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Gastrointestinal (GI) enterochromaffin (EC) cells are specialised sensors of luminal stimuli. They secrete most of the body's serotonin (5-HT), and are critical for modulating GI motility, secretion, and sensation, while also signaling satiety and intestinal discomfort. The aim of this study was to investigate mechanisms underlying the regulation of human EC cells, and the relative importance of direct nutrient stimulation compared with neuronal and paracrine regulation. Intestinal organoids from human duodenal biopsies were modified using CRISPR-Cas9 to specifically label EC cells with either the fluorescent protein Venus or the cyclic adenosine monophosphate (cAMP) sensor Epac1-S-H187. EC cells were purified by fluorescence-activated cell sorting for analysis by bulk RNA sequencing and liquid chromatography mass spectrometry peptidomics. The function of human EC cells was studied using single-cell patch clamp, calcium and cAMP imaging, and 5-hydroxytryptamine (5-HT) enzyme-linked immunosorbent assays (ELISAs). Human EC cells showed expression of receptors for nutrients (including GPR142, GPBAR1, GPR119, FFAR2, OR51E1, OR51E2), gut hormones (including SSTR1,2&5, NPY1R, GIPR) and neurotransmitters (ADRA2A, ADRB1). Functional assays revealed EC responses (calcium, cAMP, and/or secretion) to a range of stimuli, including bacterial metabolites, aromatic amino acids, and adrenergic agonists. Electrophysiological recordings showed that isovalerate increased action potential firing. 5-HT release from EC cells controls many physiological functions and is currently being targeted to treat disorders of the gut-brain axis. Studying ECs from human organoids enables improved understanding of the molecular mechanisms underlying EC cell activation, which is fundamental for the development of new strategies to target 5-HT-related gut and metabolic disorders. Show less

The contribution of glucose-dependent insulinotropic polypeptide receptor (GIPR) signalling in brown adipose tissue (BAT) remains underexplored. We studied the acute effects of exogenous acyl-GIP (1 nmol/kg) administration on whole-body lipid handling and fatty acid oxidation, using lipid tolerance tests (LTT) and indirect calorimetry, respectively. We demonstrate that in obese male mice, acute acyl-GIP administration improves lipid tolerance; however, pharmacological inhibition of GIPR, or genetic removal of GIPR globally or with the Myf5-Cre driver, completely abolishes GIP-mediated improvements in lipid tolerance, implicating GIPR in BAT. GIP-mediated improvements in lipid tolerance are associated with an increase in BAT lipid uptake, linked to increases in BAT lipoprotein lipase activity. Our data also reveal that BAT GIPR signalling is necessary for GIP-mediated increases in whole-body fatty acid oxidation, as Myf5-Cre: Gipr mice do not shift substrate oxidation upon GIP administration. Our findings suggest that BAT should be more closely considered in studies examining GIP's effects on whole-body metabolism in rodent models. Show less

A dual glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP1) receptor agonist, tirzepatide (TZPD), is a novel cardioprotective agent, particularly in metabolic disturbances-related co-morbidities, however, there is no exact study to emphasize its possible unintended action in cardiac cells. Considering a relationship between the trafficking of incretin receptors in a manner not anticipated by the standard way of cAMP as a primary actor in TZPD action, together with the role of cAMP depression in cardiac dysfunction, here, we aimed to elucidate a pattern of unintended receptor interactions of TZPD and molecular processes underlying the pleiotropic effects of TZPD through modulation of the β-adrenoceptors (β-ARs) signaling in cardiomyocytes. To establish the multifaceted cardioprotective function and underlying mechanisms of TZPD against hyperglycemia (HG)-or senescence (SC)-induced cardiac dysfunction, H9c2 cells were treated with and without TZPD. We also used β The TZPD intervention ameliorated the HG or SC phenotypes in the cardiac cells via alleviation in protein levels of GLP-1R and GIP-R as well as production of cAMP or cGMP, even in the presence of these receptor antagonisms. TZPD also increased the levels of β Our findings indicate that TZPD, with its multifaceted role, has beneficial effects on cardiac cells by positively modulating β-ARs signaling and glucose metabolism rather than on-target receptor action. Furthermore, we demonstrated how TZPD can engage the different targets with distinct signaling motifs at the sarcolemma. Show less

Dual agonists targeting glucagon-like peptide-1 receptor (GLP1R) and glucose-dependent insulinotropic polypeptide receptor (GIPR) are breakthrough treatments for patients with type 2 diabetes and obesity. Compared to GLP1R agonists, dual agonists show superior efficacy for glucose lowering and weight reduction. However, delineation of dual agonist cell targets remains challenging. Here, we develop and test daLUXendin and daLUXendin+, non-lipidated and lipidated fluorescent GLP1R/GIPR dual agonist probes, and use them to visualize cellular targets. daLUXendins are potent GLP1R/GIPR dual agonists that advantageously show less functional selectivity for mouse GLP1R over mouse GIPR. daLUXendins label rodent and human pancreatic islet cells, with a signal intensity of β cells > α cells = δ cells. Systemic administration of daLUXendin strongly labels GLP1R Show less

Glucagon-like peptide-1 (GLP-1) is a hormone known for its critical functions in managing blood sugar and offering cardiovascular benefits. Our study focuses on Glucagon Like Peptide 1 Receptor (GLP1R) agonists that act beyond glycemic control in cardiovascular and metabolic health. A comprehensive bioinformatic analysis was conducted, incorporating GLP1R, Gastric Inhibitory Polypeptide Receptor (GIPR), Gastric Inhibitory Polypeptide (GIP) and glucagon receptor (GCGR) to assess the effects of GLP1R agonists on gene and metabolite interactions. Interaction network analysis revealed 130 common genes among GLP1R, GLP1R/GIPR, GLP1R/GIP, and GLP1R/GIPR/GCGR associated with diabetes-related processes, including obesity and hyperglycemia. Enriched terms related to cardiovascular diseases, such as hypertension, calcium regulation in cardiac cells, and amino acid accumulation-induced mTOR activation. We also observed enrichment in gene sets linked to longevity and less recognized terms like fatty liver disease. In GLP1R/GIP, behavior-related terms and gastric acid secretion were identified; GLP1R/GIPR/GCGR linked to fibrosarcoma, thought/speech disturbances, and adipogenesis. The metabolite-gene layer revealed enrichment in galactose metabolism, platelet homeostasis, and nitric-oxide pathways. We found that GLP1R agonists network-level associations are stronger with heart diseases than sodium-glucose co-transporter 2 inhibitors, suggesting greater therapeutic benefits. Integrating networks with metabolites highlighted key interactors and clarified GLP1R agonists' mechanisms and therapeutic potential. Show less

Epicardial adipose tissue (EAT) is a visceral fat depot surrounding the myocardium. It contributes to coronary artery disease (CAD) through local inflammation, while its metabolic activity, including the expression of uncoupling protein-1 (UCP-1) and incretin receptors (GLP-1R, GIPR), may exert protective effects. The relationship between EAT immunohistochemical features and imaging-derived volume remains unclear. We prospectively studied 50 patients undergoing cardiac surgery: 25 with CAD undergoing coronary artery bypass grafting and 25 without CAD undergoing valve replacement. EAT samples were immunohistochemically stained for CD3, CD68, MPO, UCP-1, GLP-1R, and GIPR. Preoperative CT was used to quantify EAT volume. Patients with CAD more frequently had higher CD3 immunopositivity compared to the control group (84.0 vs. 58.3%, EAT in CAD exhibits increased T-cell infiltration and elevated UCP-1 expression, indicating an inflammatory yet metabolically active profile. Larger EAT volume was associated with UCP-1 and GLP-1R expression, underscoring the immunometabolic role of EAT in CAD. Show less

Obesity is a complex, multifactorial disease wherein the excessive accumulation of adipose tissue leads to adverse health outcomes, such as diabetes, cardiovascular disease and musculoskeletal disorders. Obesity also impacts both the risk and the clinical prognosis of heart failure (HF). The accumulation of adipose tissue results in metabolic dysregulation, including increased levels of pro-inflammatory cytokines and adipokines. These alterations are strongly associated with the development and progression of HF. Another significant comorbidity in patients with HF is sarcopenia, characterized by progressive loss of muscle mass and strength, affecting the quality of life. The study aims to critically synthesize the mechanisms by which modern pharmacological treatments-sodium-glucose cotransporter-2 (SGLT2) inhibitors, glucagon-like peptide-1 receptor (GLP-1R) agonists, and dual GIPR/GLP-1R agonists-modulate body mass composition, and to analyze the specific implications of these changes (e.g., visceral fat reduction versus lean mass loss) for heart failure (HF) prognosis and management. Show less

The incretin receptor agonists semaglutide and tirzepatide have transformed the medical management of obesity. The neural mechanisms by which incretin analogs regulate appetite remain incompletely understood, and dissecting this process is critical for the development of next-generation antiobesity drugs that are more targeted and tolerable. Moreover, the physiologic functions of incretins in appetite regulation and gut-brain communication have remained elusive. Using in vivo fiber photometry, we discovered distinct pharmacologic and physiologic roles for the incretin hormones glucose-dependent insulinotropic peptide (GIP) and glucagon-like peptide-1 (GLP-1). We showed that GIP, but not GLP-1, was required for normal nutrient-mediated inhibition of hunger-promoting AgRP neurons. By contrast, both GIP and GLP-1 analogs at pharmacologic doses were sufficient to inhibit AgRP neurons. The magnitude of neural inhibition was proportional to the effect of each incretin on food intake, and dual GIP and GLP-1 receptor agonism more potently inhibited AgRP neurons and suppressed food intake than either agonist alone. Our results have revealed a role for endogenous GIP in gut-brain appetite regulation and indicate that incretin analogs act in part via AgRP neurons to mediate their anorectic effects. Show less

Insulin secretory granule (ISG) maturation is a crucial aspect of insulin secretion and glucose homeostasis. The regulation of this maturation remains poorly understood, especially how secretory stimuli affect ISG maturity and subcellular localization. In this study, we used soft X-ray tomography (SXT) to quantitatively map ISG morphology, density, and location in single INS-1E and mouse pancreatic β cells under the effect of various secretory stimuli. We found that the activation of glucokinase (GK), gastric inhibitory polypeptide receptor (GIPR), glucagon-like peptide-1 receptor (GLP-1R), and G protein-coupled receptor 40 (GPR40) promotes ISG maturation. Each stimulus induces unique structural remodeling in ISGs, by altering size and density, depending on the specific signaling cascades activated. These distinct ISG subpopulations mobilize and redistribute in the cell, altering the overall cellular structural organization. Our results provide insight into how current diabetes and obesity therapies impact ISG maturation and may inform the development of future treatments that target maturation specifically. Show less

Metabolic dysfunction-associated steatotic liver disease (MASLD) is a leading cause of chronic liver disease globally, yet it lacks any approved pharmacological therapies. Dual glucagon-like peptide-1 receptor (GLP-1R) and glucose-dependent insulinotropic polypeptide receptor (GIPR) agonists have shown clinical promise, but their causal effect on MASLD remains unestablished. This study uses genetic evidence to evaluate the causal role of dual GLP-1R/GIPR agonists on MASLD and to explore its underlying mechanisms. Using a novel approach combining Mendelian randomization (MR) and Bayesian colocalization, we constructed a high-confidence genetic proxy for dual GLP-1R/GIPR agonists based on five genetic variants strongly associated with both mRNA expression and HbA1c levels. We then performed two-sample MR to assess the causal effect of this genetically proxied effect on MASLD and related metabolic risk abnormalities. Genetically proxied dual GLP-1R/GIPR agonists was causally associated with a substantially reduced risk of MASLD (OR: 0.24, 95 % CI: 0.08-0.75, P = 0.01). This protective effect was accompanied by significant improvements in systemic metabolic health, including increased high-density lipoprotein cholesterol (Beta: 0.39, 95 % CI: 0.13-0.66, P = 3.40 × 10 This study provides causal evidence that dual GLP-1R/GIPR agonists protects against MASLD. The mechanism likely involves broad improvements in lipid metabolism and insulin sensitivity. These findings offer strong genetic validation for this therapeutic strategy and provide a compelling rationale for its continued clinical development for the treatment of MASLD. Show less

In this study, to achieve more effective blood sugar lowering and weight-loss effects, eight glucagon-like peptide-1 (GLP-1)/glucose-dependent insulinotropic peptide (GIP)/glucagon (GCG) triple receptor agonists were designed and synthesized. Their sixteen related conjugates were obtained through Cys alkylation and Lys esterification modifications with two fatty acid side chains, respectively. After chemical structure confirmation using high-resolution mass spectrometry and peptide mapping, in vitro and in vivo biological effects of TRA01-24 were assessed. The structure-activity relationship (SAR) data on the amino acids, fatty acids, linkers and biological effects in vitro and in vivo of TRA01-24 have been summarized. Furthermore, peptide-protein molecular docking elucidated the structural basis for the biased agonist activity of TRA22 at GLP-1R, characterized by strong GLP-1R activation but weak GCGR and GIPR activation. In conclusion, a lead compound with excellent efficacy in vitro and in vivo, TRA24, was screened, which had better in vivo efficacy than tirzepatide in both normal and db/db mice. Show less

Epicardial adipose tissue (EAT), the visceral fat of the heart, is highly inflammatory fat depot with pro-inflammatory transcriptome and proteosome. EAT contributes to the development and progression of coronary artery disease (CAD) and atrial fibrillation (AF) through multifactorial inflammatory pathways. However, the paradigm linking EAT inflammation and cardiovascular risk was recently reevaluated. EAT inflammation may be also necessary process for adipose tissue remodeling and expansion to accommodate excess lipids. EAT inflammation may be also considered an adaptive response of adipose tissue to the effects of glucagon-like peptide-1 receptor (GLP-1Rs) and glucose-dependent insulinotropic polypeptide (GIP) analogs. The presence of GLP-1 (GLP-1R) and GIP receptors (GIP-R) suggest direct interaction of these agents with EAT. EAT GLP-1R and GIP-R activation can induce a beneficial balance between increased adipogenesis and reduced ectopic fat accumulation. Cardiovascular effects of liraglutide, semaglutide and tirzepatide can be mediated by EAT inflammation. Show less

G protein-coupled receptors (GPCRs) are transmembrane receptors that regulate intracellular signaling by interacting with G proteins and other effectors, influencing various physiological processes. The Glucose-dependent Insulinotropic Polypeptide Receptor (GIPR), a class B1 GPCR family member activated by GIP, regulates postprandial glycaemia by augmenting glucose-dependent insulin secretion, delaying gastric emptying, and suppressing appetite. Recent studies highlight the transmembrane domain (TMD) as the primary interface for dimerization, allowing GPCR to form homodimers or heterodimers with distinct physiological roles. However, the transient nature of these dimers challenges structural analysis, hindering experimental exploration and drug development. Computational methods now offer powerful tools for predicting such interactions. This study employs a hybrid approach, combining multiple protein docking software and dynamic structural optimization, to generate potential homodimeric models of GIPR-TMD. In addition, Next, validated models will provide insights into dimer activation mechanisms and support novel therapeutic discoveries. Show less

This patent application pertains to a novel class of peptide triagonists, generally represented by Formula I. These peptides exhibit selective activities toward GIPR (gastric inhibitory polypeptide receptor), GLP-1R (glucagon-like peptide-1 receptor), and GCGR (glucagon receptor). The unique pharma-cological profile of these triagonists offers promising therapeutic potential for the management of various conditions, including type 2 diabetes mellitus (T2DM), weight management, nonalcoholic fatty liver disease, etc. Show less

Glucose-dependent insulinotropic polypeptide (GIP) is a gut-derived incretin hormone, and pharmacologic modulation of central GIP receptors (GIPR) improves energy homeostasis. Recent reports have demonstrated that GIPR agonism is also anti-aversive. However, the mechanisms by which GIPR signaling impact food intake and aversion are incompletely understood. Here, we show that GIPR agonism abrogates the aversive and enhances the anorexigenic effects of the pro-inflammatory cytokine interleukin-1β (IL-1β). Aversion-encoding parabrachial calcitonin-gene related peptide (CGRP) neurons were required for IL-1β-induced conditioned taste avoidance (CTA) but not anorexia. Moreover, systemic IL-1β increased Show less

The use of incretin analogues has emerged as an effective approach to achieve both enhanced insulin secretion and weight loss in Type 2 diabetes (T2D) patients. Agonists which bind and stimulate multiple receptors have shown particular promise. However, off-target effects remain a complication of using these agents, and modified versions with optimised pharmacological profiles and/or biased signalling are sought. Ligand synthesis was achieved using standard solid-phase techniques. Assessments of GLP-1R-binding kinetics, G protein recruitment and receptor internalisation were performed using biochemical and imaging approaches. Insulin secretion was measured in purified mouse and human islets, and drug efficacy was assessed in hyperglycaemic db/db mice. We describe the synthesis and properties of a molecule which binds to both glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) receptors (GLP-1R and GIPR) to enhance insulin secretion. HISHS-2001 shows increased affinity at the GLP-1R, as well as a tendency towards reduced internalisation and recycling at this receptor versus FDA-approved dual GLP-1R/GIPR agonist tirzepatide. HISHS-2001 also displayed significantly greater bias towards cAMP generation versus β-arrestin 2 recruitment compared to tirzepatide. In contrast, G HISHS-2001 represents a novel dual receptor agonist with a promising pharmacological profile and actions. Future clinical studies will be needed to assess the safety and efficacy of this molecule in humans. Show less

The prevailing treatment of Parkinson's disease (PD) is not yet satisfactory. The present investigate the neuroprotective effect of the GLP-1/GIP dual agonist tirzepatide and examine the potential mechanisms involved. Analysis of GLP1 receptor (GLP1R) and GIPR expression alterations in dopaminergic neurons from PD patients in the GSE238129 dataset. The MPTP-induced subacute PD mice was treated with tirzepatide, semaglutide and levodopa. Behavioral tests and brain histopathology of mice were evaluated. The transmission electron microscopy revealed the presence of ultrastructural alterations in the mitochondrial morphology. The ATP level was assessed in substantia nigra. Western blot and immunohistochemical staining were employed to quantify Drp1 and mitophagy proteins. Furthermore, Drp1 inhibitor and mitophagy activator were used to treat MPTP-induced subacute PD mice, and lysosome inhibitor chloroquine (CQ) and the autophagy inhibitor 3-methyladenine (3-MA) were used in SY5Y cells for validation. The gene expression levels of both GLP1R and GIPR were significantly downregulated in dopaminergic neurons derived from PD patients. Tirzepatide could significantly ameliorate MPTP-induced the loss of tyrosine hydroxylase (TH) protein in the substantia nigra. There was no statistically difference observed between one-third doses of tirzepatide when compared with semaglutide and levodopa. In addition, tirzepatide not only improved mitochondrial ultrastructure, but also enhanced mitochondrial ATP content. Tirzepatide was found to reduce Drp1 expression and reverse the expressions of mitophagy-related proteins, including Pink1, Parkin, and p62. There was no statistically difference observed between one-third doses of tirzepatide compared with semaglutide in mitochondrial energy control. In addition, we observed that MPTP-induced subacute PD mice treated with a Drp1 inhibitor and mitophagy activator exhibited therapeutic effects. In SY5Y cells, lysosomal and autophagy inhibitors significantly reduced mitochondrial membrane potential, ATP levels, and the NAD+/NADH ratio. This study demonstrates that the benefits of tirzepatide extend to mitochondrial networks, achieved by means of the inhibition of mitochondrial pathological fission, the promotion of mitophagy, in MPTP-induced subacute PD mice or cells model. Show less

Glucagon-like peptide 1 receptor (GLP-1R) agonists exhibit anti-inflammatory actions, yet the importance of direct immune cell GLP-1R signaling remains uncertain. Although T cells respond to GLP-1, low receptor abundance and suboptimal antisera complicate efforts to characterize immune cell GLP-1R signaling. Here, we evaluate three frequently utilized GLP-1R antibodies, revealing that one of several antibodies, AGR-021, lack ideal specificity for detecting the GLP-1R in mice. Immunostaining with AGR-021 using tissues from two independent GLP-1R knockout mouse lines reveals persistent immunoreactive signals in GLP-1R-null pancreatic islets. Similarly, flow cytometry using AGR-021 reveals no reduction in AGR-021 immunoreactivity in GLP-1R-null splenic T cells. Moreover, western blotting detects AGR-021-immunoreactive proteins from a GLP-1R-negative cell line and fails to detect immunoreactive GLP-1R of the correct size upon overexpression of the receptor. Our findings reveal caveats governing use of multiple widely used GLP-1R antibodies, reemphasizing the importance of rigorous antibody validation for inferring accurate GLP-1R expression. Show less

Glucose-dependent insulinotropic peptide receptor (GIPR) stimulates insulin release and regulates metabolic homeostasis. GIPR function is shaped by spatiotemporal trafficking of this G protein-coupled receptor (GPCR). While GPCR endocytosis is traditionally associated with β-arrestin, GIPR internalization is only modestly dependent on this pathway. In this study, we demonstrate that GIPR engages a cytoskeletal motor, myosin VI to drive receptor endocytosis. GIPR engages the adaptor-motor complex through a PDZ-binding motif (PBM) at its C-ail. Interestingly, β-arrestin binding to phosphorylated residues upstream of the PBM enhance myosin VI recruitment and activation. GIPR internalization is dependent on both receptor phosphorylation and the PBM site to recruit β-arrestin and myosin VI, respectively. Synergistic engagement of β-arrestin and myosin VI results in desensitization of GIP-stimulated cAMP signaling while activating pERK1/2 from endosomal compartments. Blocking myosin VI activity enhances insulin release in pancreatic beta cells, demonstrating a novel role for this pathway in regulating the physiological effects of GIPR. Our findings highlight the direct convergence of two independent trafficking pathways at the level of the receptor C-tail, with implications for the nuanced regulation of individual GPCRs through the differential engagement of β-arrestin and myosin VI. GIPR has emerged as a frontline drug target in type 2 diabetes and obesity. Cellular effects of GIPR are regulated by receptor internalization and desensitization through mechanisms that are unclear. Here, we identify a novel GIPR trafficking pathway through the engagement of a cytoskeletal motor, myosin VI. Myosin VI and β-arrestin synergistically regulate GIPR endocytosis, signaling and insulin response in pancreatic beta cells. Our study highlights the convergence of two parallel trafficking mechanisms in GPCR function with potential implications in targeting metabolic disorders. Show less

The next generation of obesity medicines harness the activity of the glucose-dependent insulinotropic polypeptide and glucagon-like peptide 1 receptors (GIPR and GLP-1R), but their mechanism of action remains unclear. Here, we report that the GIPR is enriched in oligodendrocytes and GIPR signaling bidirectionally regulates oligodendrogenesis. In mice with adult-onset deletion of GIPR in oligodendrocytes, GIPR agonism fails to enhance the weight-loss effects of GLP-1R agonism. Mechanistically, GIPR agonism increases brain access of GLP-1R agonists, and GIPR signaling in oligodendrocytes is required for this effect. In addition, we show that vasopressin neurons of the paraventricular hypothalamus are necessary for the weight-loss response to GLP-1R activation, targeted by peripherally administered GLP-1R agonists via their axonal compartment, and this access is increased by activation of the GIPR in oligodendrocytes. Collectively, our findings identify a novel mechanism by which incretin therapies may function to promote synergistic weight loss in the management of excess adiposity. Show less

Unimolecular peptides targeting the receptors for glucagon-like peptide-1 (GLP-1), glucose-dependent insulinotropic polypeptide (GIP) and glucagon (GCG) have been shown to improve glycaemic management in both mice and humans. Yet the identity of the downstream signalling events mediated by these peptides remain to be elucidated. Here, we aimed to assess the mechanisms by which a validated peptide triagonist for GLP-1/GIP/GCG receptors (IUB447) stimulates insulin secretion in murine pancreatic islets. Islets were isolated from wild-type (WT), Gipr-knockout (Gipr The triagonist promoted glucose-stimulated insulin secretion (GSIS) to a greater degree than co-administration of conventional mono-agonists in WT mouse islets. The triagonist-induced increase in GSIS was unchanged in the absence of either Gipr or Gcgr. However, the triagonist failed to enhance insulin secretion in islets lacking both Glp-1r and Gipr and upon treatment with the GLP-1 receptor-specific antagonist exendin-3 (9-39). Similarly, the specific blocking of Gαq signalling with YM254890 or transient receptor potential melastatin 5 (TRPM5) with triphenylphosphine oxide (TPPO) suppressed the triagonist-induced enhancement of GSIS. In vivo assessment of high-fat-fed Trpm5 Triagonist-induced augmentation of GSIS is primarily mediated through its interaction with the GLP-1 receptor and subsequent activation of the Gαq-TRPM5 signalling pathway. Given that Gαq is a key player in the amplification of GSIS, particularly under diabetic conditions, these findings highlight a GLP-1 receptor-centric pharmacological profile that underlies the potent effects of this multi-receptor agonist. Show less

The sea cucumber collagen contains a high content of hydrophobic amino acids, which play essential roles in various bioactivities. A total of 2647 unknown active peptide fragments (2-20 amino acids) were obtained via virtual enzymolysis from 16 known collagen sequences in Apostichopus japonicus. Then, the novel bifunctional hexapeptide (DCDPRL, 717.788 Da) with hypoglycemic and antioxidant activities was identified via molecular docking and pharmacokinetics. DCDPRL revealed strong radical scavenging capacity in vitro with IC Show less

Pharmacological modulation of glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) through dual GIP/GLP-1 receptor agonists, commonly used for diabetes and obesity, shows promise in reducing alcohol consumption. We applied drug-target Mendelian randomization (MR) using genetic variation at these loci to assess their long-term effects on problematic alcohol use (PAU), binge drinking, alcohol misuse classifications, liver health, and other substance use behaviors. Genetic proxies for lowered BMI, modeling the appetite-suppressing and weight-reducing effects of variants in both the GIPR and GLP1R loci ("GIPR/GLP1R"), were linked with reduced binge drinking in the primary (β = -0.44, 95% CI [-0.72, -0.15], P = 2.42 × 10 Show less

The glucagon-like peptide-1 receptor (GLP-1R) and glucose-dependent insulinotropic polypeptide receptor (GIPR) are important incretin receptors that are therapeutic targets for the treatment of type 2 diabetes and obesity. This study extensively characterised the metabolic phenotype of mice with global deletion of either the GLP-1R or GIPR side by side under identical conditions. Age-matched male wild-type (WT) C57Bl6NTac, GLP-1RKO or GIPRKO mice were placed on a high-fat or chow diet for 12 weeks, and a range of in vivo (weight gain, food intake, glucose tolerance, insulin tolerance, and whole-body energy metabolism) and ex vivo (white adipocyte lipolysis, brown adipose tissue and liver mitochondrial function, adipocyte and islet size, and hepatic steatosis) parameters were measured. While both WT and GLP-1RKO mice gained weight similarly on a HFD, obese high-fat-fed GLP-1RKO mice had altered glucose and insulin tolerance, and exhibited hepatic steatosis, highlighting the physiological importance of the GLP-1R in the regulation of blood glucose and lipid homoeostasis. In contrast, GIPRKO mice were partially resistant to diet-induced obesity compared to the WT mice, which was associated with a small reduction in food intake and intact epididymal and subcutaneous white adipocyte β-adrenoceptor-mediated lipolysis. Similarly, WT mice treated with a GIPR antagonist prevented weight gain due to a reduction in food intake on a HFD. These findings provide further support that the GLP-1R is important for normal glycaemic control, whereas the GIPR may play a role in the regulation of body weight. Show less

Aortic valve sclerosis affects 30 % of individuals over 65 and is associated with coronary artery disease, with risk of progression to aortic stenosis. Endothelial dysfunction, mediated by oxidative stress, impaired nitric oxide (NO) signaling, inflammation, and lipoprotein deposition, plays a central role in disease initiation and progression. This study investigated whether a combination of bioactive compounds could counteract these mechanisms and support vascular health. The effects of curcuma longa, coenzyme Q10, black garlic, vitamin B1, and vitamin D3 were tested in vitro on aortic valve endothelial cells. Cell viability, reactive oxygen species (ROS), and NO levels were quantified by commercially available kits, while gene expression was analyzed by RNA sequencing. A 4-week prospective pilot clinical study in 10 healthy volunteers without cardiovascular disease evaluated endothelial function and arterial stiffness. The compounds reduced ROS production (>27 %; p < 0.05), enhanced endothelial viability (>33 %; p < 0.05; except curcuma and black garlic), and increased NO production (>6 %; p < 0.05; except black garlic). Beneficial effects were reflected in upregulation of anti-atherosclerotic (GIPR, +0.058 copies per million, CPM; p < 0.05), antioxidant (GADL1, +0.55 CPM; p < 0.001), and anti-inflammatory (IL12A, +0.17 CPM; p < 0.01) genes. Clinically, daily supplementation improved endothelial function in participants found to have pre-existing endothelial dysfunction (p = 0.0336), with 50 % achieving normal levels after 4 weeks, while all subjects exhibited reduced arterial stiffness (p = 0.0016) without hepatic toxicity. The oral supplementation of the combination of these bioactive compounds improved endothelial function and vascular health, particularly in individuals with endothelial dysfunction, offering potential therapeutic benefits for cardiovascular health. Show less

An acute increase of lipids in the upper small intestine (USI) of rodents and humans triggers lipid-sensing pathways to reduce food intake. However, USI lipid sensing does not reduce feeding in high-fat (HF) fed conditions, and the underlying mechanism remains elusive. Here, we report that HF feeding in male rats impaired USI lipid infusion to stimulate glucose-dependent insulinotropic polypeptide (GIP) secretion and decrease refeeding, and the defects of USI lipid sensing were restored by metformin. Next, we found that infusion of GIP receptor (GIPR) agonist in the nucleus of the solitary tract (NTS), but not mediobasal hypothalamus or area postrema, resulted in decreased refeeding in chow-fed rats. The anorectic effect of NTS GIPR agonist remained intact in HF rats and was inhibited by a genetic knockdown of GIPR. Finally, an inhibition of NTS GIPR also negated the ability of USI lipid sensing with metformin to decrease refeeding despite an increase in plasma GIP levels in HF rats. Thus, USI lipid sensing in HF rats is enhanced by metformin to trigger an endocrine GIP to NTS GIPR axis to reduce food intake, thereby unveiling small intestinal lipid-sensing pathways as potential targets to enhance GIP action and reduce weight in obesity. High-fat (HF) feeding in rats impairs upper small intestine (USI) lipid sensing to increase plasma glucose-dependent insulinotropic polypeptide (GIP) levels and reduce feeding. Metformin enhances USI lipids to increase GIP and reduce feeding in HF-fed rats. GIP activates the GIP receptor (GIPR) in the nucleus of the solitary tract (NTS), which reduces food intake in HF-fed rats. GIPR in the NTS is required for small intestinal lipids with metformin to reduce feeding. Show less

Multi-target peptide therapeutics targeting glucagon receptor (GCGR), glucagon-like peptide-1 receptor (GLP1R), and glucose-dependent insulinotropic polypeptide receptor (GIPR) represent a promising approach for treating diabetes and obesity. Triple agonist peptides demonstrate promising therapeutic potential compared to single-target approaches, yet rational design remains computationally challenging due to complex sequence-structure activity relationships. Existing methods, primarily based on convolutional neural networks, impose limitations including fixed sequence lengths and inadequate representation of molecular topology. Graph Attention Networks (GAT) offer advantages in capturing molecular structures and variable-length peptide sequences while providing interpretable insights into receptor-specific binding determinants. A dataset of 234 peptide sequences with experimentally determined binding affinities was compiled from multiple sources. Peptides were represented as molecular graphs with seven-dimensional node features encoding physicochemical properties and positional information. The GAT architecture employed a shared encoder with task-specific prediction heads, implementing transfer learning to address limited GIPR training data. Performance was evaluated using 5-fold cross-validation and independent validation on 24 literature-derived sequences. A genetic algorithm framework was developed for peptide sequence optimization, incorporating multi objective fitness evaluation based on predicted binding affinity, biological plausibility, and sequence novelty. Cross-validation demonstrated robust GAT performance across all receptors, with GCGR achieving high accuracy (AUC ROC: 0.915 ± 0.050), followed by GLP1R (AUC-ROC: 0.853 ± 0.059), and GIPR showing acceptable performance despite limited data (AUC-ROC: 0.907 ± 0.083). Comparative analysis revealed receptor-specific advantages: GAT significantly outperformed CNN for GCGR prediction (RMSE: 0.942 vs. 1.209, p = 0.0013), while CNN maintained superior GLP1R performance (RMSE: 0.552 vs. 0.723). Genetic algorithm optimization measurable improvement over baseline, with 4.0% fitness Enhancement and generation of 20 candidates exhibiting mean binding probabilities exceeding 0.5 across all targets. The GAT-based framework provides a computational approach in computational peptide design, demonstrating receptor-specific advantages and robust optimization capabilities. Genetic algorithm optimization enables systematic exploration of sequence space within existing agonist scaffolds while maintaining biological constraints. This approach provides a rational framework for prioritizing experimental validation efforts in triple agonist development. Show less

Chronic cold exposure in mice increases metabolic demand and food intake; the gut correspondingly expands its absorptive surface area. Gut enteroendocrine cells produce peptide hormones including glucagon-like peptide-1 (GLP-1), GLP-2, and glucose-dependent insulinotropic polypeptide (GIP) in response to a meal to facilitate nutrient absorption and post-prandial metabolism. The requirement of GLP-1, GLP-2, and GIP receptor signaling for small intestinal adaptations to chronic cold stress has not been investigated. Here, we show that male and female wild-type, double incretin receptor knockout (Glp1r Show less