Biology subjects

Jones, B.

Publications and source records attributed to Jones, B..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

Binding Kinetics, Bias, Receptor Internalization and Effects on Insulin Secretion in vitro and in vivo of a Novel GLP-1R/GIPR Dual Agonist, HISHS-2001

The use of incretin analogues has emerged in recent years 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, including nausea and diarrhoea, remain a complication of using these agents, and modified versions with optimized pharmacological profiles and/or biased signaling at the cognate receptors are increasingly sought. Here, 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 internalization 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 {beta}-arrestin 2 recruitment compared to tirzepatide. In contrast, Gs recruitment was lower versus tirzepatide at the GLP-1R, but higher at the GIPR. Administered to obese hyperglycaemic db/db mice, HISHS-2001 increased circulating insulin whilst lowering body weight and HbA1c with similar efficacy to tirzepatide at substantially lower doses. Thus, HISHS-2001 represents a novel dual receptor agonist with an improved pharmacological profile.

pharmacology and toxicology↗

bioRxiv2024

The physiological impact of an N-terminal Halo-tag on GIPR function in mice

AimsG protein-coupled receptors (GPCRs) modified with self-labelling enzymatic tags (e.g. Halo, SNAP, CLIP) have enabled the in vitro study of receptor expression and trafficking. We developed a GiprHalo/Halo mouse model to gain insights into endogenous glucose-dependent insulinotropic polypeptide (GIP) receptor (GIPR) signalling, an important modulator of glucose and appetite homeostasis. MethodsGiprHalo/Halo mice were generated through ES cell-based gene targeting and physiologically characterised via body weight, oral glucose tolerance test and intraperitoneal glucose tolerance test analysis. Live cell imaging was used to measure cAMP responses in dispersed pancreatic islets from GiprWt/Wt and GiprHalo/Halo littermates in response to GIP. cAMP accumulation and surface receptor expression were assessed in AD293 cells transiently transfected with mouse and human untagged GIPR and Halo-GIPR. Endogenous pancreatic islet expression of Halo-GIPR was determined by immunohistochemistry. ResultsGiprHalo/Halo mice displayed comparable body weights and responses to oral glucose administration as GiprHalo/Wt and GiprWt/Wt littermates. However, GiprHalo/Halo mice only responded to high dose human GIP during intraperitoneal glucose tolerance testing, and cAMP responses to GIP were impaired in GiprHalo/Halo islets. Despite unimpaired surface expression, in vitro cAMP responses were lessened in AD293 cells expressing Halo-GIPR compared to untagged GIPR. Anti-Halo staining in mouse GiprHalo/Wt pancreatic islets displayed detectable signal. ConclusionsWhile the GiprHalo/Halo mouse model could be used to study endogenous GIPR expression, due to reduced responses we advise against its use for endogenous GIPR trafficking and signalling. Our study highlights the importance of carefully phenotyping mouse models in which modifications to endogenous proteins have been introduced.

cell biology↗

bioRxiv2024

In vivo functional profiling and structural characterisation of the human Glp1r A316T variant

Glucagon-like peptide-1 receptor agonists (GLP-1RAs) are effective therapies for type 2 diabetes (T2D) and obesity, yet patient responses are variable. Variation in the human Glp1r gene might be directly linked to therapeutic responses. A naturally occurring missense variant, A316T, protects against T2D and cardiovascular disease. Here, we have generated and characterised a human Glp1r A316T mouse model. Human Glp1rA316T/A316T mice displayed lower fasting blood glucose versus wildtype littermates, even under metabolic stress, and exhibited alterations in islet cytoarchitecture and /{beta}-cell identity under a high-fat, high-sucrose diet. This was however associated with blunted responses to GLP-1RAs in vivo. Further investigations in rodent and human {beta}-cell models demonstrated that human Glp1r A316T exhibits characteristics of constitutive activation but dampened GLP-1RA responses. Results are further supported by cryo-EM analyses and molecular dynamics simulations of GLP-1R A316T structure, collectively demonstrating that the A316T variant governs basal GLP-1R activity and pharmacological responses to GLP-1R-targeting therapies. TeaserThe Glp1r A316T missense variant displays improved glucose tolerance but impaired pharmacological incretin responses in vivo.

cell biology↗

bioRxiv2024

Extending sampling approaches for great crested newt (Triturus cristatus) eDNA monitoring

O_LIEnvironmental DNA (eDNA) monitoring has been used for great crested newt (Triturus cristatus) survey in the UK since the publication of a Defra-funded trial in 2014. If eDNA results are to be used in support of a great crested newt licence, surveys must be performed during a 76-day survey window (15 April - 30 June) to coincide with peak great crested newt activity, and must follow the approved ethanol precipitation protocol. However, eDNA detection is possible in other months and filtration may be a more effective method of eDNA capture. C_LIO_LIWe investigated whether the great crested newt eDNA survey season could be extended and filtration could be used for great crested newt eDNA capture by reviewing the available evidence and conducting a field study from April to October 2022. Paired water samples for ethanol precipitation and filtration were collected from 25 ponds once a month, resulting in 124 samples of each type. All samples (N = 248) were analysed with the approved great crested newt quantitative PCR assay. C_LIO_LIOur results indicate that great crested newts can be reliably detected using both eDNA capture methods from April to August, with detection rates decreasing in September and October. Great crested newt eDNA detection was comparable or higher with filtration than ethanol precipitation. C_LIO_LIPractical implication. Acceptance of filtration for great crested newt eDNA surveys could allow more water to be processed for robust and reliable estimates of great crested newt presence. Extending the great crested newt eDNA survey season to August could allow more waterbodies to be surveyed for great crested newt presence (but not absence), and identification of sites that provide important habitat for great crested newts outside of the breeding season. This would also remove logistical challenges and costs associated with completing sampling within 11 weeks and laboratory analysis within 10 working days from sample receipt. Furthermore, great crested newt eDNA surveys could be more frequently carried out alongside monitoring for other species, which are typically surveyed from April to September/October or year-round with conventional methods or eDNA surveys using filtration. This could enable infrastructure projects to develop more effective mitigation measures as well as reduce time required from surveyors and survey costs. C_LI

ecology↗

bioRxiv2024

SARS-CoV-2 ORF3a Protein Impairs Syncytiotrophoblast Maturation, Alters ZO-1 Localization, and Shifts Autophagic Pathways in Trophoblast Cells and 3D Organoids

SARS-CoV-2 infection poses a significant risk to placental physiology, but its impact on placental homeostasis is not well understood. We and others have previously shown that SARS-CoV-2 can colonize maternal and fetal placental cells, yet the specific mechanisms remain unclear. In this study, we investigate ORF3a, a key accessory protein of SARS-CoV-2 that exhibits continuous mutations. Our findings reveal that ORF3a is present in placental tissue from pregnant women infected with SARS-CoV-2 and disrupts autophagic flux in placental cell lines and 3D stem-cell-derived trophoblast organoids (SC-TOs), impairing syncytiotrophoblast differentiation and trophoblast invasion. This disruption leads to protein aggregation in cytotrophoblasts (CTB) and activates secretory autophagy, increasing CD63+ extracellular vesicle secretion, along with ORF3a itself. ORF3a also compromises CTB barrier integrity by disrupting tight junctions via interaction with ZO-1, mediated by its PDZ-binding motif, SVPL. Colocalization of ORF3a and ZO-1 in SARS-CoV-2-infected human placental tissue supports our in vitro findings. Deleting the PDZ binding motif in the ORF3a protein (ORF3a-noPBM mutant) restored proper ZO-1 localization at the cell junctions in an autophagy-independent manner. Lastly, we demonstrate that constitutive ORF3a expression induces SC-TOs to transition towards a secretory autophagy pathway likely via the PBM motif, as the ORF3a-NoPBM mutants showed a significant lack of CD63 expression. This study demonstrates the functional impact of ORF3a on placental autophagy and reveals a new mechanism for the activation of secretory autophagy, which may lead to increased extracellular vesicle secretion. These findings provide a foundation for exploring therapeutic approaches targeting ORF3a, specifically focusing on its PBM region to block its interactions with host cellular proteins and limiting placental impact.

microbiology↗

bioRxiv2024

Potent and selective repression of SCN9A by engineered zinc finger repressors for the treatment of neuropathic pain

Peripheral neuropathies are estimated to affect several million patients in the US with no long-lasting therapy currently available. In humans, the Nav1.7 sodium channel, encoded by the SCN9A gene, is involved in a spectrum of inherited neuropathies, and has emerged as a promising target for analgesic drug development. The development of a selective Nav1.7 inhibitor has been challenging, in part due to structural similarities among other Nav channels. Here we present preclinical studies for the first genomic medicine approach using engineered zinc finger repressors (ZFRs) specifically targeting human/nonhuman primate (NHP) SCN9A. AAV-mediated delivery of ZFRs in human iPSC-derived neurons resulted in 90% reduction of SCN9A with no detectable off-target activity. To establish proof-of-concept, a ZFR targeting the mouse Scn9a was assessed in the SNI neuropathic pain mouse model, which resulted in up to 70% repression of Scn9a in mouse DRGs and was associated with reduction in pain hypersensitivity as measured by increased mechanical- and cold-induced pain thresholds. AAV-mediated intrathecal delivery of ZFR in NHPs demonstrated up to 60% repression of SCN9A in bulk DRG tissue and on single-cell levels in the nociceptors. The treatment was well tolerated in NHPs, and no dose-limiting findings were observed four weeks after a single intrathecal injection. Taken together, our results demonstrate that AAV-delivered ZFR targeting the SCN9A gene is promising and supports further development as a potential therapy for peripheral neuropathies.

neuroscience↗

bioRxiv2024

Quantitative and Kinetic Proteomics Reveal ApoE Isoform-dependent Proteostasis Adaptations in Mouse Brain

Apolipoprotein E (ApoE) polymorphisms modify the risk of neurodegenerative disease with the ApoE4 isoform increasing and ApoE2 isoform decreasing risk relative to the wild-type control ApoE3 isoform. To elucidate how ApoE isoforms alter the proteome, we measured relative protein abundance and turnover in transgenic mice expressing a human ApoE gene (isoform 2, 3, or 4). This data provides insight into how ApoE isoforms affect the in vivo synthesis and degradation of a wide variety of proteins. We identified 4849 proteins and tested for ApoE isoform-dependent changes in the homeostatic regulation of [~]2700 ontologies. In the brain, we found that ApoE4 and ApoE2 both lead to modified regulation of mitochondrial membrane proteins relative to the wild-type control ApoE3. In ApoE4 mice, this regulation is not cohesive suggesting that aerobic respiration is impacted by proteasomal and autophagic dysregulation. ApoE2 mice exhibited a matching change in mitochondrial matrix proteins and the membrane which suggests coordinated maintenance of the entire organelle. In the liver, we did not observe these changes suggesting that the ApoE-effect on proteostasis is amplified in the brain relative to other tissues. Our findings underscore the utility of combining protein abundance and turnover rates to decipher proteome regulatory mechanisms and their potential role in biology.

biochemistry↗

bioRxiv2024

Molecular mapping and functional validation of GLP-1R cholesterol binding sites in pancreatic beta cells

G protein-coupled receptors (GPCRs) are integral membrane proteins which closely interact with their plasma membrane lipid microenvironment. Cholesterol is a lipid enriched at the plasma membrane with pivotal roles in the control of membrane fluidity and maintenance of membrane microarchitecture, directly impacting on GPCR stability, dynamics and function. Cholesterol extraction from pancreatic beta cells has previously been shown to disrupt the internalisation, clustering and cAMP responses of the glucagon-like peptide-1 receptor (GLP-1R), a class B1 GPCR with key roles in the control of blood glucose levels via the potentiation of insulin secretion in beta cells and weight reduction via the modulation of brain appetite control centres. Here, we unveil the detrimental effect of a high cholesterol diet on GLP-1R-dependent glucoregulation in vivo, and the improvement in GLP-1R function that a reduction in cholesterol synthesis using simvastatin exerts in pancreatic islets. We next identify and map sites of cholesterol high occupancy and residence time on active versus inactive GLP-1Rs using coarse-grained molecular dynamics (cgMD) simulations, followed by a screen of key residues selected from these sites and detailed analyses of the effects of mutating one of these residues, Val229, to alanine on GLP-1R interactions with cholesterol, plasma membrane behaviours, clustering, trafficking and signalling in pancreatic beta cells and primary islets, unveiling an improved insulin secretion profile for the V229A mutant receptor. This study 1) highlights the role of cholesterol in regulating GLP-1R responses in vivo; 2) provides a detailed map of GLP-1R - cholesterol binding sites in model membranes; 3) validates their functional relevance in beta cells; and 4) highlights their potential as locations for the rational design of novel allosteric modulators with the capacity to fine-tune GLP-1R responses.

cell biology↗

bioRxiv2024

Spatial transcriptomics identifies novel P. aeruginosa virulence factors

To holistically unravel the complexity of pathogen-host interactions within infected tissues we leverage a dual spatial transcriptomic approach that, for the first time, simultaneously captures the expression of Pseudomonas aeruginosa genes alongside the entire host transcriptome in a model of ocular infection. This innovative method reveals differential pathogen and host-specific gene expression patterns across specific anatomical regions generating a unified transcriptional map of infection. By integrating these data, we developed a predictive ridge regression model trained on images from infected tissues. The model achieved an R{superscript 2} score of 0.923 in predicting bacterial burden distributions by using host features thereby predicting novel biomarkers associated with disease severity. Our analysis revealed a complex interplay between P. aeruginosa nutritional requirements and protective host responses and identified novel interactions between bacterial metabolite transport proteins and host autophagy. Among an array of iron acquisition gene transcripts that showed significant enrichment at the host-pathogen interface, we discovered a novel virulence mediator PA2590. This study highlights the power of spatial transcriptomics, particularly in combining bacterial and host transcriptomes, to uncover novel host-pathogen interactions, advance our understanding of bacterial virulence mechanisms, and point to druggable molecules.

microbiology↗

bioRxiv2024

A type II secreted subtilase from commensal rhizobacteria disarms the immune elicitor peptide flg22

Plant roots grow in association with a community of microorganisms collectively known as the rhizosphere microbiome. Immune activation in response to elicitors like the flagellin-derived epitope flg22 restricts bacteria on plant roots but also inhibits plant growth. Some commensal root-associated bacteria are capable of suppressing the plant immune response to elicitors. In this study, we investigated the ability of 165 root-associated bacteria to suppress flg22-induced immune activation and growth restriction. We demonstrate that a type II secreted subtilase, which we term Immunosuppressive Subtilase A (IssA), from Dyella japonica strain MF79 cleaves the immune eliciting peptide flg22 and contributes to immune suppression. IssA homologs are found in other plant-associated commensals, with particularly high conservation in the order Xanthomonadales. This represents a novel mechanism by which commensal microbes modulate flg22-induced immunity in the rhizosphere microbiome.

microbiology↗

bioRxiv2024

An inter-organelle contact between endosomal GLP-1R, ER VAP-B, and the mitochondrial AKAP SPHKAP triggers PKA-dependent MIC19 phosphorylation and β-cell mitochondrial remodelling

Glucagon-like peptide-1 receptor (GLP-1R) agonists (GLP-1RAs) ameliorate mitochondrial health by increasing mitochondrial turnover in metabolically relevant tissues. Mitochondrial adaptation to metabolic stress is crucial to maintain pancreatic {beta}-cell function and prevent type 2 diabetes (T2D) progression. While the GLP-1R is well-known to stimulate cAMP production leading to Protein Kinase A (PKA) and Exchange Protein Activated by cyclic AMP 2 (Epac2) activation, there is a lack of understanding of the molecular mechanisms linking GLP-1R signalling with mitochondrial and {beta}-cell functional adaptation. Here, we present a comprehensive study in {beta}-cell lines and primary islets that demonstrates that, following GLP-1RA stimulation, GLP-1R-positive endosomes associate with the endoplasmic reticulum (ER) membrane contact site (MCS) tether VAPB at ER-mitochondria MCSs (ERMCSs), where active GLP-1R engages with SPHKAP, an A-kinase anchoring protein (AKAP) previously linked to T2D and adiposity risk in genome-wide association studies (GWAS). The inter-organelle complex formed by endosomal GLP-1R, ER VAPB and SPHKAP triggers a pool of ERMCS-localised cAMP/PKA signalling via the formation of a PKA-RI biomolecular condensate which leads to changes in mitochondrial contact site and cristae organising system (MICOS) complex phosphorylation, mitochondrial remodelling, and {beta}-cell functional adaptation, with important consequences for the regulation of {beta}-cell insulin secretion and survival to stress.

cell biology↗

bioRxiv2024

Medulloblastoma oncogene aberrations are not involved in tumor initiation, but essential for disease progression and therapy resistance

Despite recent advances in understanding disease biology, treatment of Group 3/4 medulloblastoma remains a therapeutic challenge in pediatric neuro-oncology. Bulk-omics approaches have identified considerable intertumoral heterogeneity in Group 3/4 medulloblastoma, including the presence of clear single-gene oncogenic drivers in only a subset of cases, whereas in the majority of cases, large-scale copy-number aberrations prevail. However, intratumoral heterogeneity, the role of oncogene aberrations, and broad CNVs in tumor evolution and treatment resistance remain poorly understood. To dissect this interplay, we used single-cell technologies (snRNA-seq, snATAC-seq, spatial transcriptomics) on a cohort of Group 3/4 medulloblastoma with known alterations in the oncogenes MYC, MYCN, and PRDM6. We show that large-scale chromosomal aberrations are early tumor initiating events, while the single-gene oncogenic events arise late and are typically sub-clonal, but MYC can become clonal upon disease progression to drive further tumor development and therapy resistance. We identify that the subclones are mostly interspersed across tumor tissue using spatial transcriptomics, but clear segregation is also present. Using a population genetics model, we estimate medulloblastoma initiation in the cerebellar unipolar brush cell-lineage starting from the first gestational trimester. Our findings demonstrate how single-cell technologies can be applied for early detection and diagnosis of this fatal disease.

cancer biology↗

bioRxiv2024

Gene regulatory network landscape of Group 3/4 medulloblastoma

Cellular heterogeneity in Group 3 and Group 4 medulloblastomas is a major driver of therapeutic intractability. Here, we describe transcription factor-driven mechanisms underlying cellular diversity in these tumors using a comprehensive single-nucleus multi-omics atlas. Our analysis reveals that rather than fixed entities, these tumors exist along a continuum of cell states defined by four molecular identity axes. We show that perturbing transcription factor activity drives cellular plasticity, a key contributor to tumor heterogeneity. Strikingly, modulation of the lineage determinant PAX6 redirects tumor cells along both Group 3- and Group 4-like differentiation trajectories, modeling a bi-lineage medulloblastoma, and reduces aggressiveness of MYC-driven tumor models. Together, our findings reveal how oncogenic signals co-opt cerebellar unipolar brush cell programs to rewire tumor cell states and identify cellular plasticity as a potential targetable vulnerability in medulloblastoma.

cancer biology↗

bioRxiv2024

Microfluidic platform enables shear-less aerosolization of lipid nanoparticles for messenger RNA inhalation

Leveraging the extensive surface area of the lungs for gene therapy, inhalation route offers distinct advantages for delivery. Clinical nebulizers that employ vibrating mesh technology are the standard choice for converting liquid medicines into aerosols. However, they have limitations when it comes to delivering mRNA through inhalation, including severe damage to nanoparticles due to shearing forces. Here, we introduce a novel microfluidic aerosolization platform (MAP) that preserves the structural and physicochemical integrity of lipid nanoparticles, enabling safe and efficient mRNA delivery to the respiratory system. Our results demonstrated the superiority of the novel MAP over the conventional vibrating mesh nebulizer, as it avoided problems such as particle aggregation, loss of mRNA encapsulation, and deformation of nanoparticle morphology. Notably, aerosolized nanoparticles generated by the microfluidic device led to enhanced transfection efficiency across various cell lines. In vivo experiments with mice that inhaled these aerosolized nanoparticles revealed successful, lung-specific mRNA transfection without observable signs of toxicity. This pioneering MAP represents a significant advancement for the pulmonary gene therapy, enabling precise and effective delivery of aerosolized nanoparticles.

bioengineering↗

bioRxiv2023

Adventitial macrophage accumulation impairs perivascular nerve function in mesenteric arteries with inflammatory bowel disease

1.Introduction: Inflammatory bowel disease (IBD) involves aberrant immune responses and is associated with both cardiovascular disease risk and altered intestinal blood flow. However, little is known about how IBD affects regulation of perivascular nerves that mediate blood flow. Previous work found perivascular nerve function is impaired in mesenteric arteries with IBD. The purpose of this study was to determine the mechanism of impaired perivascular nerve function. Methods: RNA sequencing was performed on mesenteric arteries from IL10-/- mice treated with H.hepaticus to induce disease (IBD) or left non-gavaged (Control). For all other studies, Control and IBD mice received either saline or clodronate liposome injections to study the effect of macrophage depletion. Perivascular nerve function was assessed using pressure myography and electrical field stimulation. Leukocyte populations, and perivascular nerves, and adventitial neurotransmitter receptors were labeled using fluorescent immunolabeling. Results: IBD was associated with increased in macrophage-associated gene expression, and immunolabeling showed accumulation of adventitial macrophages. Clodronate liposome injection eliminated adventitial macrophages, which reversed significant attenuation of sensory vasodilation, sympathetic vasoconstriction and sensory inhibition of sympathetic constriction in IBD. Acetylcholine-mediated dilation was impaired in IBD and restored after macrophage depletion, but sensory dilation remained nitric oxide independent regardless of disease and/or macrophage presence. Conclusion: Altered neuro-immune signaling between macrophages and perivascular nerves in the arterial adventitia contributes to impaired vasodilation, particularly via dilatory sensory nerves. Targeting the adventitial macrophage population may help preserve intestinal blood flow in IBD patients.

physiology↗

bioRxiv2023

Control of human pancreatic beta cell kinome by GLP-1R biased agonism

AimTo determine the kinase activity profiles of human pancreatic beta cells downstream of GLP-1R balanced versus biased agonist stimulations. Materials and methodsThis study analysed the kinomic profiles of human EndoC-{beta}h1 cells following vehicle and glucagon-like peptide-1 receptor (GLP-1R) stimulation with the pharmacological agonist exendin-4, as well as exendin-4-based biased derivatives exendin-phe1 and exendin-asp3 for acute (10-minute) versus sustained (120-minute) responses, using PamChip(R) protein tyrosine kinase (PTK) and serine/threonine kinase (STK) assays. The raw data were filtered and normalised using BioNavigator. The kinase analyses were conducted with R, mainly including kinase-substrate mapping and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. ResultsThe present analysis reveals that kinomic responses are distinct for acute versus sustained GLP-1R agonist (GLP-1RA) exposure, with individual responses associated with agonists presenting specific bias profiles. According to pathway analysis, several kinases, including JNKs, PKCs, INSR and LKB1, are important GLP-1R signalling mediators, constituting potential targets for further research on biased GLP-1R downstream signalling. ConclusionResults from this study suggest that differentially biased exendin-phe1 and exendin-asp3 can modulate distinct kinase interaction networks. Further understanding of these mechanisms will have important implications for the selection of appropriate anti-T2D therapies with optimised downstream kinomic profiles.

cell biology↗

bioRxiv2022

Intra-islet glucagon signalling regulates pulsatile insulin secretion and glucose homeostasis

BackgroundType 2 diabetes (T2D) is characterised by the loss of pulsatile insulin secretion. We studied mice with {beta}-cell specific loss of the glucagon receptor (Gcgr fl/fl X Ins-1Cre), to investigate the role of intra-islet glucagon receptor signalling on pan-islet calcium oscillations and insulin pulsatility. MethodsFrequently sampled intravenous glucose tolerance tests were conducted on Gcgr {beta}-cell-/- and littermate controls. Crossing with GCaMP6f (STOP flox) animals further allowed for {beta}-cell specific expression of a fluorescent calcium indicator. These islets were functionally imaged in vitro and in vivo. Wild-type mice were transplanted with islets expressing GCaMP6f in {beta}-cells into the anterior eye chamber and placed on a high fat diet. Part of the cohort received a glucagon analogue (GCG-analogue) for 40 days and the control group were fed to achieve weight matching. Calcium imaging was performed regularly during the development of hyperglycaemia and in response to GCG-analogue treatment. ResultsGcgr {beta}-cell-/- mice exhibited impaired glucose tolerance following intraperitoneal glucose challenge (control 12.7mmol/L {+/-}0.6 vs. Gcgr {beta}-cell-/- 15.4mmol/L {+/-}0.0 at 15 min, p=0.002); fasting glycaemia was not different to controls. In vitro, Gcgr {beta}-cell-/- islets showed profound loss of synchronised calcium waves in response to glucose which was only partially rescued in vivo. First-phase insulin pulsatility on peripheral blood sampling (n=5) was significantly disordered in Gcgr {beta}-cell-/- mice (burst mass Gcgr {beta}-cell-/- 0.30 {+/-}0.03 versus 0.84 {+/-}0.23 for controls p=0.04). Diet induced obesity and hyperglycaemia resulted in a loss of co-ordinated [Ca2+]I waves in transplanted islets. This was reversed with GCG-analogue treatment, independently of weight-loss (n=8). ConclusionThese data provide novel evidence for the role of intra-islet GCGR signalling in sustaining synchronised calcium oscillations and support a possible therapeutic role for glucagonergic agents to restore the insulin pulsatility lost in T2D.

physiology↗

bioRxiv2022

An examination of the divergent spatiotemporal signaling of GLP-1R versus GIPR in pancreatic beta cells

The incretin receptors, glucagon-like peptide-1 receptor (GLP-1R) and glucose-dependent insulinotropic polypeptide receptor (GIPR), are class B GPCRs and prime therapeutic targets for the treatment of type 2 diabetes (T2D) and obesity. They are expressed in pancreatic beta cells where they potentiate insulin release in response to food intake. Despite GIP being the main incretin in healthy individuals, GLP-1R has been favoured versus GIPR as a therapeutic target due to GIPR responses being blunted in T2D patients and the conflicting effects of GIPR agonists and antagonists in improving glucose tolerance and preventing weight gain. There is, however, a recently renewed interest in GIPR biology following the realisation that GIPR responses can be restored after an initial period of blood glucose normalization and the recent development of dual GLP-1R-GIPR agonists with superior capacity for the control of blood glucose levels and weight. The importance of GLP-1R trafficking and subcellular signaling in the control of receptor outputs is well established, but little is known about the pattern of spatiotemporal signaling from the GIPR in beta cells. Here we have directly compared the main trafficking and signaling characteristics of both receptors in pancreatic beta cells, finding striking differences in their propensities for internalization, recycling, and degradation, as well as plasma membrane versus endosomal activity, with potential implications for receptor-specific control of beta cell function.

cell biology↗