despite similar food intake along with lower blood leptin levels and high energy expenditure. The DEGs of iWAT, epididymal (gonadal) WAT, brown adipose tissue, muscle, liver, hypothalamus, and hippocampus common to these low-weight gain mice were designated as candidate genes associated with metabolism. One such gene tetraspanin 7 (Tspan7) from the iWAT was validated using knockout and overexpressing mouse models. Mice with low Tspan7 expression gained more weight, while those with high Tspan7 expression gained less weight, confirming the involvement of the Tspan7 gene in weight regulation. Collectively, these findings suggest that the candidate gene list generated in this study contains potential target molecules for obesity regulation. Further validation and additional data from low-weight gain mice will aid in understanding the molecular mechanisms associated with obesity.

Reduced incretin receptor trafficking upon activation enhances glycemic control and reverses obesity in diet-induced obese mice.

Activation of incretin receptors by their cognate agonist augments sustained cAMP generation both from the plasma membrane as well as from the endosome. To address the functional outcome of this spatiotemporal signaling, we developed a nonacylated glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) receptor dual agonist I-M-150847 that reduced receptor internalization following activation of the incretin receptors. The incretin receptor dual agonist I-M-150847 was developed by replacing the tryptophan cage of exendin-4 tyrosine substituted at the amino terminus with the C-terminal undecapeptide sequence of oxyntomodulin that placed lysine 30 of I-M-150847 in frame with the corresponding lysine residue of GIP. The peptide I-M-150847 is a partial agonist of GLP-1R and GIPR; however, the receptors, upon activation by I-M-150847, undergo reduced internalization that promotes agonist-mediated iterative cAMP signaling and augments glucose-stimulated insulin exocytosis in pancreatic ß cells. Chronic administration of I-M-150847 improved glycemic control, enhanced insulin sensitivity, and provided profound weight loss in diet-induced obese (DIO) mice. Our results demonstrated that despite being a partial agonist, I-M-150847, by reducing the receptor internalization upon activation, enhanced the incretin effect and reversed obesity.NEW & NOTEWORTHY Replacement of the tryptophan cage (Trp-cage) with the C-terminal oxyntomodulin undecapeptide along with the tyrosine substitution at the amino terminus converts the selective glucagon-like peptide-1 receptor (GLP-1R) agonist exendin-4 to a novel GLP-1R and GIPR dual agonist I-M-150847. Reduced internalization of incretin receptors upon activation by the GLP-1R and GIPR dual agonist I-M-150847 promotes iterative receptor signaling that enhances the incretin effect and reverses obesity.

DNA Methylation Pattern in Somatotroph Pituitary Neuroendocrine Tumors.

INTRODUCTION: Growth hormone secretion by sporadic somatotroph neuroendocrine pituitary tumors (PitNETs) is a major cause of acromegaly. These tumors are relatively heterogenous in terms of histopathological and molecular features. Our previous transcriptomic profiling of somatotroph tumors revealed three distinct molecular subtypes. This study aimed to investigate the difference in DNA methylation patterns in subtypes of somatotroph PitNETs and its role in distinctive gene expression. METHODS: Genome-wide DNA methylation was investigated in 48 somatotroph PitNETs with EPIC microarrays. Gene expression was assessed with RNAseq. Bisulfite pyrosequencing and qRT-PCR were used for verifying the results of DNA methylation and gene expression. RESULTS: Clustering tumor samples based on methylation data reflected the transcriptome-related classification. Subtype 1 tumors are densely granulated without GNAS mutation, characterized by high expression of NR5A1 (SF-1) and GIPR. The expression of both genes is correlated with specific methylation of the gene body and promoter. This subtype has a lower methylation level of 5' gene regions and CpG islands than the remaining tumors. Subtype 2 PitNETs are densely granulated and frequently GNAS-mutated, while those in subtype 3 are mainly sparsely granulated. Methylation/expression analysis indicates that ∼50% genes located in differentially methylated regions are those differentially expressed between tumor subtypes. Correlation analysis revealed DNA methylation-controlled genes, including CDKN1B, CCND2, EBF3, CDH4, CDH12, MGMT, STAT5A, PLXND1, PTPRE, and MMP16, and genes encoding ion channels and semaphorins. CONCLUSION: DNA methylation profiling confirmed the existence of three molecular subtypes of somatotroph PitNETs. High expression of NR5A1 and GIPR in subtype 1 tumors is correlated with specific methylation of both genes.

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

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

KDM1A inactivation causes hereditary food-dependent Cushing syndrome.

PURPOSE: This study aimed to investigate the genetic cause of food-dependent Cushing syndrome (FDCS) observed in patients with primary bilateral macronodular adrenal hyperplasia (PBMAH) and adrenal ectopic expression of the glucose-dependent insulinotropic polypeptide receptor. Germline ARMC5 alterations have been reported in about 25% of PBMAH index cases but are absent in patients with FDCS. METHODS: A multiomics analysis of PBMAH tissues from 36 patients treated by adrenalectomy was performed (RNA sequencing, single-nucleotide variant array, methylome, miRNome, exome sequencing). RESULTS: The integrative analysis revealed 3 molecular groups with different clinical features, namely G1, comprising 16 patients with ARMC5 inactivating variants; G2, comprising 6 patients with FDCS with glucose-dependent insulinotropic polypeptide receptor ectopic expression; and G3, comprising 14 patients with a less severe phenotype. Exome sequencing revealed germline truncating variants of KDM1A in 5 G2 patients, constantly associated with a somatic loss of the KDM1A wild-type allele on 1p, leading to a loss of KDM1A expression both at messenger RNA and protein levels (P = 1.2 × 10-12 and P < .01, respectively). Subsequently, KDM1A pathogenic variants were identified in 4 of 4 additional index cases with FDCS. CONCLUSION: KDM1A inactivation explains about 90% of FDCS PBMAH. Genetic screening for ARMC5 and KDM1A can now be offered for most PBMAH operated patients and their families, opening the way to earlier diagnosis and improved management.

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.

Discordance between GLP-1R gene and protein expression in mouse pancreatic islet cells.

The insulinotropic actions of glucagon-like peptide 1 receptor (GLP-1R) in ß-cells have made it a useful target to manage type 2 diabetes. Metabolic stress reduces ß-cell sensitivity to GLP-1, yet the underlying mechanisms are unknown. We hypothesized that Glp1r expression is heterogeneous among ß-cells and that metabolic stress decreases the number of GLP-1R-positive ß-cells. Here, analyses of publicly available single-cell RNA-Seq sequencing (scRNASeq) data from mouse and human ß-cells indicated that significant populations of ß-cells do not express the Glp1r gene, supporting heterogeneous GLP-1R expression. To check these results, we used complementary approaches employing FACS coupled with quantitative RT-PCR, a validated GLP-1R antibody, and flow cytometry to quantify GLP-1R promoter activity, gene expression, and protein expression in mouse α-, ß-, and δ-cells. Experiments with Glp1r reporter mice and a validated GLP-1R antibody indicated that >90% of the ß-cells are GLP-1R positive, contradicting the findings with the scRNASeq data. α-cells did not express Glp1r mRNA and δ-cells expressed Glp1r mRNA but not protein. We also examined the expression patterns of GLP-1R in mouse models of metabolic stress. Multiparous female mice had significantly decreased ß-cell Glp1r expression, but no reduction in GLP-1R protein levels or GLP-1R-mediated insulin secretion. These findings suggest caution in interpreting the results of scRNASeq for low-abundance transcripts such as the incretin receptors and indicate that GLP-1R is widely expressed in ß-cells, absent in α-cells, and expressed at the mRNA, but not protein, level in δ-cells.

Cryo-EM structure of the dual incretin receptor agonist, peptide-19, in complex with the glucagon-like peptide-1 receptor.

Dual agonists that can activate both the glucagon-like peptide-1 receptor (GLP-1R) and the gastric inhibitory polypeptide receptor (GIPR) have demonstrated high efficacy for the treatment of metabolic disease. Peptide-19 is a prototypical dual agonist that has high potency at both GLP-1R and GIPR but has a distinct signalling profile relative to the native peptides at the cognate receptors. In this study, we solved the structure of peptide-19 bound to the GLP-1R in complex with Gs protein, and compared the structure and dynamics of this complex to that of published structures of GLP-1R:Gs in complex with other receptor agonists. Unlike other peptide-bound receptor complexes, peptide-19:GLP-1R:Gs demonstrated a more open binding pocket where transmembrane domain (TM) 6, TM7 and the interconnecting extracellular loop 3 (ECL3) were located away from the peptide, with no interactions between peptide-19 and TM6/ECL3. Analysis of conformational variance of the complex revealed that peptide-19 was highly dynamic and underwent binding and unbinding motions facilitated by the more open TM binding pocket. Both the consensus structure of the GLP-1R complex with peptide-19 and the dynamics of this complex were distinct from previously described GLP-1R structures providing unique insights into the mode of GLP-1R activation by this dual agonist.

The pathogenic role of the GIP/GIPR axis in human endocrine tumors: emerging clinical mechanisms beyond diabetes.

The glucose-dependent insulinotropic polypeptide (GIP) is an incretin hormone produced in the gastrointestinal tract in response to nutrients. GIP has a variety of effects on different systems, including the potentiation of insulin secretion from pancreatic ß-cells after food intake (i.e. incretin effect), which is probably the most important. GIP effects are mediated by the GIP receptor (GIPR), a G protein-coupled receptor expressed in several tissues, including islet ß-cells, adipocytes, bone cells, and brain. As well as its involvement in metabolic disorders (e.g. it contributes to the impaired postprandial insulin secretion in type 2 diabetes (T2DM), and to the pathogenesis of obesity and associated insulin resistance), an inappropriate GIP/GIPR axis activation of potential diagnostic and prognostic value has been reported in several endocrine tumors in recent years. The ectopic GIPR expression seen in patients with overt Cushing syndrome and primary bilateral macronodular adrenal hyperplasia or unilateral cortisol-producing adenoma has been associated with an inverse rhythm of cortisol secretion, with low fasting morning plasma levels that increase after eating. On the other hand, most acromegalic patients with an unusual GH response to oral glucose suppression have GIPR-positive somatotropinomas, and a milder phenotype, and are more responsive to medical treatment. Neuroendocrine tumors are characterized by a strong GIPR expression that may correlate positively or inversely with the proliferative index MIB-1, and that seems an attractive target for developing novel radioligands. The main purpose of this review is to summarize the role of the GIP/GIPR axis in endocrine neoplasia, in the experimental and the clinical settings.

Evaluation of a rare glucose-dependent insulinotropic polypeptide receptor variant in a patient with diabetes.

AIMS: Glucose-dependent insulinotropic polypeptide (GIP) is an incretin hormone that augments insulin secretion in pancreatic ß-cells via its glucose-dependent insulinotropic polypeptide receptor (GIPR). Recent genome-wide association studies identified a single nucleotide variant (SNV) in the GIPR encoding gene (GIPR), rs1800437, that is associated with obesity and insulin resistance. In the present study, we tested whether GIPR variants contribute to obesity and disturb glucose homeostasis or diabetes in specific patient populations. MATERIALS AND METHODS: Exon sequencing of GIPR was performed in 164 children with obesity and insulin resistance and in 80 children with paediatric-onset diabetes of unknown origin. The Study of Health in Pomerania (SHIP) cohort, comprising 8320 adults, was screened for the GIPR variant Arg217Leu. GIPR variants were expressed in COS-7 cells and cAMP production was measured upon stimulation with GIP. Cell surface expression was determined by ELISA. Protein homology modelling of the GIPR variants was performed to extract three-dimensional information of the receptor. RESULTS: A heterozygous missense GIPR variant Arg217Leu (rs200485112) was identified in a patient of Asian ancestry. Functional characterization of Arg217Leu revealed reduced surface expression and signalling after GIP challenge. The homology model of the GIPR structure supports the observed functional relevance of Arg217Leu. CONCLUSION: In vitro functional studies and protein homology modelling indicate a potential relevance of the GIPR variant Arg217Leu in receptor function. The heterozygous variant displayed partial co-segregation with diabetes. Based on these findings, we suggest that GIPR variants may play a role in disturbed glucose homeostasis and may be of clinical relevance in homozygous patients.

Investigation of gene-diet interactions in the incretin system and risk of type 2 diabetes: the EPIC-InterAct study.

AIMS/HYPOTHESIS: The gut incretin hormones glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP) have a major role in the pathophysiology of type 2 diabetes. Specific genetic and dietary factors have been found to influence the release and action of incretins. We examined the effect of interactions between seven incretin-related genetic variants in GIPR, KCNQ1, TCF7L2 and WFS1 and dietary components (whey-containing dairy, cereal fibre, coffee and olive oil) on the risk of type 2 diabetes in the European Prospective Investigation into Cancer and Nutrition (EPIC)-InterAct study. METHODS: The current case-cohort study included 8086 incident type 2 diabetes cases and a representative subcohort of 11,035 participants (median follow-up: 12.5 years). Prentice-weighted Cox proportional hazard regression models were used to investigate the associations and interactions between the dietary factors and genes in relation to the risk of type 2 diabetes. RESULTS: An interaction (p = 0.048) between TCF7L2 variants and coffee intake was apparent, with an inverse association between coffee and type 2 diabetes present among carriers of the diabetes risk allele (T) in rs12255372 (GG: HR 0.99 [95% CI 0.97, 1.02] per cup of coffee; GT: HR 0.96 [95% CI 0.93, 0.98]); and TT: HR 0.93 [95% CI 0.88, 0.98]). In addition, an interaction (p = 0.005) between an incretin-specific genetic risk score and coffee was observed, again with a stronger inverse association with coffee in carriers with more risk alleles (0-3 risk alleles: HR 0.99 [95% CI 0.94, 1.04]; 7-10 risk alleles: HR 0.95 [95% CI 0.90, 0.99]). None of these associations were statistically significant after correction for multiple testing. CONCLUSIONS/INTERPRETATION: Our large-scale case-cohort study provides some evidence for a possible interaction of TCF7L2 variants and an incretin-specific genetic risk score with coffee consumption in relation to the risk of type 2 diabetes. Further large-scale studies and/or meta-analyses are needed to confirm these interactions in other populations.

Structural and pharmacological characterization of novel potent and selective monoclonal antibody antagonists of glucose-dependent insulinotropic polypeptide receptor.

Glucose-dependent insulinotropic polypeptide (GIP) is an endogenous hormonal factor (incretin) that, upon binding to its receptor (GIPr; a class B G-protein-coupled receptor), stimulates insulin secretion by beta cells in the pancreas. There has been a lack of potent inhibitors of the GIPr with prolonged in vivo exposure to support studies on GIP biology. Here we describe the generation of an antagonizing antibody to the GIPr, using phage and ribosome display libraries. Gipg013 is a specific competitive antagonist with equally high potencies to mouse, rat, dog, and human GIP receptors with a Ki of 7 nm for the human GIPr. Gipg013 antagonizes the GIP receptor and inhibits GIP-induced insulin secretion in vitro and in vivo. A crystal structure of Gipg013 Fab in complex with the human GIPr extracellular domain (ECD) shows that the antibody binds through a series of hydrogen bonds from the complementarity-determining regions of Gipg013 Fab to the N-terminal α-helix of GIPr ECD as well as to residues around its highly conserved glucagon receptor subfamily recognition fold. The antibody epitope overlaps with the GIP binding site on the GIPr ECD, ensuring competitive antagonism of the receptor. This well characterized antagonizing antibody to the GIPr will be useful as a tool to further understand the biological roles of GIP.

GIPR expression in gastric and duodenal neuroendocrine tumors.

BACKGROUND: Compounds targeting somatostatin-receptor-type-2 (SSTR2) are useful for small bowel neuroendocrine tumor (SBNET) and pancreatic neuroendocrine tumor (PNET) imaging and treatment. We recently characterized expression of 13 cell surface receptor genes in SBNETs and PNETs, identifying three drug targets (GIPR, OXTR, and OPRK1). This study set out to characterize expression of this gene panel in the less common neuroendocrine tumors of the stomach and duodenum (gastric and duodenal neuroendocrine tumors [GDNETs]). METHODS: Primary tumors and adjacent normal tissue were collected at surgery, RNA was extracted, and expression of 13 target genes was determined by quantitative polymerase chain reaction. Expression was normalized to GAPDH and POLR2A internal control genes. Expression relative to normal tissue (ddCT) and absolute expression (dCT) were calculated. Wilcoxon tests compared median expression with false discovery rate correction for multiple comparisons. RESULTS: Gene expression was similar in two gastric and seven duodenal tumors, and these were analyzed together. Like SBNETs (n = 63) and PNETs (n = 51), GDNETs showed significant overexpression compared with normal tissue of BRS3, GIPR, GRM1, GPR113, OPRK1, and SSTR2 (P < 0.05 for all). Of these, SSTR2 had the highest absolute expression in GDNETs (median dCT 4.0). Absolute expression of BRS3, GRM1, GPR113, and OPRK1 was significantly lower than SSTR2 in GDNETs (P < 0.05 for all), whereas expression of GIPR was similar to SSTR2 (median 4.3, P = 0.4). CONCLUSIONS: As in SBNETs and PNETs, GIPR shows absolute expression close to SSTR2 but has greater overexpression relative to normal tissue (21.1 versus 3.5-fold overexpression). We conclude that GIPR could provide an improved signal-to-noise ratio for imaging versus SSTR2 and represents a promising novel therapeutic target in GDNETs.

Obesity Variants in the GIPR Gene Are not Associated With Risk of Fracture or Bone Mineral Density.

CONTEXT: It is not clear if antagonizing the GIP (glucose-dependent insulinotropic polypeptide) receptor (GIPR) for treatment of obesity is likely to increase the risk of fractures, or to lower bone mineral density (BMD) beyond what is expected with rapid weight loss. OBJECTIVE: The objective of this study was to investigate the risk of fracture and BMD of sequence variants in GIPR that reduce the activity of the GIP receptor and have been associated with reduced body mass index (BMI). METHODS: We analyzed the association of 3 missense variants in GIPR, a common variant, rs1800437 (p.Glu354Gln), and 2 rare variants, rs139215588 (p.Arg190Gln) and rs143430880 (p.Glu288Gly), as well as a burden of predicted loss-of-function (LoF) variants with risk of fracture and with BMD in a large meta-analysis of up to 1.2 million participants. We analyzed associations with fractures at different skeletal sites in the general population: any fractures, hip fractures, vertebral fractures and forearm fractures, and specifically nonvertebral and osteoporotic fractures in postmenopausal women. We also evaluated associations with BMD at the lumbar spine, femoral neck, and total body measured with dual-energy x-ray absorptiometry (DXA), and with BMD estimated from heel ultrasound (eBMD). RESULTS: None of the 3 missense variants in GIPR was significantly associated with increased risk of fractures or with lower BMD. Burden of LoF variants in GIPR was not associated with fractures or with BMD measured with clinically validated DXA, but was associated with eBMD. CONCLUSION: Missense variants in GIPR, or burden of LoF variants in the gene, are not associated with risk of fractures or with lower BMD.

Regulation of energy metabolism through central GIPR signaling.

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

Molecular dynamics-guided optimization of BGM0504 enhances dual-target agonism for combating diabetes and obesity.

The dual activation of glucagon-like peptide-1 receptor (GLP-1R) and glucose-dependent insulinotropic polypeptide receptor (GIPR) has emerged as a promising therapeutic strategy for managing type 2 diabetes and obesity. Tirzepatide, a dual agonist peptide, has exhibited superior clinical efficacy in glycemic and weight control compared to selective GLP-1R agonists. Nevertheless, the structural basis of Tirzepatide's extended half-life, attributed to an acylation side chain on the parent peptide, raises questions regarding its partial agonistic activity. Employing molecular dynamics simulations, we explored the dynamic processes of peptide-receptor interactions. We uncovered a crucial salt bridge between parent peptide and GLP-1R/GIPR at K20, a feature not discernible in cryo-electron microscopy structures. Building upon these insights, we developed an optimization strategy based on the parent peptide which involved repositioning the acylation side chain. The results of both in vitro and in vivo experiments demonstrated that the optimized peptide has twofold to threefold increase in agonistic activity compared to Tirzepatide while maintaining its extended half-life in plasma. This led to the design of BGM0504, which proved to be more effective than its predecessor, Tirzepatide, in both laboratory and animal studies.

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

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

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.

Gastric Inhibitory Polypeptide Receptor (GIPR) Overexpression Reduces the Tumorigenic Potential of Retinoblastoma Cells.

Retinoblastoma (RB) is the most common malignant intraocular tumor in early childhood. Gene expression profiling revealed that the gastric inhibitory polypeptide receptor (GIPR) is upregulated following trefoil factor family peptide 1 (TFF1) overexpression in RB cells. In the study presented, we found this G protein-coupled transmembrane receptor to be co-expressed with TFF1, a new diagnostic and prognostic RB biomarker for advanced subtype 2 RBs. Functional analyses in two RB cell lines revealed a significant reduction in cell viability and growth and a concomitant increase in apoptosis following stable, lentiviral GIPR overexpression, matching the effects seen after TFF1 overexpression. In chicken chorioallantoic membrane (CAM) assays, GIPR-overexpressing RB cells developed significantly smaller CAM tumors. The effect of GIPR overexpression in RB cells was reversed by the GIPR inhibitor MK0893. The administration of recombinant TFF1 did not augment GIPR overexpression effects, suggesting that GIPR does not serve as a TFF1 receptor. Investigations of potential GIPR up- and downstream mediators suggest the involvement of miR-542-5p and p53 in GIPR signaling. Our results indicate a tumor suppressor role of GIPR in RB, suggesting its pathway as a new potential target for future retinoblastoma therapy.

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.