Expression Of Glucose-dependent Insulinotropic Polypeptide Receptor Research Articles

Primary unilateral macronodular adrenal hyperplasia with concomitant glucocorticoid and androgen excess and KDM1A inactivation.

Primary bilateral macronodular adrenal hyperplasia (PBMAH) is a rare cause of Cushing's syndrome. Individuals with PBMAH and glucose-dependent insulinotropic polypeptide (GIP)-dependent Cushing's syndrome due to ectopic expression of the GIP receptor (GIPR) typically harbor inactivating KDM1A sequence variants. Primary unilateral macronodular adrenal hyperplasia (PUMAH) with concomitant glucocorticoid and androgen excess has never been encountered or studied. We investigated a woman with a large, heterogeneous adrenal mass and severe adrenocorticotropic hormone-independent glucocorticoid and androgen excess, a biochemical presentation typically suggestive of adrenocortical carcinoma. The patient presented during pregnancy (22nd week of gestation) and reported an 18-month history of oligomenorrhea, hirsutism, and weight gain. We undertook an exploratory study with detailed histopathological and genetic analysis of the resected adrenal mass and leukocyte DNA collected from the patient and her parents. Histopathology revealed benign macronodular adrenal hyperplasia. Imaging showed a persistently normal contralateral adrenal gland. Whole-exome sequencing of 4 representative nodules detected KDM1A germline variants, benign NM_001009999.3:c.136G > A:p.G46S, and likely pathogenic NM_001009999.3:exon6:c.865_866del:p.R289Dfs*7. Copy number variation analysis demonstrated an additional somatic loss of the KDM1A wild-type allele on chromosome 1p36.12 in all nodules. RNA sequencing of a representative nodule showed low/absent KDM1A expression and increased GIPR expression compared with 52 unilateral sporadic adenomas and 4 normal adrenal glands. Luteinizing hormone/chorionic gonadotropin receptor expression was normal. Sanger sequencing confirmed heterozygous KDM1A variants in both parents (father: p.R289Dfs*7 and mother: p.G46S) who showed no clinical features suggestive of glucocorticoid or androgen excess. We investigated the first PUMAH associated with severe Cushing's syndrome and concomitant androgen excess, suggesting pathogenic mechanisms involving KDM1A.

GIP receptor antagonism eliminates paradoxical growth hormone secretion in some patients with acromegaly.

About 30% of patients with active acromegaly experience paradoxically increased growth hormone (GH) secretion during the diagnostic oral glucose tolerance test (OGTT). Endogenous glucose-dependent insulinotropic polypeptide (GIP) is implicated in this paradoxical secretion. We used the GIP receptor (GIPR) antagonist GIP(3-30)NH2 to test the hypothesis that GIP mediates this paradoxical response when GIPR is abundantly expressed in somatotropinomas. 25 treatment-naïve patients with acromegaly were enrolled. Each patient underwent one OGTT during simultaneous placebo infusion and one OGTT during a GIP(3-30)NH2 infusion. Blood samples were drawn at baseline and regularly after infusions to measure GH. We assessed pituitary adenoma size by magnetic resonance imaging and GIPR expression by immunohistochemistry on resected somatotropinomas. For mechanistic confirmation, we applied in vitro and ex vivo approaches. The effect of GIP(3-30)NH2 on paradoxical GH secretion during OGTT as a measure of GIP involvement. In four of seven patients with paradoxical GH secretion, GIP(3-30)NH2 infusion completely abolished the paradoxical response (P = 0.0003). Somatotrophs were available from three of four of these patients, all showing abundant GIPR expression. Adenoma size did not differ between patients with and without paradoxical GH secretion. Of 25 patients with acromegaly, seven had paradoxical GH secretion during OGTT, and pharmaceutical GIPR blockade abolished this secretion in four. Corresponding somatotroph adenomas abundantly expressed GIPR, suggesting a therapeutic target in this subpopulation of patients. In vitro and ex vivo analyses confirmed the role of GIP and the effects of the antagonist.

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.

KDM1A genotyping and expression in 146 sporadic somatotroph pituitary adenomas.

A paradoxical increase of growth hormone (GH) following oral glucose load has been described in ∼30% of patients with acromegaly and has been related to the ectopic expression of the glucose-dependent insulinotropic polypeptide (GIP) receptor (GIPR) in somatotropinomas. Recently, we identified germline pathogenic variants and somatic loss of heterozygosity of lysine demethylase 1A (KDM1A) in patients with GIP-dependent primary bilateral macronodular adrenal hyperplasia with Cushing's syndrome. The ectopic expression of GIPR in both adrenal and pituitary lesions suggests a common molecular mechanism. We aimed to analyze KDM1A gene sequence and KDM1A and GIPR expressions in somatotroph pituitary adenomas. We conducted a cohort study at university hospitals in France and in Italy. We collected pituitary adenoma specimens from acromegalic patients who had undergone pituitary surgery. We performed targeted exome sequencing (gene panel analysis) and array-comparative genomic hybridization on somatic DNA derived from adenomas and performed droplet digital PCR on adenoma samples to quantify KDM1A and GIPR expressions. One hundred and forty-six patients with sporadic acromegaly were studied; 72.6% presented unsuppressed classical GH response, whereas 27.4% displayed a paradoxical rise in GH after oral glucose load. We did not identify any pathogenic variant in the KDM1A gene in the adenomas of these patients. However, we identified a recurrent 1p deletion encompassing the KDM1A locus in 29 adenomas and observed a higher prevalence of paradoxical GH rise (P = .0166), lower KDM1A expression (4.47 ± 2.49 vs 8.56 ± 5.62, P < .0001), and higher GIPR expression (1.09 ± 0.92 vs 0.43 ± 0.51, P = .0012) in adenomas from patients with KDM1A haploinsufficiency compared with those with 2 KDM1A copies. Unlike in GIP-dependent primary bilateral macronodular adrenal hyperplasia, KDM1A genetic variations are not the cause of GIPR expression in somatotroph pituitary adenomas. Recurrent KDM1A haploinsufficiency, more frequently observed in GIPR-expressing adenomas, could be responsible for decreased KDM1A function resulting in transcriptional derepression on the GIPR locus.

OR17-05 Lysine Demethylase KDM1A And Ectopic Expression Of GIP-Receptor In Somatotropinomas Of Patients With Paradoxical Response To Oral Glucose

Abstract Disclosure: F. Chasseloup: None. L. Tosca: None. D. Regazzo: None. A. Proust: None. M. Hage: None. E. Kuhn: None. C. Jublanc: None. K. Mokhtari: None. S. Salenave: None. S. Gaillard: None. F. Parker: None. A. Boch: None. G. Tachdjian: None. P. Chanson: None. J. Bouligand: None. G. Occhi: None. P. Kamenicky: None. Introduction: Paradoxical increase of GH following oral glucose load has been described in ∼30% of patients with acromegaly and has been related to the ectopic expression of the glucose-dependent insulinotropic polypeptide (GIP) receptor (GIPR) in somatotropinomas. Recently, we identified germline pathogenic variants of lysine demethylase 1A (KDM1A) in patients with GIP-dependent primary bilateral macronodular adrenal hyperplasia with Cushing’s syndrome. Most patients also displayed a deletion of chromosome 1p, including the KDM1A locus in their adrenal tissues, resulting in complete loss of KDM1A expression. The ectopic expression of GIPR in both adrenal and pituitary lesions suggests a common molecular mechanism. The aim of our study was to search for genetic abnormalities of KDM1A in somatotroph pituitary adenomas. Methods: We collected somatotropinoma specimens from acromegalic patients followed in two tertiary endocrine centers in France and one in Italy. Somatic DNA was studied by targeted exome NGS and array-CGH. GIPR and KDM1A expression was quantified in the tumors using digital droplet PCR. Results: We included 186 patients: 108 patients (70.6 %) had a classic pathological GH response after oral glucose load, whereas 45 patients (29.4%) displayed a paradoxical rise of GH concentrations. Patients with a paradoxical response displayed higher IGF-1 levels (360 ± 111.8 % above ULN vs. 309.8 ± 107.3 %, p= 0.0130) and less invasive and smaller tumors (14.5 ± 5.58 mm vs. 18.5 ± 8.73 m, p= 0.0066). Amongst the 146 somatotropinoma specimens analyzed by targeted-NGS, no tumor harbored a KDM1A pathogenic variant, and 35 tumors harbored GNAS mutations (6 with and 29 without paradoxical GH responses). We identified a recurrent 1p deletion encompassing the KDM1A locus in 26 tumors, which were more frequently but not exclusively found in patients with paradoxical GH response compared to those with classic GH response. Somatic deletion of one KMD1A allele was associated with lower KDM1A expression (p=4.5e-5) and higher GIPR expression (p=0.0005). Discussion: Unlike in GIP-dependent PBMAH, we did not identify KDM1A genetic variants in a large cohort of acromegalic patients, independently of their GH response pattern to oral glucose loading. We identified recurrent 1p deletion in some tumors. Pituitary adenomas with a loss of one KDM1A copy due to chromosome 1p deletion harbored higher levels of GIPR transcripts than adenomas diploid for the KDM1A locus. If KDM1A haploinsufficiency leads to partial transcriptional derepression at the GIPR locus and a paradoxical rise of GH after glucose load warrants further investigations. Presentation: Saturday, June 17, 2023

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.

GIPR expression is induced by thiazolidinediones in a PPARγ-independent manner and repressed by obesogenic stimuli

Adipose tissue (AT) dysfunctions are associated with the onset of insulin resistance (IR) and type 2 diabetes mellitus (T2DM). Targeting glucose-dependent insulinotropic peptide receptor (GIPR) is a valid option to increase the efficacy of glucagon-like peptide 1 (GLP-1) receptor agonists in T2DM treatment. Nevertheless, the therapeutic potential of targeting the GIP/GIPR axis and its effect on the AT are controversial. In this work, we explored the expression and regulation of GIPR in precursor cells and mature adipocytes, investigating if and how obesogenic stimuli and thiazolidinediones perturb GIPR expression. Using publicly available gene expression datasets, we assessed that, among white adipose tissue (WAT) cells, adipocytes express lower levels of GIPR compared to cells of mesothelial origin, pericytes, dendritic and NK/T cells. However, we report that GIPR levels markedly increase during the in vitro differentiation of both murine and human adipocytes, from 3T3-L1 and human mesenchymal precursor cells (MSCs), respectively. Notably, we demonstrated that thiazolidinediones – ie. synthetic PPARγ agonists widely used as anti-diabetic drugs and contained in the adipogenic mix – markedly induce GIPR expression. Moreover, using multiple in vitro systems, we assessed that thiazolidinediones induce GIPR in a PPARγ-independent manner. Our results support the hypothesis that PPARγ synthetic agonists may be used to increase GIPR levels in AT, potentially affecting in turn the targeting of GIP system in patients with metabolic dysfunctions. Furthermore, we demonstrate in vitro and in vivo that proinflammatory stimuli, and especially the TNFα, represses GIPR both in human and murine adipocytes, even though discordant results were obtained between human and murine cellular systems for other cytokines. Finally, we demonstrated that GIPR is negatively affected also by the excessive lipid engulfment. Overall, we report that obesogenic stimuli - ie. pro-inflammatory cytokines and the increased lipid accumulation – and PPARγ synthetic ligands oppositely modulate GIPR expression, possibly influencing the effectiveness of GIP agonists.

Safflower yellow and its main component hydroxysafflor yellow A alleviate hyperleptinemia in diet-induced obesity mice through a dual inhibition of the GIP-GIPR signaling axis.

Glucose-dependent insulinotropic polypeptide (GIP) is a gastrointestinal hormone secreted by K cells in the small intestine and is considered an obesity-promoting factor. In this study, we systematically investigated the anti-obesity effects of intragastric safflower yellow (SY)/hydroxysafflor yellow A (HSYA) and the underlying mechanism for the first time. Our results showed that intragastric SY/HSYA, rather than an intraperitoneal injection, notably decreased serum GIP levels and GIP staining in the small intestine in diet-induced obese (DIO) mice. Moreover, intragastric SY/HSYA was also first found to significantly suppress GIP receptor (GIPR) signaling in both the hypothalamus and subcutaneous White adipose tissue. Our study is the first to show that intragastric SY/HSYA obviously reduced food intake and body weight gain in leptin sensitivity experiments and decreased serum leptin levels in DIO mice. Further experiments demonstrated that SY treatment also significantly reduced leptin levels, whereas the inhibitory effect of SY on leptin levels was reversed by activating GIPR in 3T3-L1 adipocytes. In addition, intragastric SY/HSYA had already significantly reduced serum GIP levels and GIPR expression before the serum leptin levels were notably changed in high-fat-diet-fed mice. These findings suggested that intragastric SY/HSYA may alleviate diet-induced obesity in mice by ameliorating hyperleptinemia via dual inhibition of the GIP-GIPR axis.

Extensive expertise in endocrinology: glucose-dependent insulinotropic peptide-dependent Cushing's syndrome.

Thirty years ago, we identified that cortisol secretion in some patients with unilateral adenoma or primary bilateral macronodular adrenal hyperplasia (PBMAH) was stimulated by food intake; this was secondary to the abnormal adrenocortical responsiveness to physiological post-prandial increase in glucose-dependent insulinotropic peptide (GIP). This resulted from the ectopic expression of non-mutated GIP receptor in the pathological adrenal tissues of those patients. Although ectopic GIP receptor (GIPR) was confirmed in a relatively limited number of cases to date, its elucidation leads to the identification of a wide diversity of aberrant G-protein-coupled receptors regulating steroidogenesis and cell proliferation in a high proportion of patients with PBMAH or cortisol-secreting adenomas. In addition, ectopic GIPR was identified in other endocrine tumors including somatotroph pituitary tumors with paradoxical growth hormone response to oral glucose, medullary thyroid carcinomas, and other neuroendocrine tumors. The first molecular pathogenic mechanism responsible for ectopic GIPR expression was elucidated in unilateral GIP-dependent adenomas in which somatic duplication and rearrangements in chromosome region 19q13.32 containing the GIPR locus lead to increased expression of GIPR which was enhanced by the activity of a glucocorticoid response element. Recently, germline lysine demythylase 1A (KDMIA) mutations combined with somatic chromosome 1p deletions were found to be specifically responsible for ectopic GIPR in sporadic or familial GIP-dependent PBMAH and can be associated with adrenal myelolipoma, monoclonal gammopathy of unknown significance (MGUS), or multiple myeloma. Screening for ectopic GIPR should be conducted in all patients with PBMAH; genetic studies to identify KDM1A mutations should be offered to such patients in order to detect affected members and provide early detection of PBMAH and other potential associated neoplasias. The elucidation of GIP-dependent Cushing's syndrome (CS) illustrates that careful bedside phenotyping of rare conditions can lead to identification of genetically determined diseases requiring personalized approaches to investigation and therapy.

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

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. 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. 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. GIP inhibits bone resorption and improves survival of human osteoblasts, indicating that drugs targeting GIPR may impair bone resorption, whilst preserving bone formation.

OR04-4 Loss of KDM1A in Bilateral Macronodular Adrenal Hyperplasia With GIP-Dependent Cushing's Syndrome and in Acromegaly With Paradoxical GH Response to Oral Glucose

Abstract Context Primary bilateral macronodular adrenal hyperplasia (PBMAH) with glucose-dependent insulinotropic polypeptide (GIP)-dependent Cushing's syndrome is caused by ectopic expression of GIP receptor (GIPR) in the adrenal lesions. Such ectopic expression of GIPR was also reported in other endocrine neoplasm, notably in somatotroph pituitary adenomas from acromegalic patients with paradoxical increase of GH after oral glucose load, suggesting a common molecular pathogenesis. We aimed to identify the driver event responsible for GIP-dependent PBMAH with Cushing's syndrome and ectopic GIPR expression in somatotropinomas. Methods We conducted an international, multicenter, cohort study. We collected blood and adrenal samples from patients who had undergone unilateral or bilateral adrenalectomy for GIP-dependent PBMAH with Cushing's syndrome. Adrenal samples from patients with PBMAH and Cushing's syndrome without food-dependent cortisol production were used as controls. We further collected somatotropinoma specimens from acromegaly patients followed at two expert endocrine centers in France. Results 17 patients with familial or sporadic GIP-dependent PBMAH with Cushing's syndrome were studied. We identified germline heterozygous mutations in the lysine demethylase 1A (KDM1A) gene in all 17 patients. We further identified a recurrent deletion of the short arm of chromosome 1 harboring the KDM1A locus in the adrenal lesions of affected patients. None of the 25 patients in the control group had KDM1A germline or somatic alterations. Concomitant genetic inactivation of both KDM1A alleles resulted in loss of KDM1A expression in the adrenal lesions. RNA-sequencing revealed the global impact of KDM1A loss in adrenal tissue on gene transcription and identified differentially regulated genes including those encoding for GIPR and other G-Protein-Coupled Receptors that may be involved in adrenal tumorigenesis and regulation of steroidogenesis. In vitro pharmacologic inhibition, silencing and knock-out by CRISPR-Cas9 genome editing of KDM1A led to an increase in GIPR transcripts and protein in human adrenocortical H295R cells. Somatotropinoma samples from 78 patients with acromegaly were studied. 24% of these patients presented with a paradoxical rise of GH after oral glucose load and expressed ectopically GIP-receptor in their somatotropinoma. None of the somatotropinomas harbored KDM1A pathogenic variants, but those from patients with paradoxical GH response displayed a recurrent chromosome 1p loss. Discussion We identified germline inactivating KDM1A mutations and loss of heterozygosity as a genetic predisposition to GIP-dependent PBMAH with Cushing's syndrome following a tumor suppressor gene model of tumorigenesis. We currently perform genetic screening in first-degree relatives of patients with GIP-dependent PBMAH with Cushing's syndrome and clinical examination with biochemical testing in asymptomatic KDM1A variant carriers. We did not identify somatic KDM1A mutations in somatotropinomas expressing GIPR ectopically, however their recurrent 1p chromosome loss suggests that KDM1A haploinsufficiency may contribute to GIPR expression in those tumors. Presentation: Saturday, June 11, 2022 12:15 p.m. - 12:30 p.m.

Characterization of sporadic somatotropinomas with high GIP receptor expression

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. 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 +). 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). 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.

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

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

The GIP/GIPR axis in medullary thyroid cancer: clinical and molecular findings.

The improper expression of glucose-dependent insulinotropic polypeptide receptor (GIPR) and the GIP/GIPR axis activation has been increasingly recognized in endocrine tumors, with a potential diagnostic and prognostic value. A high tumor-to-normal tissue ratio (T/N ratio) of GIPR was reported both in humans' and in rats' medullary thyroid cancer (MTC), suggesting a direct link between the neoplastic transformation and the mechanism of receptor overexpression. In this study, we evaluated the potential diagnostic and prognostic significance of GIPR expression in a large cohort of MTC patients by correlating GIPR mRNA steady-state levels to clinical phenotypes. The molecular effect of GIP/GIPR axis stimulation in MTC-derived cells was also determined. We detected GIPR expression in ~80% of tumor specimens, especially in sporadic, larger, advanced-stage cancers with higher Ki-67 values. GIPR stimulation induced cAMP elevation in MTC-derived cells and a small but significant fluctuation in Ca2+, both likely associated with increased calcitonin secretion. On the contrary, the effects on PI3K-Akt and MAPK-ERK1/2 signaling pathways were marginal. To conclude, our data confirm the high T/N GIPR ratio in MTC tumors and suggest that it may represent an index for the degree of advancement of the malignant process. We have also observed a functional coupling between GIP/GIPR axis and calcitonin secretion in MTC models. However, the molecular mechanisms underlying this process and the possible implication of GIP/GIPR axis activation in MTC diagnosis and prognosis need further evaluation.

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

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

GIP Receptor Agonism Attenuates GLP-1 Receptor Agonist-Induced Nausea and Emesis in Preclinical Models.

Glucagon-like peptide 1 receptor (GLP-1R) agonists decrease body weight and improve glycemic control in obesity and diabetes. Patient compliance and maximal efficacy of GLP-1 therapeutics are limited by adverse side effects, including nausea and emesis. In three different species (i.e., mice, rats, and musk shrews), we show that glucose-dependent insulinotropic polypeptide receptor (GIPR) signaling blocks emesis and attenuates illness behaviors elicited by GLP-1R activation, while maintaining reduced food intake, body weight loss, and improved glucose tolerance. The area postrema and nucleus tractus solitarius (AP/NTS) of the hindbrain are required for food intake and body weight suppression by GLP-1R ligands and processing of emetic stimuli. Using single-nuclei RNA sequencing, we identified the cellular phenotypes of AP/NTS cells expressing GIPR and GLP-1R on distinct populations of inhibitory and excitatory neurons, with the greatest expression of GIPR in γ-aminobutyric acid-ergic neurons. This work suggests that combinatorial pharmaceutical targeting of GLP-1R and GIPR will increase efficacy in treating obesity and diabetes by reducing nausea and vomiting.

Single-Cell Mapping of GLP-1 and GIP Receptor Expression in the Dorsal Vagal Complex.

The dorsal vagal complex (DVC) in the hindbrain, composed of the area postrema, nucleus of the solitary tract, and dorsal motor nucleus of the vagus, plays a critical role in modulating satiety. The incretins glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) act directly in the brain to modulate feeding, and receptors for both are expressed in the DVC. Given the impressive clinical responses to pharmacologic manipulation of incretin signaling, understanding the central mechanisms by which incretins alter metabolism and energy balance is of critical importance. Here, we review recent single-cell approaches used to detect molecular signatures of GLP-1 and GIP receptor–expressing cells in the DVC. In addition, we discuss how current advancements in single-cell transcriptomics, epigenetics, spatial transcriptomics, and circuit mapping techniques have the potential to further characterize incretin receptor circuits in the hindbrain.

MANAGEMENT OF ENDOCRINE DISEASE: Etiology and outcome of acromegaly in patients with a paradoxical GH response to glucose.

To gain more insight on the pathogenesis of somatotroph pituitary adenomas, recent studies have focused on a subgroup of patients with acromegaly displaying a paradoxical growth hormone (GH) response during oral glucose tolerance test (OGTT). The paradoxical rise of GH after oral glucose intake occurs in about one-third of acromegaly patients and has been pathogenetically linked, by analogy to glucose-dependent insulinotropic polypeptide (GIP)-dependent Cushing's syndrome, to the ectopic expression of GIP receptor (GIPR) in somatotroph adenoma cells. GIPR-expressing adenomas are negative for activating GNAS gene mutations and display distinct cytogenetic and DNA methylation profiles, highlighting their unique molecular pathogenesis. Acromegaly patients with a paradoxical GH response pattern seem to display higher insulin-like growth factor-1 (IGF-1) concentrations and harbour smaller adenomas that are more often of the densely granulated phenotype. They seem also to show a better response to somatostatin receptor ligands. In addition, persistent paradoxical GH response after surgery may be a biological marker of the residual disease postoperatively. Targeted therapy to antagonize GIP receptor on GIPR-expressing somatotroph adenomas could be a new treatment approach for acromegaly patients with a paradoxical pattern of GH response to OGTT.

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 β-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 β-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.

Upregulation of spinal glucose-dependent insulinotropic polypeptide receptor induces membrane translocation of PKCγ and synaptic target of AMPA receptor GluR1 subunits in dorsal horns in a rat model of incisional pain

It is unclear whether glucose-dependent insulinotropic polypeptide receptor (GIPR) signaling plays an important role in spinal nociception. We hypothesized that the spinal GIPR is implicated in central sensitization of postoperative pain. Our data showed that the cumulative pain scores peaked at 3 h, kept at a high level at 1 d after incision, gradually decreased afterwards and returned to the baseline values at 5 d after incision. Correspondingly, the expression of GIPR in spinal cord dorsal horn peaked at 1 d after incision, and returned to the baseline value at 5 d after incision. The double-labeling immunofluorescence demonstrated that spinal GIPR was expressed in dorsal horn neurons, but not in astrocyte or microglial cells. At 1 d after incision, the effects of intrathecal saline, GIPR antagonist (Pro3)GIP on pain behaviors were investigated. Our data showed that at 30 min and 60 min following intrathecal treatments of 300 ng (Pro3)GIP, the cumulative pain scores were decreased and paw withdrawal thresholds to mechanical stimuli were increased when compared to those immediately before intrathecal treatments. Accordingly, at 30 min after intrathecal injections, the membrane translocation levels of PKCγ and the GluR1 expression in postsynaptic membrane in ipsilateral dorsal horns to the incision were significantly upregulated in rats with intrathecal saline injections, as compared to normal control group. At 30 min after intrathecal treatment, (Pro3)GIP inhibited the membrane translocation levels of PKCγ and the GluR1 expression in postsynaptic membrane in ipsilateral dorsal horns. Our study indicates that upregulation of spinal GIPR may contribute to pain hypersensitivity through inducing membrane translocation level of PKCγ and synaptic target of AMPA receptor GluR1 subunits in ipsilateral dorsal horns of rats with plantar incision.