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Beta-cell Receptors in Diabetes Therapy

Final Report Summary - BRIDIT (Beta-cell Receptors in Diabetes Therapy)

Publishable summary
The main objectives of this MCIEF were to investigate the role of the G-protein coupled receptors (GPCRs) FFAR2 and FFAR3 in regulating β-cell function in response to short-chain fatty acids (SCFAs), with a particular focus on the regulation of mouse and human islet hormone secretion and the impact on β-cell apoptosis and proliferation. The contribution of other islet-expressed GPCRs to the regulation of islet hormone secretion was a subsidiary aim of the project. To achieve these tasks, the fellow’s initial skills in isolation of mouse islets of Langerhans and measurements of cell function were widened in order for him to become proficient in the assessment of dynamic islet hormone secretion, quantification of islet second messenger generation and identification of β-cell proliferation following in vivo BrdU delivery (months 0-3).
Since the beginning of the project the fellow has performed several experiments aimed to:
1. Investigate the role of short-chain fatty acids in mouse and human islets in terms of hormone secretion (i) and impact on β-cell apoptosis and proliferation (ii) [months 4-24]; 2. identify and elucidate the role of other GPCRs and their ligands in mouse and human islets such as GPR55 (i), GPR56 (ii), Peptide YY (iii) [months 13-24].
In agreement with the spirit of this MCIEF to improve and potentiate the abilities of the awarded fellow, Dr. Pingitore has also participated in training courses for personal and professional development, teaching and mentoring activities, international conferences as well as outreach activities, for all the duration of his fellowship.
1. Investigation of the role of short-chain fatty acids in mouse and human islets (months 4-24)
i. Hormone secretion. The effects of SCFAs on insulin secretion from isolated mouse and human islets were investigated and acetate (2C) and propionate (3C) were identified as being able to potentiate glucose-induced insulin secretion from human islets (Figure 1a and 1b). This stimulatory effect most likely occurs via activation of FFAR2 since FFAR3, the other GPCR that is activated by SCFAs, couples to pathways that inhibit insulin secretion. This led to a focus on the role of FFAR2 in mediating SCFA stimulation of insulin release, providing further support for the use of this receptor system as a pharmacological target. Data generated using the FFAR2 knockout mouse model (FFAR2-/-) support this conclusion as the stimulatory effects mediated by acetate and propionate are lost in islets isolated from mice lacking FFAR2, while there is no reduction in insulin content of FFAR2-/- islets (Figure 2). Single cell microfluorimetry techniques were applied to complement the insulin secretion data that implicate SCFAs acting in β-cells at FFAR2, which is coupled to Gq to mobilise intracellular Ca2+. In these experiments stimulation of Fura-2-loaded human and mouse islets with 100µM-1mM of acetate or propionate, in the presence of 20mM glucose, resulted in an increase in the intracellular concentration of free Ca2+ (Figure 3). This increase in Ca2+ was lost in islets isolated from FFAR2-/- mice (Figure 4), consistent with a role for Gq-coupled FFAR2 activation in SCFA-induced elevations in β-cell calcium. Ca2+ events that are Gq-triggered induce the activation of PLC to generate IP3, which mobilises Ca2+ from intracellular stores, and DAG, which activates PKC. We have used inhibitors of PLC (U73122) and depletion of PKC from islets pharmacologically to analyse the role played by these effectors on SCFA-mediated Ca2+ mobilisation. As shown in Figure 5a, treatment with U73122 prevents the potentiation of glucose-induced insulin secretion by both acetate in human islets. Similarly, depletion of PKC by exposure of islets to 200nM PMA for 24h, abrogates both the increase in insulin secretion triggered by acetate and propionate in human islets (Figure 5b, c ). Both FFAR2 and FFAR3 have been reported to couple to Gi to inhibit cAMP generation. While FFAR3 is only Gi coupled FFAR2 also has a Gq component, as confirmed by the stimulatory effects of SCFAs on insulin secretion and [Ca2+]i that are lost following FFAR2 deletion. Our findings support the hypothesis that FFAR2 preferentially signals via Gq. To further support this, human islets were exposed to acetate (used as lead compound for SCFAs) after being treated with the pertussis toxin, that abrogates Gi signalling. It is clearly shown in figure 6a, that no Gi activity is recruited upon treatment with acetate, as no differences exists between PTX-treated islets exposed or not to 1mM acetate, in terms of potentiation of insulin secretion. However, it was important to effectively quantify intracellular islet cAMP levels in response to SCFAs and this was carried out using a cAMP ELISA, which had been set up by the fellow during his first year. No reduction of cAMP was observed upon treatment of human islets with SCFAs (Figure 6b). Of course the presence of FFAR2 in human islets was verified by western blotting and its specific localisation within the distinct islet cell populations (and β-cell in particular) was achieved via immunohistochemistry (Figure 7).
iii. β-cell apoptosis and proliferation. The research fellow has conducted experiments aimed at determining whether acetate and/or propionate protects islets from cytokine- and palmitate-induced apoptosis, in vitro, by using a luminescence assay that quantifies caspase 3/7 activities. He generated a large amount of data in his second year to support a role for SCFAs in promoting survival of human islets (Figure 8a and b). This anti-apoptotic feature of SCFAs appears to be strictly mediated by FFA2 as demonstrated by islets isolated from FFAR2-/- mice, where the protective effects of acetate and propionate is lost. (Figure 8c and d).

2. Investigate the role of other GPCRs and their ligands in mouse and human islets (months 13-24)
The project was extended to investigate other islet GPCRs and ligands that were previously identified in the host laboratory as being highly expressed by both mouse and human islets, and for which a specific role in terms of hormone secretion had not yet been defined. A particular focus was on the signalling downstream of GPR55, GPR56 and peptide YY (PYY) in isolated islets.
i) GPR55 and the cannabinoid receptor system: Activation of GPR55 is regulated by endogenous and pharmacological cannabinoid ligands, and this GPCR has been proposed as a novel cannabinoid receptor. We have found that activation of GPR55 is coupled to stimulation of insulin secretion so we have aimed at further characterising the role of GPR55 in islet physiology, in terms of intracellular signaling downstream its activation and its role in protecting islets from cell death. The use of a GPR55-/- mouse model in the host laboratory has allowed discrimination between GPR55-dependent and -independent mechanisms upon stimulation with pharmacological agonists belonging to the cannabinoid family. In particular, in collaboration with Dr. Ruz-Maldonado and Dr. Liu, the fellow has characterized the Gq-dependent coupling of GPR55 activation to changes in [Ca2+]i via microfluorimetry using the selective GPR55 agonist O-1602 (figure 9 a-d) and has identified LH-21 as a GPR55 agonist in islets that potentiates glucose-dependent insulin secretion (figure 10) and protects islets from undergoing apoptosis (figure 11).
ii) PYY: Peptide YY (PYY) is a 36 amino acid peptide synthesised by enteroendocrine L-cells in the distal gut. It is also present in the pancreas, but its role in this organ is not well understood. PYY exists endogenously in two forms: PYY1-36 and PYY3-36 both of which act via the neuropeptide Y family of GPCRs: PYY1-36 binds with similar affinity to Y1, Y2, Y4 and Y5, while PYY3-36 is selective for the Y2 receptor subtype. Our lab had previously demonstrated that ablation of PYY-expressing cells in the adult mouse causes a catastrophic disruption of islet architecture and β-cell destruction, with consequent development of insulin-dependent diabetes mellitus. The synthesis of PYY by islet cells and expression of its receptors by islets suggests a potential paracrine role for this peptide in regulating islet function. Dr. Pingitore therefore conducted experiments aimed at characterising the insulin secretory responses and insulin content of islets isolated from PYY-/- mice available in the host laboratory. As can be seen in Figure 12a, dynamic insulin secretion under glucose challenge in PYY-/- is significantly reduced compared to secretion of islets from age- or weight-matched wild-type mice. This reduction was evident both in terms of first and second phases of insulin secretion, and also when the muscarinic agonist carbachol is used, that potentiates the glucose-induced insulin exocytosis via activation of a Gq-coupled GPCR. Considering the difference in terms of the quantity of insulin being secreted, the islet content of insulin was measured, and this indicated that the PYY-/- islets contain less insulin than wild-type ones (Fig 12b). This aspect could be partially responsible for the diminished secretory capability of PYY-/- islets, confirming the importance of this peptide in terms of proper development of the pancreas.
iii) GPR56: GPR56 is a member of the adhesion class GPCRs. It possesses an exceptionally long N-terminal extracellular domain of 393 residues that contributes to its cell adhesion property. The host laboratory has demonstrated that it is the most abundant GPCR mRNA in human and mouse islets and it may therefore serve as a key modulator of islet functions. Nothing is known about its function in islets but in the brain its endogenous ligand is the extracellular matrix protein collagen III and it is G12/13 coupled to activate RhoA signalling. GPR56 is also coupled to Gq in the presence of CD81. Since collagen III is also present within the islet environment, it is possible that GPR56 may be activated in a paracrine manner to regulate islet functions. The fellow has worked in collaboration with Edward Olaniru, a final year PhD in the host laboratory to identify the role of collagen III in insulin release from islets and intracellular signalling, making use of islets isolated from a GPR56-/- model available through a collaborating institution at Harvard (USA). This study is still ongoing, but Dr. Pingitore demonstrated that deletion of GPR56 causes a small reduction in glucose-induced insulin secretion, but paradoxically increases glucose-stimulated elevation in intracellular calcium (Figure 13). There is no alteration in insulin content following deletion of GPR56.

Taken together, the data generated by Dr. Pingitore in his 24 month fellowship, demonstrate a role for SCFAs in glucose homeostasis via direct stimulaton of FFAR2 (GPR43) receptors on islets of Langerhans. His extensive work making use of isolatd human islets, sheds light on the ability of acetate and propionate to potentiate insulin secretion, to protect islets from cytotoxic and lipotoxic insults, which are all hallmarks of a succesfull therapy for type 2 diabetes. His results obtained with the FFAR2-/- mouse model confer an important role to FFAR2 in mediating the effects of acetate and propionate, thus identifying this receptor as a potential target for therapeutic intervention. His studies on other GPCRs and their related ligands, provide further information on the importance of this class of receptors as potential pharmacological targets in the treatment of diabetes, and the studies with GPR55 in particular suggest that this is a receptor that should be the focus of future studies.