Stimulating intestinal GIP release reduces food intake and body weight in mice.

OBJECTIVE: Glucose dependent insulinotropic polypeptide (GIP) is well established as an incretin hormone, boosting glucose-dependent insulin secretion. However, whilst anorectic actions of its sister-incretin glucagon-like peptide-1 (GLP-1) are well established, a physiological role for GIP in appetite regulation is controversial, despite the superior weight loss seen in preclinical models and humans with GLP-1/GIP dual receptor agonists compared with GLP-1R agonism alone. METHODS: We generated a mouse model in which GIP expressing K-cells can be activated through hM3Dq Designer Receptor Activated by Designer Drugs (DREADD, GIP-Dq) to explore physiological actions of intestinally-released GIP. RESULTS: In lean mice, Dq-stimulation of GIP expressing cells increased plasma GIP to levels similar to those found postprandially. The increase in GIP was associated with improved glucose tolerance, as expected, but also triggered an unexpected robust inhibition of food intake. Validating that this represented a response to intestinally-released GIP, the suppression of food intake was prevented by injecting mice peripherally or centrally with antagonistic GIPR-antibodies, and was reproduced in an intersectional model utilising Gip-Cre / Villin-Flp to limit Dq transgene expression to K-cells in the intestinal epithelium. The effects of GIP cell activation were maintained in diet induced obese mice, in which chronic K-cell activation reduced food intake and attenuated body weight gain. CONCLUSIONS: These studies establish a physiological gut-brain GIP-axis regulating food intake in mice, adding to the multi-faceted metabolic effects of GIP which need to be taken into account when developing GIPR-targeted therapies for obesity and diabetes.

Fasting and post prandial pancreatic and enteroendocrine hormone levels in obese and non-obese participants.

Circulating insulin levels are known to be increased in people with higher body mass index (BMI) due to effects of adiposity on insulin resistance, whilst gut hormones have a more complex relationship, with fasting peptideYY (PYY) reported to be inversely related to BMI. This study aimed to further explore fasting and post prandial pancreatic and gut hormone concentrations in plasma samples from obese and non-obese participants. Participants with healthy BMI (n=15), overweight BMI (n=29) and obesity (n=161) had samples taken fasting and 30 min post mixed liquid meal for analysis of glucagon-like peptide-1 (GLP-1), PYY, glucose-dependent insulinotropic polypeptide (GIP), insulin and glucagon. Data visualiation used linear discriminant analysis for dimensionality reduction, to visualise the data and assess scaling of each hormone. Fasting levels of insulin, GIP and PYY were shown to be key classifiers between the 3 groups on ANCOVA analysis, with an observation of increased GIP levels in overweight, but not obese participants. In non-obese subjects, fasting GIP, PYY and insulin correlated with BMI, whereas in subjects with obesity only the pancreatic hormones glucagon and insulin correlated with BMI. Concentrations of total GLP-1 in the fasting state correlated strongly with glucagon levels, highlighting potential assay cross-reactivities. The study, which included a relatively large number of subjects with severe obesity, supported previous evidence of BMI correlating negatively with fasting PYY and positively with fasting insulin. The observation of increased fasting GIP levels in overweight but not obese participants deserves further validation and mechanistic investigation.

Motilin fluctuations in healthy volunteers determined by liquid chromatography mass spectrometry.

Introduction: Motilin is a hormone secreted by specialised enteroendocrine cells in the small intestine, and is known to modulate gastrointestinal motility in humans, regulating the migratory motor complex. It is understudied at least in part due to the lack of commercially available immunoassays. Method: A multiplexed liquid chromatography mass spectrometry (LC-MS/MS) method was optimised to measure motilin, insulin, C-peptide, GIP (1-42) and GIP (3-42). Corresponding active ghrelin concentrations were determined by immunoassay. Ten healthy volunteers with no prior history of gastroenterological or endocrine condition attended after overnight fast and had blood samples taken every 15 minutes for 4 hours whilst continuing to fast, and then further sampling for 2 hours following a liquid mixed meal. Hunger scores were taken at each time point using a visual analogue scale. Normal bowel habit was confirmed by 1 week stool diary. Results: Motilin levels fluctuated in the fasting state with an average period between peaks of 109.5 mins (SD:30.0), but with no evidence of a relationship with either ghrelin levels or hunger scores. The mixed meal interrupted cyclical motilin fluctuations, increased concentrations of motilin, insulin, C-peptide, GIP(1-42) and GIP(3-42), and suppressed ghrelin levels. Discussion: This study highlights the utility of LC-MS/MS for parallel measurement of motilin alongside other peptide hormones, and supports previous reports of the cyclical nature of motilin levels in the fasting state and interruption with feeding. This analytical method has utility for further clinical studies into motilin and gut hormone physiology in human volunteers.

Cellular mechanisms of incretin hormone secretion.

Enteroendocrine cells located along the gastrointestinal epithelium sense different nutrients/luminal contents that trigger the secretion of a variety of gut hormones with different roles in glucose homeostasis and appetite regulation. The incretin hormones glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are involved in the regulation of insulin secretion, appetite, food intake and body weight after their nutrient-induced secretion from the gut. GLP-1 mimetics have been developed and used in the treatment of type 2 diabetes mellitus and obesity. Modulating the release of endogenous intestinal hormones may be a promising approach for the treatment of obesity and type 2 diabetes without surgery. For that reason, current understanding of the cellular mechanisms underlying intestinal hormone secretion will be the focus of this review. The mechanisms controlling hormone secretion depend on the nature of the stimulus, involving a variety of signalling pathways including ion channels, nutrient transporters and G-protein-coupled receptors.

Towards Novel Nutritional Strategies in Gestational Diabetes: Eating Behaviour and Obesity in Women with Gestational Diabetes Compared with Non-Pregnant Adults.

BACKGROUND: Gestational diabetes is associated with increased risk of obesity, type 2 diabetes and cardiovascular disease. Effective nutritional strategies are needed to reduce BMI and improve long-term maternal cardiometabolic health, but the relative contribution of maternal eating behaviour, a potential barrier to dietary change, has not been explored. We compared eating behaviour in women with gestational diabetes with that of men and non-pregnant women with comparable risk factors, and tested associations between eating behaviour traits and BMI in women with gestational diabetes. We hypothesized that eating behaviour would be unfavourable in gestational diabetes and would be associated with BMI. METHODS: Participants (n = 417) including 53 men, 164 non-pregnant women and 200 women with gestational diabetes (singleton pregnancy; 29 weeks' gestation) were recruited into three prospective studies assessing weight loss interventions, with similar entry criteria. The three-factor eating questionnaire (TFEQ-R18) assessed uncontrolled eating, emotional eating and cognitive restraint at study enrolment. Associations between BMI at study enrolment and TFEQ-R18 (% maximum score) were assessed using linear regression. RESULTS: Women with gestational diabetes had significantly lower uncontrolled eating scores vs. men (53% vs. 65%; p < 0.001) and non-pregnant women (53% vs. 66%; p < 0.001), lower emotional eating scores vs. non-pregnant women (60% vs. 71%; p < 0.001) and higher cognitive restraint (p < 0.001 vs. men and non-pregnant women). In women with gestational diabetes, emotional eating scores were positively associated with BMI at study enrolment (beta coefficient 7.8 (95% CI 3.9 to 11.7), p < 0.001). CONCLUSIONS: Women with gestational diabetes have favourable eating behaviour compared with other population groups. Because BMI at study enrolment was associated with emotional eating, nutritional strategies which reduce emotional eating may provide new opportunities to improve long-term maternal health after gestational diabetes.

The intestine as an endocrine organ and the role of gut hormones in metabolic regulation.

Gut hormones orchestrate pivotal physiological processes in multiple metabolically active tissues, including the pancreas, liver, adipose tissue, gut and central nervous system, making them attractive therapeutic targets in the treatment of obesity and type 2 diabetes mellitus. Most gut hormones are derived from enteroendocrine cells, but bioactive peptides that are derived from other intestinal epithelial cell types have also been implicated in metabolic regulation and can be considered gut hormones. A deeper understanding of the complex inter-organ crosstalk mediated by the intestinal endocrine system is a prerequisite for designing more effective drugs that are based on or target gut hormones and their receptors, and extending their therapeutic potential beyond obesity and diabetes mellitus. In this Review, we present an overview of gut hormones that are involved in the regulation of metabolism and discuss their action in the gastrointestinal system and beyond.

Optimized LC-MS/MS Method for the Detection of ppCCK(21-44): A Surrogate to Monitor Human Cholecystokinin Secretion.

The hormone cholecystokinin (CCK) is secreted postprandially from duodenal enteroendocrine cells and circulates in the low picomolar range. Detection of this digestion and appetite-regulating hormone currently relies on the use of immunoassays, many of which suffer from insufficient sensitivity in the physiological range and cross-reactivity problems with gastrin, which circulates at higher plasma concentrations. As an alternative to existing techniques, a liquid chromatography and mass spectrometry-based method was developed to measure CCK-derived peptides in cell culture supernatants. The method was initially applied to organoid studies and was capable of detecting both CCK8 and an N-terminal peptide fragment (prepro) ppCCK(21-44) in supernatants following stimulation. Extraction optimization was performed using statistical modeling software, enabling a quantitative LC-MS/MS method for ppCCK(21-44) capable of detecting this peptide in the low pM range in human plasma and secretion buffer solutions. Plasma samples from healthy individuals receiving a standardized meal (Ensure) after an overnight fast were analyzed; however, the method only had sensitivity to detect ppCCK(21-44). Secretion studies employing human intestinal organoids and meal studies in healthy volunteers confirmed that ppCCK(21-44) is a suitable surrogate analyte for measuring the release of CCK in vitro and in vivo.

Enteroendocrine cell lineages that differentially control feeding and gut motility.

Enteroendocrine cells are specialized sensory cells of the gut-brain axis that are sparsely distributed along the intestinal epithelium. The functions of enteroendocrine cells have classically been inferred by the gut hormones they release. However, individual enteroendocrine cells typically produce multiple, sometimes apparently opposing, gut hormones in combination, and some gut hormones are also produced elsewhere in the body. Here, we developed approaches involving intersectional genetics to enable selective access to enteroendocrine cells in vivo in mice. We targeted FlpO expression to the endogenous Villin1 locus (in Vil1-p2a-FlpO knock-in mice) to restrict reporter expression to intestinal epithelium. Combined use of Cre and Flp alleles effectively targeted major transcriptome-defined enteroendocrine cell lineages that produce serotonin, glucagon-like peptide 1, cholecystokinin, somatostatin, or glucose-dependent insulinotropic polypeptide. Chemogenetic activation of different enteroendocrine cell types variably impacted feeding behavior and gut motility. Defining the physiological roles of different enteroendocrine cell types provides an essential framework for understanding sensory biology of the intestine.

Rapid and Quantitative Enrichment of Peptides from Plasma for Mass Spectrometric Analysis.

Mass spectrometric analysis of peptides enables the assignment of their exact mass and confirmation of all or a significant portion of the peptide's amino acid sequence. LC-MS/MS analysis has proven invaluable in peptidomics research and can identify new biomarkers and assign their circulatory concentrations to aid research into disease processes. However, due to the high background plasma protein content, which masks the presence of the naturally low abundance circulatory peptidome, extraction of peptides from plasma prior to mass spectrometric analysis is therefore crucial. Organic solvents efficiently precipitate these high molecular weight plasma proteins while leaving small molecular weight peptides in solution, providing a rapid and effective technique for separating peptides from the contaminating plasma proteins. A secondary cleanup step involving solid phase extraction is required to remove lipids and highly hydrophobic contaminants before LC-MS/MS analysis. The method described within this chapter is effective at enriching circulatory plasma peptides prior to LC-MS/MS analysis and has been used in multiple peptidomic studies to improve peptide detection and quantification. Peptides studied using this methodology include insulin, C-peptide, glucagon, PYY, GIP, and a number of other challenging gut peptide hormones. Quantitative analyses of peptides using the described method showed good correlation with existing immunoassays.

Obesity medication lorcaserin activates brainstem GLP-1 neurons to reduce food intake and augments GLP-1 receptor agonist induced appetite suppression.

OBJECTIVE: Overweight and obesity are endemic in developed countries, with a substantial negative impact on human health. Medications developed to treat obesity include agonists for the G-protein coupled receptors glucagon-like peptide-1 (GLP-1R; e.g. liraglutide), serotonin 2C (5-HT2CR; e.g, lorcaserin), and melanocortin4 (MC4R) which reduce body weight primarily by suppressing food intake. However, the mechanisms underlying the therapeutic food intake suppressive effects are still being defined and were investigated here. METHODS: We profiled PPG neurons in the nucleus of the solitary tract (PPGNTS) using single nucleus RNA sequencing (Nuc-Seq) and histochemistry. We next examined the requirement of PPGNTS neurons for obesity medication effects on food intake by virally ablating PPGNTS neurons. Finally, we assessed the effects on food intake of the combination of liraglutide and lorcaserin. RESULTS: We found that 5-HT2CRs, but not GLP-1Rs or MC4Rs, were widespread in PPGNTS clusters and that lorcaserin significantly activated PPGNTS neurons. Accordingly, ablation of PPGNTS neurons prevented the reduction of food intake by lorcaserin but not MC4R agonist melanotan-II, demonstrating the functional significance of PPGNTS 5-HT2CR expression. Finally, the combination of lorcaserin with GLP-1R agonists liraglutide or exendin-4 produced greater food intake reduction as compared to either monotherapy. CONCLUSIONS: These findings identify a necessary mechanism through which obesity medication lorcaserin produces its therapeutic benefit, namely brainstem PPGNTS neurons. Moreover, these data reveal a strategy to augment the therapeutic profile of the current frontline treatment for obesity, GLP-1R agonists, via coadministration with 5-HT2CR agonists.

Evidence for Involvement of GIP and GLP-1 Receptors and the Gut-Gonadal Axis in Regulating Female Reproductive Function in Mice.

Substantial evidence suggests crosstalk between reproductive and gut-axis but mechanisms linking metabolism and reproduction are still unclear. The present study evaluated the possible role of glucose-dependent-insulinotropic-polypeptide (GIP) and glucagon-like-peptide-1 (GLP-1) in reproductive function by examining receptor distribution and the effects of global GIPR and GLP-1R deletion on estrous cycling and reproductive outcomes in mice. GIPR and GLP-1R gene expression were readily detected by PCR in female reproductive tissues including pituitary, ovaries and uterine horn. Protein expression was confirmed with histological visualisation of incretin receptors using GIPR-Cre and GLP1R-Cre mice in which the incretin receptor expressing cells were fluorescently tagged. Functional studies revealed that female GIPR-/- and GLP-1R-/- null mice exhibited significantly (p < 0.05 and p < 0.01) deranged estrous cycling compared to wild-type controls, indicative of reduced fertility. Furthermore, only 50% and 16% of female GIPR-/- and GLP-1R-/- mice, respectively produced litters with wild-type males across three breeding cycles. Consistent with a physiological role of incretin receptors in pregnancy outcome, litter size was significantly (p < 0.001-p < 0.05) decreased in GIPR-/- and GLP-1R-/- mice. Treatment with oral metformin (300 mg/kg body-weight), an agent used clinically for treatment of PCOS, for a further two breeding periods showed no amelioration of pregnancy outcome except that litter size in the GIPR-/- group was approximately 2 times greater in the second breeding cycle. These data highlight the significance of incretin receptors in modulation of female reproductive function which may provide future targets for pharmacological intervention in reproductive disorders.

LC-MS/MS based detection of circulating proinsulin derived peptides in patients with altered pancreatic beta cell function.

Routine immunoassays for insulin and C-peptide have the potential to cross-react with partially processed proinsulin products, although in healthy patients these are present at such low levels that the interference is insignificant. Elevated concentrations of proinsulin and des-31,32 proinsulin arising from pathological conditions, or injected insulin analogues, however can cause significant assay interferences, complicating interpretation. Clinical diagnosis and management therefore sometimes require methods that can distinguish true insulin and C-peptide from partially processed proinsulin or injected insulin analogues. In this scenario, the high specificity of mass spectrometric analysis offers potential benefit for patient care. A high throughput targeted LC-MS/MS method was developed as a fit for purpose investigation of insulin, insulin analogues, C-peptide and proinsulin processing intermediates in plasma samples from different patient groups. Using calibration standards and bovine insulin as an internal standard, absolute concentrations of insulin and C-peptide were quantified across a nominal human plasma postprandial range and correlated strongly with immunoassay-based measurements. The ability to distinguish between insulin, insulin analogues and proinsulin intermediates in a single extraction is an improvement over existing immunological based techniques, offering the advantage of exact identification of the species being measured. The method promises to aid in the detection of circulating peptides which have previously been overlooked but may interfere with standard insulin and C-peptide immunoassays.

Relaxin/insulin-like family peptide receptor 4 (Rxfp4) expressing hypothalamic neurons modulate food intake and preference in mice.

OBJECTIVE: Insulin-like peptide 5 (INSL5) signalling, through its cognate receptor relaxin/insulin-like family peptide receptor 4 (RXFP4), has been reported to be orexigenic, and the high fat diet (HFD) preference observed in wildtype mice is altered in Rxfp4 knock-out mice. In this study, we used a new Rxfp4-Cre mouse model to investigate the mechanisms underlying these observations. METHODS: We generated transgenic Rxfp4-Cre mice and investigated central expression of Rxfp4 by RT-qPCR, RNAscope and intraparenchymal infusion of INSL5. Rxfp4-expressing cells were chemogenetically manipulated in global Cre-reporter mice using designer receptors exclusively activated by designer drugs (DREADDs) or after stereotactic injection of a Cre-dependent AAV-DIO-Dq-DREADD targeting a population located in the ventromedial hypothalamus (RXFP4VMH). Food intake and feeding motivation were assessed in the presence and absence of a DREADD agonist. Rxfp4-expressing cells in the hypothalamus were characterised by single-cell RNA-sequencing (scRNAseq) and the connectivity of RXFP4VMH cells was investigated using viral tracing. RESULTS: Rxfp4-Cre mice displayed Cre-reporter expression in the hypothalamus. Active expression of Rxfp4 in the adult mouse brain was confirmed by RT-qPCR and RNAscope. Functional receptor expression was supported by cyclic AMP-responses to INSL5 application in ex vivo brain slices and increased HFD and highly palatable liquid meal (HPM), but not chow, intake after intra-VMH INSL5 infusion. scRNAseq of hypothalamic RXFP4 neurons defined a cluster expressing VMH markers, alongside known appetite-modulating neuropeptide receptors (Mc4r, Cckar and Nmur2). Viral tracing demonstrated RXFP4VMH neural projections to nuclei implicated in hedonic feeding behaviour. Whole body chemogenetic inhibition (Di-DREADD) of Rxfp4-expressing cells, mimicking physiological INSL5-RXFP4 Gi-signalling, increased intake of the HFD and HPM, but not chow, whilst activation (Dq-DREADD), either at whole body level or specifically within the VMH, reduced HFD and HPM intake and motivation to work for the HPM. CONCLUSION: These findings identify RXFP4VMH neurons as regulators of food intake and preference, and hypothalamic RXFP4 signalling as a target for feeding behaviour manipulation.

Intestinal Enteroendocrine Cell Signaling: Retinol-binding Protein 2 and Retinoid Actions.

Retinol-binding protein 2-deficient (Rbp2-/-) mice are more prone to obesity, glucose intolerance, and hepatic steatosis than matched controls. Glucose-dependent insulinotropic polypeptide (GIP) blood levels are dysregulated in these mice. The present studies provide new insights into these observations. Single cell transcriptomic and immunohistochemical studies establish that RBP2 is highly expressed in enteroendocrine cells (EECs) that produce incretins, either GIP or glucagon-like peptide-1. EECs also express an enzyme needed for all-trans-retinoic acid (ATRA) synthesis, aldehyde dehydrogenase 1 family member A1, and retinoic acid receptor-alpha, which mediates ATRA-dependent transcription. Total and GIP-positive EECs are significantly lower in Rbp2-/- mice. The plasma transport protein for retinol, retinol-binding protein 4 (RBP4) is also expressed in EECs and is cosecreted with GIP upon stimulation. Collectively, our data support direct roles for RBP2 and ATRA in cellular processes that give rise to GIP-producing EECs and roles for RBP2 and RBP4 within EECs that facilitate hormone storage and secretion.

Glucose-Dependent Insulinotropic Polypeptide-A Postprandial Hormone with Unharnessed Metabolic Potential.

Glucose-dependent insulinotropic polypeptide (GIP) is released from the upper small intestine in response to food intake and contributes to the postprandial control of nutrient disposition, including of sugars and fats. Long neglected as a potential therapeutic target, the GIPR axis has received increasing interest recently, with the emerging data demonstrating the metabolically favorable outcomes of adding GIPR agonism to GLP-1 receptor agonists in people with type 2 diabetes and obesity. This review examines the physiology of the GIP axis, from the mechanisms underlying GIP secretion from the intestine to its action on target tissues and therapeutic development.

Expression of the relaxin family peptide 4 receptor by enterochromaffin cells of the mouse large intestine.

The gastrointestinal hormone, insulin-like peptide 5 (INSL5), is found in large intestinal enteroendocrine cells (EEC). One of its functions is to stimulate nerve circuits that increase propulsive activity of the colon through its receptor, the relaxin family peptide 4 receptor (RXFP4). To investigate the mechanisms that link INSL5 to stimulation of propulsion, we have determined the localisation of cells expressing Rxfp4 in the mouse colon, using a reporter mouse to locate cells expressing the gene. The fluorescent signal indicating the location of Rxfp4 expression was in EEC, the greatest overlap of Rxfp4-dependent labelling being with cells containing 5-HT. In fact, > 90% of 5-HT cells were positive for Rxfp4 labelling. A small proportion of cells with Rxfp4-dependent labelling was 5-HT-negative, 11-15% in the distal colon and rectum, and 35% in the proximal colon. Of these, some were identified as L-cells by immunoreactivity for oxyntomodulin. Rxfp4-dependent fluorescence was also found in a sparse population of nerve endings, where it was colocalised with CGRP. We used the RXFP4 agonist, INSL5-A13, to activate the receptor and probe the role of the 5-HT cells in which it is expressed. INSL5-A13 administered by i.p. injection to conscious mice caused an increase in colorectal propulsion that was antagonised by the 5-HT3 receptor blocker, alosetron, also given i.p. We conclude that stimuli that excite INSL5-containing colonic L-cells release INSL5 that, through RXFP4, excites 5-HT release from neighbouring endocrine cells, which in turn acts on 5-HT3 receptors of enteric sensory neurons to elicit propulsive reflexes.

Targeting the Enteroendocrine System for Treatment of Obesity.

Mimetics of the anorexigenic gut hormone glucagon-like peptide 1 (GLP-1) were originally developed as insulinotropic anti-diabetic drugs but also evoke significant weight loss, leading to their recent approval as obesity therapeutics. Co-activation of receptors for GLP-1 and other gut hormones which reduce food intake - peptide YY (PYY3-36), cholecystokinin (CCK) and glucose-dependent insulinotropic peptide (GIP) - is now being explored clinically to enhance efficacy. An alternative approach involves pharmacologically stimulating endogenous secretion of these hormones from enteroendocrine cells (EECs) to recapitulate the metabolic consequences of bariatric surgery, where highly elevated postprandial levels of GLP-1 and PYY3-36 are thought to contribute to improved glycaemia and weight loss.

The Enteroendocrine System in Obesity.

The enteroendocrine system coordinates the physiological response to food intake by regulating rates of digestion, nutrient absorption, insulin secretion, satiation and satiety. Gut hormones with important anorexigenic and/or insulinotropic roles include glucagon-like peptide 1 (GLP-1), peptide YY (PYY3-36), cholecystokinin (CCK) and glucose-dependent insulinotropic peptide (GIP). High BMI or obesogenic diets do not markedly disrupt this enteroendocrine system, which represents a critical target for inducing weight loss and treating co-morbidities in individuals with obesity.

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

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