Rho Family GTPases and Rho GEFs in Glucose Homeostasis.

Dysregulation of glucose homeostasis leading to metabolic syndrome and type 2 diabetes is the cause of an increasing world health crisis. New intriguing roles have emerged for Rho family GTPases and their Rho guanine nucleotide exchange factor (GEF) activators in the regulation of glucose homeostasis. This review summates the current knowledge, focusing in particular on the roles of Rho GEFs in the processes of glucose-stimulated insulin secretion by pancreatic β cells and insulin-stimulated glucose uptake into skeletal muscle and adipose tissues. We discuss the ten Rho GEFs that are known so far to regulate glucose homeostasis, nine of which are in mammals, and one is in yeast. Among the mammalian Rho GEFs, P-Rex1, Vav2, Vav3, Tiam1, Kalirin and Plekhg4 were shown to mediate the insulin-stimulated translocation of the glucose transporter GLUT4 to the plasma membrane and/or insulin-stimulated glucose uptake in skeletal muscle or adipose tissue. The Rho GEFs P-Rex1, Vav2, Tiam1 and β-PIX were found to control the glucose-stimulated release of insulin by pancreatic β cells. In vivo studies demonstrated the involvement of the Rho GEFs P-Rex2, Vav2, Vav3 and PDZ-RhoGEF in glucose tolerance and/or insulin sensitivity, with deletion of these GEFs either contributing to the development of metabolic syndrome or protecting from it. This research is in its infancy. Considering that over 80 Rho GEFs exist, it is likely that future research will identify more roles for Rho GEFs in glucose homeostasis

A GLP-1:CCK fusion peptide harnesses the synergistic effects on metabolism of CCK-1 and GLP-1 receptor agonism in mice.

Combination approaches for the treatment of metabolic diseases such as obesity and diabetes are becoming increasingly relevant. Co-administration of a glucagon-like peptide-1 receptor (GLP-1R) agonist with a cholecystokinin receptor-1 (CCKR1) agonist exert synergistic effects on weight loss in obese rodents. Here, we report on the effects of a novel fusion peptide (C2816) comprised of a stabilized GLP-1R agonist, AC3174, and a CCKR1-selective agonist, AC170222. C2816 was constructed such that AC3174 was linked to the N-terminus of AC170222, thus preserving the C-terminal amide of the CCK moiety. In functional in vitro assays C2816 retained full agonism at GLP-1R and CCKR1 at lower potency compared to parent molecules, whereas a previously reported fusion peptide in the opposite orientation, (pGlu-Gln)-CCK-8/exendin-4, exhibited no activity at either receptor. Acutely, in vivo, C2816 increased cFos in key central nuclei relevant to feeding behavior, and reduced food intake in wildtype (WT), but less so in GLP-1R-deficient (GLP-1RKO), mice. In sub-chronic studies in diet-induced obese (DIO) mice, C2816 exerted superior reduction in body weight compared to co-administration of AC3174 and AC170222 albeit at a higher molar dose. These data suggest that the synergistic pharmacological effects of GLP-1 and CCK pathways can be harnessed in a single therapeutic peptide

P-Rex1 Controls Sphingosine 1-Phosphate Receptor Signalling, Morphology, and Cell-Cycle Progression in Neuronal Cells.

P-Rex1 is a guanine-nucleotide exchange factor (GEF) that activates Rac-type small G proteins in response to the stimulation of a range of receptors, particularly G protein-coupled receptors (GPCRs), to control cytoskeletal dynamics and other Rac-dependent cell responses. P-Rex1 is mainly expressed in leukocytes and neurons. Whereas its roles in leukocytes have been studied extensively, relatively little is known about its functions in neurons. Here, we used CRISPR/Cas9-mediated P-Rex1 deficiency in neuronal PC12 cells that stably overexpress the GPCR S1PR1, a receptor for sphingosine 1-phosphate (S1P), to investigate the role of P-Rex1 in neuronal GPCR signalling and cell responses. We show that P-Rex1 is required for the S1P-stimulated activation of Rac1 and Akt, basal Rac3 activity, and constitutive cAMP production in PC12-S1PR1 cells. The constitutive cAMP production was not due to increased expression levels of major neuronal adenylyl cyclases, suggesting that P-Rex1 may regulate adenylyl cyclase activity. P-Rex1 was required for maintenance of neurite protrusions and spreading in S1P-stimulated PC12-S1PR1 cells, as well as for cell-cycle progression and proliferation. In summary, we identified novel functional roles of P-Rex1 in neuronal Rac, Akt and cAMP signalling, as well as in neuronal cell-cycle progression and proliferation

P-Rex1 Controls Sphingosine 1-Phosphate Receptor Signalling, Morphology, and Cell-Cycle Progression in Neuronal Cells

From MDPI via Jisc Publications RouterHistory: accepted 2021-09-15, pub-electronic 2021-09-18Publication status: PublishedFunder: Biotechnology and Biological Sciences Research Council; Grant(s): BB/P013384/1P-Rex1 is a guanine-nucleotide exchange factor (GEF) that activates Rac-type small G proteins in response to the stimulation of a range of receptors, particularly G protein-coupled receptors (GPCRs), to control cytoskeletal dynamics and other Rac-dependent cell responses. P-Rex1 is mainly expressed in leukocytes and neurons. Whereas its roles in leukocytes have been studied extensively, relatively little is known about its functions in neurons. Here, we used CRISPR/Cas9-mediated P-Rex1 deficiency in neuronal PC12 cells that stably overexpress the GPCR S1PR1, a receptor for sphingosine 1-phosphate (S1P), to investigate the role of P-Rex1 in neuronal GPCR signalling and cell responses. We show that P-Rex1 is required for the S1P-stimulated activation of Rac1 and Akt, basal Rac3 activity, and constitutive cAMP production in PC12-S1PR1 cells. The constitutive cAMP production was not due to increased expression levels of major neuronal adenylyl cyclases, suggesting that P-Rex1 may regulate adenylyl cyclase activity. P-Rex1 was required for maintenance of neurite protrusions and spreading in S1P-stimulated PC12-S1PR1 cells, as well as for cell-cycle progression and proliferation. In summary, we identified novel functional roles of P-Rex1 in neuronal Rac, Akt and cAMP signalling, as well as in neuronal cell-cycle progression and proliferation

Longitudinal evaluation of quality of life in 288 patients with Neurofibromatosis 2

Advances in molecular biology have resulted in novel therapy for neurofibromatosis 2-related (NF2) tumours, highlighting the need for robust outcome measures. The disease-focused NF2 impact on quality of life (NFTI-QOL) patient questionnaire was assessed as an outcome measure for treatment in a multi-centre study. NFTI-QOL was related to clinician-rated severity (ClinSev) and genetic severity (GenSev) over repeated visits. Data were evaluated for 288 NF2 patients (n = 464 visits) attending the English national NF2 clinics from 2010 to 2012. The male-to-female ratio was equal and the mean age was 42.2 (SD 17.8) years. The analysis included NFTI-QOL eight-item score, ClinSev graded as mild, moderate, or severe, and GenSev as a rank order of the number of NF2 mutations (graded as mild, moderate, severe). The mean (SD) 8.7 (5.4) score for NFTI-QOL for either a first visit or all visits 9.2 (5.4) was similar to the published norm of 9.4 (5.5), with no significant relationships with age or gender. NFTI-QOL internal reliability was good, with a Cronbach’s alpha score of 0.85 and test re-test reliability r = 0.84. NFTI related to ClinSev (r = 0.41, p < 0.001; r = 0.46 for all visits), but weakly to GenSev (r = 0.16, p < 0.05; r = 0.15 for all visits). ClinSev related to GenSev (r = 0.41, p < 0.001; r = 0.42 for all visits). NFTI-QOL showed a good reliability and ability to detect significant longitudinal changes in the QOL of individuals. The moderate relationships of NFTI-QOL with clinician- and genetic-rated severity suggest that NFTI-QOL taps into NF2 patient experiences that are not encompassed by ClinSev rating or genotype

Evaluation of quality of life in adults with neurofibromatosis 1 (NF1) using the Impact of NF1 on Quality Of Life (INF1-QOL) questionnaire

Background Neurofibromatosis 1 (NF1) is an inherited, multi-system, tumour suppressor disorder with variable complications that cause psychological distress and social isolation. The study aim was to develop and validate a disease-specific questionnaire to measure quality of life (QOL) in NF1 that is suitable both as an assessment tool in clinical practice and in clinical trials of novel therapy. Methods The Impact of NF1 on Quality of Life (INF1-QOL) questionnaire was developed by a literature search for common terms, focus group (n=6), semi-structured interviews (n=21), initial drafts (n =50) and final 14 item questionnaire (n=50). Bivariate correlations between items, exploratory factor analysis, correlations with severity and EuroQol were employed. Results INF1-QOL showed good internal reliability (Cronbach’s alpha 0.87), mean total INF1-QOL score was 8.64 (SD 6.3), median 7.00, range 0-30 (possible range 0-42); no significant correlations with age or gender. The mean total EuroQol score was 7.38 (SD 2.87), median 6.5, mean global EuroQol score was 76.34 (SD 16.56), median 80. Total INF1-QOL score correlated with total EuroQol r=0.82, p<0.0001. The highest impact on QOL was moderate or severe problems with anxiety and depression (32%) and negative effects of NF1 on role and outlook on life (42%). The mean inter-relater reliability for grading of clinical severity scores was 0.71 (range 0.65-0.79), and intra-class correlation was 0.92. The mean clinical severity score was 1.95 (SD 0.65) correlating r=0.34 with total INF1-QOL score p<0.05 and correlated 0.37 with total EuroQol score p<0.01. The clinical severity score was mild in 17 (34%), moderate in 16 (32%) and 17 (34%) individuals had severe disease. Conclusions INF1-QOL is a validated, reliable disease specific questionnaire that is easy and quick to complete. Role and outlook on life and anxiety and depression have the highest impact on QOL indicating the variability, severity and unpredictability of NF1. INFI-QOL correlates moderately with clinical severity. The moderate relationship between INF1-QOL and physician rated severity emphasizes the difference between clinical and patient perception. INFI-QOL will be useful in individual patient assessment and as an outcome measure for clinical trials

Development and characterisation of a novel glucagon like peptide-1 receptor antibody.

AIMS/HYPOTHESIS: Glucagon like peptide-1 (GLP-1) enhances glucose-dependent insulin secretion by binding to GLP-1 receptors (GLP1Rs) on pancreatic beta cells. GLP-1 mimetics are used in the clinic for the treatment of type 2 diabetes, but despite their therapeutic success, several clinical effects of GLP-1 remain unexplained at a mechanistic level, particularly in extrapancreatic tissues. The aim of this study was to generate and characterise a monoclonal antagonistic antibody for the GLP1R for use in vivo. METHODS: A naive phage display selection strategy was used to isolate single-chain variable fragments (ScFvs) that bound to GLP1R. The ScFv with the highest affinity, Glp1R0017, was converted into a human IgG1 and characterised further. In vitro antagonistic activity was assessed in a number of assays: a cAMP-based homogenous time-resolved fluorescence assay in GLP1R-overexpressing cell lines, a live cell cAMP imaging assay and an insulin secretion assay in INS-1 832/3 cells. Glp1R0017 was further tested in immunostaining of mouse pancreas, and the ability of Glp1R0017 to block GLP1R in vivo was assessed by both IPGTT and OGTT in C57/Bl6 mice. RESULTS: Antibodies to GLP1R were selected from naive antibody phage display libraries. The monoclonal antibody Glp1R0017 antagonised mouse, human, rat, cynomolgus monkey and dog GLP1R. This antagonistic activity was specific to GLP1R; no antagonistic activity was found in cells overexpressing the glucose-dependent insulinotropic peptide receptor (GIPR), glucagon like peptide-2 receptor or glucagon receptor. GLP-1-stimulated cAMP and insulin secretion was attenuated in INS-1 832/3 cells by Glp1R0017 incubation. Immunostaining of mouse pancreas tissue with Glp1R0017 showed specific staining in the islets of Langerhans, which was absent in Glp1r knockout tissue. In vivo, Glp1R0017 reversed the glucose-lowering effect of liraglutide during IPGTTs, and reduced glucose tolerance by blocking endogenous GLP-1 action in OGTTs. CONCLUSIONS/INTERPRETATION: Glp1R0017 is a monoclonal antagonistic antibody to the GLP1R that binds to GLP1R on pancreatic beta cells and blocks the actions of GLP-1 in vivo. This antibody holds the potential to be used in investigating the physiological importance of GLP1R signalling in extrapancreatic tissues where cellular targets and signalling pathways activated by GLP-1 are poorly understood

Important Role of the GLP-1 Axis for Glucose Homeostasis after Bariatric Surgery.

Bariatric surgery is widely used to treat obesity and improves type 2 diabetes beyond expectations from the degree of weight loss. Elevated post-prandial concentrations of glucagon-like peptide 1 (GLP-1), peptide YY (PYY), and insulin are widely reported, but the importance of GLP-1 in post-bariatric physiology remains debated. Here, we show that GLP-1 is a major driver of insulin secretion after bariatric surgery, as demonstrated by blocking GLP-1 receptors (GLP1Rs) post-gastrectomy in lean humans using Exendin-9 or in mice using an anti-GLP1R antibody. Transcriptomics and peptidomics analyses revealed that human and mouse enteroendocrine cells were unaltered post-surgery; instead, we found that elevated plasma GLP-1 and PYY correlated with increased nutrient delivery to the distal gut in mice. We conclude that increased GLP-1 secretion after bariatric surgery arises from rapid nutrient delivery to the distal gut and is a key driver of enhanced insulin secretion.RNA-sequencing was undertaken at the CRUK Cambridge Institute Genomics Core. Cell sorting was performed at the NIHR Cambridge BRC Cell Phenotyping Hub. PL received a Society for Endocrinology Early Career Grant. GR received an Addenbrooke’s Charitable Trust / Evelyn Trust Cambridge Clinical Research Fellowship [16-69] and a Royal College of Surgeons Research Fellowship. The work was partially funded by a project grant from the EFSD/Novo Nordisk Programme for Diabetes Research in Europe

The Rac-GEF Tiam1 controls integrin-dependent neutrophil responses

Rac GTPases are required for neutrophil adhesion and migration, and for the neutrophil effector responses that kill pathogens. These Rac-dependent functions are impaired when neutrophils lack the activators of Rac, Rac-GEFs from the Prex, Vav, and Dock families. In this study, we demonstrate that Tiam1 is also expressed in neutrophils, governing focal complexes, actin cytoskeletal dynamics, polarisation, and migration, in a manner depending on the integrin ligand to which the cells adhere. Tiam1 is dispensable for the generation of reactive oxygen species but mediates degranulation and NETs release in adherent neutrophils, as well as the killing of bacteria. In vivo, Tiam1 is required for neutrophil recruitment during aseptic peritonitis and for the clearance of Streptococcus pneumoniae during pulmonary infection. However, Tiam1 functions differently to other Rac-GEFs. Instead of promoting neutrophil adhesion to ICAM1 and stimulating β2 integrin activity as could be expected, Tiam1 restricts these processes. In accordance with these paradoxical inhibitory roles, Tiam1 limits the fMLP-stimulated activation of Rac1 and Rac2 in adherent neutrophils, rather than activating Rac as expected. Tiam1 promotes the expression of several regulators of small GTPases and cytoskeletal dynamics, including αPix, Psd4, Rasa3, and Tiam2. It also controls the association of Rasa3, and potentially αPix, Git2, Psd4, and 14-3-3ζ/δ, with Rac. We propose these latter roles of Tiam1 underlie its effects on Rac and β2 integrin activity and on cell responses. Hence, Tiam1 is a novel regulator of Rac-dependent neutrophil responses that functions differently to other known neutrophil Rac-GEFs

Phosphatidylinositol 3-kinase (PI3K) pathway activation in bladder cancer

The phosphatidylinositol 3-kinase (PI3K) pathway is a critical signal transduction pathway that regulates multiple cellular functions. Aberrant activation of this pathway has been identified in a wide range of cancers. Several pathway components including AKT, PI3K and mTOR represent potential therapeutic targets and many small molecule inhibitors are in development or early clinical trials. The complex regulation of the pathway, together with the multiple mechanisms by which it can be activated, make this a highly challenging pathway to target. For successful inhibition, detailed molecular information on individual tumours will be required and it is already clear that different tumour types show distinct combinations of alterations. Recent results have identified alterations in pathway components PIK3CA, PTEN, AKT1 and TSC1 in bladder cancer, some of which are significantly related to tumour phenotype and clinical behaviour. Co-existence of alterations to several PI3K pathway genes in some bladder tumours indicates that these proteins may have functions that are not related solely to the known canonical pathway