Inhibitory effect of UDP-glucose on cAMP generation and insulin secretion.

Type-2 diabetes (T2D) is a global disease caused by the inability of pancreatic ß-cells to secrete adequate insulin. However, the molecular mechanisms underlying the failure of ß-cells to respond to glucose in T2D remains unknown. Here, we investigated the relative contribution of UDP-glucose (UDP-G), a P2Y14-specific agonist, in the regulation of insulin release using human isolated pancreatic islets and INS-1 cells. P2Y14 was expressed in both human and rodent pancreatic ß-cells. Dose-dependent activation of P2Y14 by UDP-G suppressed glucose-stimulated insulin secretion (GSIS) and knockdown of P2Y14 abolished the UDP-G effect. 12-h pretreatment of human islets with pertussis-toxin (PTX) improved GSIS and prevented the inhibitory effect of UDP-G on GSIS. UDP-G on GSIS suppression was associated with suppression of cAMP in INS-1 cells. UDP-G decreased the reductive capacity of nondiabetic human islets cultured at 5 mm glucose for 72 h and exacerbated the negative effect of 20 mm glucose on the cell viability during culture period. T2D donor islets displayed a lower reductive capacity when cultured at 5 mm glucose for 72 h that was further decreased in the presence of 20 mm glucose and UDP-G. Presence of a nonmetabolizable cAMP analog during culture period counteracted the effect of glucose and UDP-G. Islet cultures at 20 mm glucose increased apoptosis, which was further amplified when UDP-G was present. UDP-G modulated glucose-induced proliferation of INS-1 cells. The data provide intriguing evidence for P2Y14 and UDP-G's role in the regulation of pancreatic ß-cell function.

CXCL14 Inhibits Insulin Secretion Independently of CXCR4 or CXCR7 Receptor Activation or cAMP Inhibition.

BACKGROUND/AIMS: CXCL14, a secreted chemokine peptide that promotes obesity-induced insulin resistance, is expressed by islets, but its effects on islet function are unknown. The aim of this study was to determine the role of CXCL14 in ß-cells and investigate how it transduces these effects. METHODS: Cxcl14 and Cxc-receptor mRNA expression was quantified by qPCR and CXCL14 expression in the pancreas was determined by immunohistochemistry. The putative function of CXCL14 at CXCR4 and CXCR7 receptors was determined by ß-arrestin recruitment assays. The effects of CXCL14 on glucose-stimulated insulin secretion, cAMP production, glucose-6-phosphate accumulation, ATP generation, apoptosis and proliferation were determined using standard techniques. RESULTS: CXCL14 was present in mouse islets, where it was mainly localised to islet δ-cells. Cxc-receptor mRNA profiling indicated that Cxcr4 and Cxcr7 are the most abundant family members in islets, but CXCL14 did not promote ß-arrestin recruitment at CXCR4 or CXCR7 or antagonise CXCL12 activation of these receptors. CXCL14 induced a concentration-dependent inhibition of glucose-stimulated insulin secretion, which was not coupled to Gαi signalling. However, CXCL14 inhibited glucose-6-phosphate generation and ATP production in mouse islets. CONCLUSION: CXCL14 is expressed by islet δ-cells where it may have paracrine effects to inhibit insulin secretion in a CXCR4/CXCR7-independent manner through reductions in ß-cell ATP levels. These observations, together with the previously reported association of CXCL14 with obesity and impaired glucose homeostasis, suggest that inhibition of CXCL14 signalling could be explored to treat type 2 diabetes.

C3aR and C5aR1 act as key regulators of human and mouse ß-cell function.

AIMS: Complement components 3 and 5 (C3 and C5) play essential roles in the complement system, generating C3a and C5a peptides that are best known as chemotactic and inflammatory factors. In this study we characterised islet expression of C3 and C5 complement components, and the impact of C3aR and C5aR1 activation on islet function and viability. MATERIALS AND METHODS: Human and mouse islet mRNAs encoding key elements of the complement system were quantified by qPCR and distribution of C3 and C5 proteins was determined by immunohistochemistry. Activation of C3aR and C5aR1 was determined using DiscoverX beta-arrestin assays. Insulin secretion from human and mouse islets was measured by radioimmunoassay, and intracellular calcium ([Ca2+]i), ATP generation and apoptosis were assessed by standard techniques. RESULTS: C3 and C5 proteins and C3aR and C5aR1 were expressed by human and mouse islets, and C3 and C5 were mainly localised to ß- and α-cells. Conditioned media from islets exposed for 1 h to 5.5 and 20 mM glucose stimulated C3aR and C5aR1-driven beta-arrestin recruitment. Activation of C3aR and C5aR1 potentiated glucose-induced insulin secretion from human and mouse islets, increased [Ca2+]i and ATP generation, and protected islets against apoptosis induced by a pro-apoptotic cytokine cocktail or palmitate. CONCLUSIONS: Our observations demonstrate a functional link between activation of components of the innate immune system and improved ß-cell function, suggesting that low-level chronic inflammation may improve glucose homeostasis through direct effects on ß-cells.

Defining G protein-coupled receptor peptide ligand expressomes and signalomes in human and mouse islets.

INTRODUCTION: Islets synthesise and secrete numerous peptides, some of which are known to be important regulators of islet function and glucose homeostasis. In this study, we quantified mRNAs encoding all peptide ligands of islet G protein-coupled receptors (GPCRs) in isolated human and mouse islets and carried out in vitro islet hormone secretion studies to provide functional confirmation for the species-specific role of peptide YY (PYY) in mouse islets. MATERIALS AND METHODS: GPCR peptide ligand mRNAs in human and mouse islets were quantified by quantitative real-time PCR relative to the reference genes ACTB, GAPDH, PPIA, TBP and TFRC. The pathways connecting GPCR peptide ligands with their receptors were identified by manual searches in the PubMed, IUPHAR and Ingenuity databases. Distribution of PYY protein in mouse and human islets was determined by immunohistochemistry. Insulin, glucagon and somatostatin secretion from islets was measured by radioimmunoassay. RESULTS: We have quantified GPCR peptide ligand mRNA expression in human and mouse islets and created specific signalomes mapping the pathways by which islet peptide ligands regulate human and mouse GPCR signalling. We also identified species-specific islet expression of several GPCR ligands. In particular, PYY mRNA levels were ~ 40,000-fold higher in mouse than human islets, suggesting a more important role of locally secreted Pyy in mouse islets. This was confirmed by IHC and functional experiments measuring insulin, glucagon and somatostatin secretion. DISCUSSION: The detailed human and mouse islet GPCR peptide ligand atlases will allow accurate translation of mouse islet functional studies for the identification of GPCR/peptide signalling pathways relevant for human physiology, which may lead to novel treatment modalities of diabetes and metabolic disease.

Identifying Signalling Pathways Regulated by GPRC5B in ß-Cells by CRISPR-Cas9-Mediated Genome Editing.

BACKGROUND/AIMS: CRISPR-Cas9, a RNA-guided targeted genome editing tool, has revolutionized genetic engineering by offering the ability to precisely modify DNA. GPRC5B is an orphan receptor belonging to the group C family of G protein-coupled receptors (GPCRs). In this study, we analysed the functional roles of the Gprc5b receptor in MIN6 ß-cells using CRISPR-Cas9 and transient over-expression of Gprc5b. METHODS: The optimal transfection reagent for use in MIN6 ß-cells was determined by analysing efficiency of GFP plasmid delivery by cell sorting. A MIN6 ß-cell line in which Gprc5b expression was knocked down (Gprc5b KD) was generated using CRISPR-Cas9 technology. Gprc5b receptor mRNA expression, proliferation, apoptosis, Cignal 45-Pathway Reporter Array signalling and western blot assays were carried out using Gpcr5b KD MIN6 ß-cells that had been transiently transfected with different concentrations of mouse Gprc5b plasmid to over-express Gprc5b. RESULTS: JetPRIME® was the best candidate for MIN6 ß-cell transfection, providing approximately 30% transfection efficiency. CRISPR-Cas9 technology targeting Gprc5b led to stable knock-down of this receptor in MIN6 ß-cells and its re-expression induced proliferation and potentiated cytokine- and palmitate-induced apoptosis. The Cignal 45 Reporter analysis indicated Gprc5b-dependent regulation of apoptotic and proliferative pathways, and western blotting confirmed activation of signalling via TGF-ß and IFNγ. CONCLUSION: This study provides evidence of CRISPR-Cas9 technology being used to down-regulate Gprc5b expression in MIN6 ß-cells. This strategy allowed us to identify signalling pathways linking GPRC5B receptor expression to ß-cell proliferation and apoptosis.

Mesenchymal stromal cell secretory factors induce sustained improvements in islet function pre- and post-transplantation.

BACKGROUND AIMS: Mesenchymal stromal cells (MSCs) enhance islet function both in vitro and in vivo, at least in part by secreting ligands that activate islet G-protein coupled receptors (GPCRs). We assessed whether pre-treatment with a defined "cocktail" of MSC-secreted GPCR ligands enhances islet functional survival in vitro and improves the outcomes of islet transplantation in an experimental model of diabetes. METHODS: Isolated islets were cultured for 48 h with ANXA1, SDF-1 or C3a, alone or in combination. Glucose-stimulated insulin secretion (GSIS) and cytokine-induced apoptosis were measured immediately after the 48 h culture period and at 24 h or 72 h following removal of the ligands from the culture media. Islets were syngeneically transplanted underneath the kidney capsule of streptozotocin-induced diabetic C57BL/6 mice and blood glucose levels monitored for 28 days. RESULTS: Pre-culturing islets with a cocktail of ANXA1/SDF-1/C3a potentiated GSIS and protected islet cells from cytokine-induced apoptosis in vitro. These effects were maintained for up to 72 h after the removal of the factors from the culture medium, suggesting a sustained protection of islet graft functional survival during the immediate post-transplantation period. Islets pre-treated with the cocktail of MSC secretory factors were more effective in reducing blood glucose in diabetic mice, consistent with their improved functional survival in vivo. DISCUSSION: Pre-culturing islets with a cocktail of MSC secretory products offers a well-defined, cell-free approach to improve clinical islet transplantation outcomes while avoiding many of the safety, regulatory and logistical hurdles of incorporating MSCs into transplantation protocols.

Anti-diabetic action of all-trans retinoic acid and the orphan G protein coupled receptor GPRC5C in pancreatic ß-cells.

Pancreatic islets express high levels of the orphan G-protein coupled receptor C5C (GPRC5C), the function of which remains to be established. Here we have examined the role of GPRC5C in the regulation of insulin secretion and ß-cell survival and proliferation using human and mouse pancreatic islets. The expression of GPRC5C was analysed by RNA-sequencing, qPCR, western blotting and confocal microscopy. Insulin secretion and cell viability were determined by RIA and MTS assays, respectively. GPRC5C mRNA expression and protein level were reduced in the islets from type-2 diabetic donors. RNA sequencing in human islets revealed GPRC5C expression correlated with the expression of genes controlling apoptosis, cell survival and proliferation. A reduction in Gprc5c mRNA and protein expression was observed in islets isolated from old mice (>46 weeks of age) compared to that in islets from newborn (<3 weeks) mice. Down-regulation of Gprc5c led to both moderately reduced glucose-stimulated insulin release and also reduced cAMP content in mouse islets. Potentiation of glucose-stimulated insulin secretion concomitant with enhanced islet cAMP level by all-trans retinoic acid (ATRA) was attenuated upon Gprc5c-KD. ATRA also increased [Ca+2]i in Huh7-cells. Gprc5c over expression in Huh7 cells was associated with increased ERK1/2 activity. Gprc5c-KD in clonal MIN6c4 cells reduced cell proliferation and in murine islets increased apoptosis and the sensitivity of primary islet cells to a cocktail of pro-apoptotic cytokines. Our results demonstrate that agents activating GPRC5C represent a novel modality for the treatment and/or prevention of diabetes by restoring and/or maintaining functional ß-cell mass.

The novel chemokine receptor, G-protein-coupled receptor 75, is expressed by islets and is coupled to stimulation of insulin secretion and improved glucose homeostasis.

AIMS/HYPOTHESIS: Chemokine (C-C motif) ligand 5 (CCL5) acts at C-C chemokine receptors (CCRs) to promote immune cell recruitment to sites of inflammation, but is also an agonist at G-protein-coupled receptor 75 (GPR75), which has very limited homology with CCRs. GPR75 is coupled to Gq to elevate intracellular calcium, so we investigated whether islets express this receptor and whether its activation by CCL5 increases beta cell calcium levels and insulin secretion. METHODS: Islet CCL5 receptor mRNA expression was measured by quantitative RT-PCR and GPR75 was detected in islets by western blotting and immunohistochemistry. In some experiments GPR75 was downregulated by transient transfection with small interfering RNA. Real-time changes in intracellular calcium were determined by single-cell microfluorimetry. Dynamic insulin secretion from perifused islets was quantified by radioimmunoassay. Glucose homeostasis in lean and obese mice was determined by measuring glucose and insulin tolerance, and insulin secretion in vivo. RESULTS: Mouse and human islets express GPR75 and its ligand CCL5. Exogenous CCL5 reversibly increased intracellular calcium in beta cells via GPR75, this phenomenon being dependent on phospholipase C activation and calcium influx. CCL5 also stimulated insulin secretion from mouse and human islets in vitro, and improved glucose tolerance in lean mice and in a mouse model of hyperglycaemia and insulin resistance (ob/ob). The improvement in glucose tolerance was associated with enhanced insulin secretion in vivo, without changes in insulin sensitivity. CONCLUSIONS/INTERPRETATION: Although CCL5 is implicated in the pathogenesis of diabetes through activation of CCRs, it has beneficial effects on beta cells through GPR75 activation.

GPRC5B a putative glutamate-receptor candidate is negative modulator of insulin secretion.

GPRC5B is an orphan receptor belonging to the group C family of G protein-coupled receptors (GPCRs). GPRC5B is abundantly expressed in both human and mouse pancreatic islets, and both GPRC5B mRNA and protein are up-regulated 2.5-fold in islets from organ donors with type 2 diabetes. Expression of Gprc5b is 50% lower in islets isolated from newborn (<3 weeks) than in adult (>36 weeks) mice. Lentiviral shRNA-mediated down-regulation of Gprc5b in intact islets from 12 to 16 week-old mice strongly (2.5-fold) increased basal (1 mmol/l) and moderately (40%) potentiated glucose (20 mmol/l) stimulated insulin secretion and also enhanced the potentiating effect of glutamate on insulin secretion. Downregulation of Gprc5b protected murine insulin-secreting clonal MIN6 cells against cytokine-induced apoptosis. We propose that increased expression of GPRC5B contributes to the reduced insulin secretion and b-cell viability observed in type-2 diabetes. Thus, pharmacological targeting of GPRC5B might provide a novel means therapy for the treatment and prevention of type-2 diabetes.

Platelet transcriptional profile and protein expression in patients with systemic lupus erythematosus: up-regulation of the type I interferon system is strongly associated with vascular disease.

Patients with systemic lupus erythematosus (SLE) have a markedly increased risk to develop cardiovascular disease, and traditional cardiovascular risk factors fail to account for this increased risk. We used microarray to probe the platelet transcriptome in patients with SLE and healthy controls, and the gene and protein expression of a subset of differentially expressed genes was further investigated and correlated to platelet activation status. Real-time PCR was used to confirm a type I interferon (IFN) gene signature in patients with SLE, and the IFN-regulated proteins PRKRA, IFITM1 and CD69 (P < .0001) were found to be up-regulated in platelets from SLE patients compared with healthy volunteers. Notably, patients with a history of vascular disease had increased expression of type I IFN-regulated proteins as well as more activated platelets compared with patients without vascular disease. We suggest that interferogenic immune complexes stimulate production of IFNα that up-regulates the megakaryocytic type I IFN-regulated genes and proteins. This could affect platelet activation and contribute to development of vascular disease in SLE. In addition, platelets with type I IFN signature could be a novel marker for vascular disease in SLE.

Kv1.3 is the exclusive voltage-gated K+ channel of platelets and megakaryocytes: roles in membrane potential, Ca2+ signalling and platelet count.

A delayed rectifier voltage-gated K(+) channel (Kv) represents the largest ionic conductance of platelets and megakaryocytes, but is undefined at the molecular level. Quantitative RT-PCR of all known Kv alpha and ancillary subunits showed that only Kv1.3 (KCNA3) is substantially expressed in human platelets. Furthermore, megakaryocytes from Kv1.3(/) mice or from wild-type mice exposed to the Kv1.3 blocker margatoxin completely lacked Kv currents and displayed substantially depolarised resting membrane potentials. In human platelets, margatoxin reduced the P2X(1)- and thromboxaneA(2) receptor-evoked [Ca(2+)](i) increases and delayed the onset of store-operated Ca(2+) influx. Megakaryocyte development was normal in Kv1.3(/) mice, but the platelet count was increased, consistent with a role of Kv1.3 in apoptosis or decreased platelet activation. We conclude that Kv1.3 forms the Kv channel of the platelet and megakaryocyte, which sets the resting membrane potential, regulates agonist-evoked Ca(2+) increases and influences circulating platelet numbers.

Uridine diphosphate (UDP) stimulates insulin secretion by activation of P2Y6 receptors.

We examined the transcriptional expression and functional effects of receptors for the extracellular pyrimidines uridine triphosphate (UTP) and uridine diphosphate (UDP), on insulin and glucagon secretion in isolated mouse pancreatic islets and purified beta-cells. Using real-time PCR, the UDP receptor P2Y(6) was found to be highly expressed in both whole islets and beta-cells purified by repeated counter-flow elutriation, whereas no mRNA expression for UTP receptors P2Y(4) and P2Y(2) could be detected. Functional in vitro experiments revealed that the P2Y(6) agonist UDPbetaS dose-dependently enhanced insulin and glucagon release during short-term incubation (1h), while P2Y(6) activation during a longer period (24h), selectively increased insulin release, especially at high glucose levels. The corresponding EC(50) value for UDPbetaS ranged from 3.2 x 10(-8)M to 1.6 x 10(-8)M for both glucose concentrations. The P2Y(6) antagonist MRS2578 inhibited the effects of UDPbetaS, supporting a P2Y(6) specific effect. In addition to negative RT-PCR results, the lack of response to UTPgammaS a selective P2Y(2/4) agonist further rule out the involvement of P2Y(2/4) receptors in the islet hormone release. Our results suggest a modulatory role for UDP via a functional active P2Y(6) receptor in the regulation of islet hormone release.

Gene expression profiling for the identification of G-protein coupled receptors in human platelets.

UNLABELLED: INTRODUCTION AND MATERIALS AND METHODS: G-protein coupled receptors (GPCRs) play an important role in platelet aggregation. To identify new platelet GPCRs, a platelet gene expression profile was generated and validated using quantitative real-time PCR. RESULTS: In total, mRNA of 28 GPCR genes was detected in human platelets. The 12 most abundant platelet GPCR transcripts were: thrombin receptor PAR1 (1865+/-178%), ADP receptor P2Y(12) (459+/-88%), succinate receptor 1 (257+/-48%), ADP receptor P2Y(1) (100%), orphan P2RY(10) (68.2+/-3.3%), lysophosphatidic acid receptors GPR23 (48.2+/-11%) and GPR92 (26.1+/-3.3%), adrenergic receptor alpha(2A) (18.4+/-4.4%), orphan EBI2 (6.31+/-0.42), adenosine receptors A(2A) (2.94+/-0.24%) and A(2B) (2.88+/-0.16%) and lysophosphatidic acid receptor LPA(1) (2.59+/-0.39%) (% relative to the chosen calibrator P2Y(1)). A surprising G-protein coupled receptor redundancy was found: two ADP receptors (P2Y(1) and P2Y(12)), three adenosine receptors (A(2A), A(2B), and A(1)), four lysophosphatidic acid receptors (LPA(1), LPA(3), GPR23 and GPR92), two l-glutamate receptors (mGlu(3) and mGlu(4)) and two serotonin receptors (5-HT(1F) and 5-HT(4)). The adenosine receptor A(2B) gene expression was validated with protein expression and functional studies. Western blot confirmed A(2B) receptor protein expression and platelet flow cytometry demonstrated inhibition of the effect of NECA by the adenosine A(2B)-antagonist MRS1754. CONCLUSIONS: We have detected several GPCRs not previously known to be expressed in platelets, including a functional adenosine A(2B) receptor. The findings could improve our understanding of platelet aggregation and provide new targets for drug development.

Expression profiling and electrophysiological studies suggest a major role for Orai1 in the store-operated Ca2+ influx pathway of platelets and megakaryocytes.

Store-operated Ca2+ influx represents a major route by which cytosolic Ca2+ can be elevated during platelet activation, yet its molecular identity in this cell type remains highly controversial. Using quantitative RT-PCR analysis of candidate receptor-operated cation entry pathways in human platelets, we show a >30-fold higher expression of message for the recently discovered Orai1 store-operated Ca2+ channel, and also the store Ca2+ sensor STIM1, when compared to the non-selective cation channels TRPC1, TRPC6 and TRPM2. Orai1 and STIM1 gene transcripts were also detected at higher levels than TRPC1, TRPC6 and TRPM2 in primary murine megakaryocytes and human megakaryocytic cell lines. In direct electrophysiological recordings from murine megakaryocytes, Ca2+ ionophore-induced store depletion stimulated CRAC currents, which are known to require Orai1, and these overlapped with TRPC6-like currents following P2Y receptor activation. Together with recent transgenic studies, these data provide evidence for STIM1:Orai1 as a primary pathway for agonist-evoked Ca2+ influx in the platelet and megakaryocyte.

Activation of imidazoline receptor I2, and improved pancreatic ß-cell function in human islets.

AIM: The impact of BL11282, an imidazoline receptor (NISCH) agonist, on potentiation of glucose-stimulated insulin secretion (GSIS) from isolated human non-diabetic (ND) and type 2 diabetic (T2D) islets was investigated. METHODS: Analysis of mRNA was performed by RNA-sequencing and qPCR. Insulin and cAMP by RIA and ELISA respectively. RESULTS: RNA-sequencing data revealed that NISCH is highly expressed in fat tissues, islets, liver and muscles, with eight detectable splice variants of transcripts in islets. NISCH had a positive correlation with GLP-1 (GLP1R) and GIP (GIPR) receptor transcripts. The expression of NISCH was confirmed by qPCR in human islets. NISCH and GLP1R were comparably higher expressed in mouse islets compared to human islets. GSIS was dose-dependently potentiated by BL11282 from incubated islets of ND and T2D human islet donors. The insulinotropic action of BL11282 was associated with increased cAMP. While the harmful effect of high glucose on reductive capacity of islet cells was enhanced by glibenclamide during long-term culture, it was counteracted by BL11282 or Bt2-cAMP. BL11282 also increased proliferation of INS-1 cells during long-time culture. CONCLUSION: Our data suggest that BL11282 potentiates GSIS by an action involving cAMP/PKA system and BL11282 could be an attractive insulinotropic and ß-cell protective agent.

A comparative analysis of human and mouse islet G-protein coupled receptor expression.

G-protein coupled receptors (GPCRs) are essential for islet function, but most studies use rodent islets due to limited human islet availability. We have systematically compared the GPCR mRNA expression in human and mouse islets to determine to what extent mouse islets can be used as surrogates for human islets to study islet GPCR function, and we have identified species-specific expression of several GPCRs. The A3 receptor (ADORA3) was expressed only in mouse islets and the A3 agonist MRS 5698 inhibited glucose-induced insulin secretion from mouse islets, with no effect on human islets. Similarly, mRNAs encoding the galanin receptors GAL1 (GALR1), GAL2 (GALR2) and GAL3 GALR3) were abundantly expressed in mouse islets but present only at low levels in human islets, so that it reads (GALR3) and galanin inhibited insulin secretion only from mouse islets. Conversely, the sst1 receptor (SSTR1) was abundant only in human islets and its selective activation by CH 275 inhibited insulin secretion from human islets, with no effect on mouse islets. Our comprehensive human and mouse islet GPCR atlas has demonstrated that species differences do exist in islet GPCR expression and function, which are likely to impact on the translatability of mouse studies to the human context.

Quantification of the mRNA expression of G protein-coupled receptors in human adipose tissue.

G protein-coupled receptors (GPCRs) are important regulators of human physiology and therefore the targets of a large number of modern therapeutics. Although GPCRs are important regulators of adipose tissue endocrine and energy storage functions, the expression and function of a majority of GPCRs in adipose tissue is poorly characterized. A first step in the functional characterization of adipose tissue GPCRs is to accurately quantify the expression of GPCRs in adipose tissue. In this methods chapter, a detailed, step-by-step protocol is presented for the isolation of adipose tissue total RNA, its conversion into cDNA and the real-time PCR quantification of human GPCR mRNA expression relative to the mRNA expression of the stable adipose tissue housekeeping gene peptidylprolyl isomerase A (PPIA). A comprehensive list of 377 manually validated, commercially available GPCR qPCR primers allows facilitated swift quantification of either the entire human GPCRome or individual GPCRs, thus providing a sensitive, flexible, and cost-effective means of determining the mRNA expression of GPCRs in adipose tissue.

Transcriptomic analysis of the ion channelome of human platelets and megakaryocytic cell lines.

Ion channels have crucial roles in all cell types and represent important therapeutic targets. Approximately 20 ion channels have been reported in human platelets; however, no systematic study has been undertaken to define the platelet channelome. These membrane proteins need only be expressed at low copy number to influence function and may not be detected using proteomic or transcriptomic microarray approaches. In our recent work, quantitative real-time PCR (qPCR) provided key evidence that Kv1.3 is responsible for the voltage-dependent K+ conductance of platelets and megakaryocytes. The present study has expanded this approach to assess relative expression of 402 ion channels and channel regulatory genes in human platelets and three megakaryoblastic/erythroleukaemic cell lines. mRNA levels in platelets are low compared to other blood cells, therefore an improved method of isolating platelets was developed. This used a cocktail of inhibitors to prevent formation of leukocyte-platelet aggregates, and a combination of positive and negative immunomagnetic cell separation, followed by rapid extraction of mRNA. Expression of 34 channel-related transcripts was quantified in platelets, including 24 with unknown roles in platelet function, but that were detected at levels comparable to ion channels with established roles in haemostasis or thrombosis. Trace expression of a further 50 ion channel genes was also detected. More extensive channelomes were detected in MEG-01, CHRF-288-11 and HEL cells (195, 185 and 197 transcripts, respectively), but lacked several channels observed in the platelet. These "channelome" datasets provide an important resource for further studies of ion channel function in the platelet and megakaryocyte.

Adhesion G Protein-Coupled Receptor G1 (ADGRG1/GPR56) and Pancreatic ß-Cell Function.

CONTEXT: Adhesion G protein-coupled receptor (GPCR)-G1 (ADGRG1) is the most abundant GPCR in human pancreatic islets, but its role in islet function is unclear. OBJECTIVE: Investigate how ADGRG1 expression and activation by its ligand, collagen III, impacts ß-cell function in normal and type 2 diabetic (T2D) islets. DESIGN: Genes associated with the ADGRG1 in human islets was probed by RNA-sequencing of human pancreatic islet isolated from cadaveric donors, followed by functional studies on ß-cell proliferation, apoptosis, and insulin secretion in human and mouse islets and in INS-1 cells. MAIN OUTCOME MEASURES: Changes in ß-cell gene expression, proliferation, apoptosis, and insulin secretion were quantified by RNA-sequencing, qPCR, Thymidine incorporation, Western blotting, and RIA, respectively. RESULTS: ADGRG1 is the most abundant GPCR mRNA in both human and mouse islets, and its expression in human islets strongly correlates with genes important for ß-cell function and T2D risk. The expression of ADGRG1 was reduced in islets of T2D donors, in db/db mouse islets, and in isolated human islets exposed to chronic hyperglycemia. Beneficial effects of collagen type III on ß-cell function via activation of the cAMP/protein kinase A pathway, suppression of RhoA and caspase-3 activity, increased ß-cell viability, and proliferation were abolished when ADGRG1 was down-regulated in ß-cells. CONCLUSIONS: We demonstrate a mechanistic link between ADGRG1 expression and ß-cell function. Pharmacological agents that promote expression or activation of the ADGRG1 receptor may represent a novel approach for the treatment of T2D.

Annexin A1 Is a Key Modulator of Mesenchymal Stromal Cell-Mediated Improvements in Islet Function.

We have previously demonstrated that coculture of islets with mesenchymal stromal cells (MSCs) enhanced islet insulin secretory capacity in vitro, correlating with improved graft function in vivo. To identify factors that contribute to MSC-mediated improvements in islet function, we have used an unbiased quantitative RT-PCR screening approach to identify MSC-derived peptide ligands of G-protein-coupled receptors that are expressed by islets cells. We demonstrated high expression of annexin A1 (ANXA1) mRNA by MSCs and confirmed expression at the protein level in lysates and MSC-conditioned media by Western blot analysis and ELISA. Preculturing islets with exogenous ANXA1 enhanced glucose-stimulated insulin secretion (GSIS), thereby mimicking the beneficial influence of MSC preculture in vitro. Small interfering RNA-mediated knockdown of ANXA1 in MSCs reduced their capacity to potentiate GSIS. MSCs derived from ANXA1(-/-) mice had no functional capacity to enhance GSIS, in contrast to wild-type controls. Preculturing islets with ANXA1 had modest effects on their capacity to regulate blood glucose in streptozotocin-induced diabetic mice, indicating that additional MSC-derived factors are required to fully mimic the beneficial effects of MSC preculture in vivo. These findings demonstrate the feasibility of harnessing the MSC secretome as a defined, noncellular strategy to improve the efficiency of clinical islet transplantation protocols.