Islets of Langerhans control glucose homeostasis and metabolism by sensing blood glucose and nutrients. Factors secreted within the islet create a parallel tuning, defined autocrine-paracrine signaling, that modulates hormone release and cell viability (Barker et al., 2013). Studying the molecular actors of this signaling and their physiological significance may offer help in understanding diabetes pathogenesis and in providing novel therapeutic targets. G-protein coupled receptors (GPCRs) are expressed in islets of Langerhans, regulate hormone secretion and cell survival, and are emerging as new targets for type-2 diabetes therapies. However, the type of GPCRs expressed by human islets, as well as their function, is poorly understood. In this work, we focused on the expression and role of the atypical orphan receptor GPR17 (Ciana et al., 2006), which is activated by uracil nucleotides and can be partially antagonized by ATP, a well known paracrine/autocrine signal in islet of Langerhans. We first verified the GPR17 expression in endocrine cell lines (αTC3, βTC3 and RIN14B) and in human islets, then we investigated its possible role in islet physiology. By means of RT-PCR analysis, we detected GPR17 mRNA in human islets, in α and δ cell lines, but not in β cell lines (n=5). We confirmed GPR17 protein expression in α and δ cell lines and in human islet by western blot experiments (n=4). To investigate the specific endocrine cells where GPR17 is expressed in vivo, we carried out double immunofluorescence and confocal analysis on human pancreas slices, using hormones as markers of the different cell populations and we confirmed the expression of GPR17 in a sub-population of δ cells. No expression was detected in β- cells in physiological conditions. Furthermore, chronic incubation of human islets with 16 mM glucose, known to cause β-cell death, causes a two-fold increase in GPR17 expression (p<0.05; n=4), thus indicating that the receptor may sense stress conditions. To understand the possible role of GPR17 in islet physiology we activated GPR17 with the agonist UDP-glucose, and we measured its effect on cell viability and hormone release in cell lines and human isolated islets. 3-day incubation of αTC3 with 100 µM UDP-G causes a significant increase in cell viability, measured by MTT test (108±2.56% UDP-G vs ctr, p<0.005; n= 10). A similar behavior was observed in human islets, where we found a reduction of apoptosis in presence of 100 µM UDP-G (38±7% UDP-G vs ctr). Preliminary data on hormone release in static conditions, indicate that acute incubation of human islets with 100 µM UDP-G causes a decrease in glucagon and somatostatin release in normal glucose (5 mM) (Elisa assay, 2 different islets preparations, triplicate). In conclusion, we demonstrate for the first time that GPR17 is expressed and is functional in human islets of Langerhans. Studies are in progress to better define its role in islet cell physiology and pathology.

Expression and function of the atypical purinergic receptor GPR17 in the endocrine pancreas / E. Di Cairano, V. Meraviglia, A. Ulivi, P. Rosa, S. Moretti, F. Daniele, F. Bertuzzi, S. La Rosa, V. Sacchi, C. Perego. ((Intervento presentato al convegno Physiology tenutosi a London nel 2014.

Expression and function of the atypical purinergic receptor GPR17 in the endocrine pancreas

E. Di Cairano;A. Ulivi;S. Moretti;V. Sacchi;C. Perego
2014-03-31

Abstract

Islets of Langerhans control glucose homeostasis and metabolism by sensing blood glucose and nutrients. Factors secreted within the islet create a parallel tuning, defined autocrine-paracrine signaling, that modulates hormone release and cell viability (Barker et al., 2013). Studying the molecular actors of this signaling and their physiological significance may offer help in understanding diabetes pathogenesis and in providing novel therapeutic targets. G-protein coupled receptors (GPCRs) are expressed in islets of Langerhans, regulate hormone secretion and cell survival, and are emerging as new targets for type-2 diabetes therapies. However, the type of GPCRs expressed by human islets, as well as their function, is poorly understood. In this work, we focused on the expression and role of the atypical orphan receptor GPR17 (Ciana et al., 2006), which is activated by uracil nucleotides and can be partially antagonized by ATP, a well known paracrine/autocrine signal in islet of Langerhans. We first verified the GPR17 expression in endocrine cell lines (αTC3, βTC3 and RIN14B) and in human islets, then we investigated its possible role in islet physiology. By means of RT-PCR analysis, we detected GPR17 mRNA in human islets, in α and δ cell lines, but not in β cell lines (n=5). We confirmed GPR17 protein expression in α and δ cell lines and in human islet by western blot experiments (n=4). To investigate the specific endocrine cells where GPR17 is expressed in vivo, we carried out double immunofluorescence and confocal analysis on human pancreas slices, using hormones as markers of the different cell populations and we confirmed the expression of GPR17 in a sub-population of δ cells. No expression was detected in β- cells in physiological conditions. Furthermore, chronic incubation of human islets with 16 mM glucose, known to cause β-cell death, causes a two-fold increase in GPR17 expression (p<0.05; n=4), thus indicating that the receptor may sense stress conditions. To understand the possible role of GPR17 in islet physiology we activated GPR17 with the agonist UDP-glucose, and we measured its effect on cell viability and hormone release in cell lines and human isolated islets. 3-day incubation of αTC3 with 100 µM UDP-G causes a significant increase in cell viability, measured by MTT test (108±2.56% UDP-G vs ctr, p<0.005; n= 10). A similar behavior was observed in human islets, where we found a reduction of apoptosis in presence of 100 µM UDP-G (38±7% UDP-G vs ctr). Preliminary data on hormone release in static conditions, indicate that acute incubation of human islets with 100 µM UDP-G causes a decrease in glucagon and somatostatin release in normal glucose (5 mM) (Elisa assay, 2 different islets preparations, triplicate). In conclusion, we demonstrate for the first time that GPR17 is expressed and is functional in human islets of Langerhans. Studies are in progress to better define its role in islet cell physiology and pathology.
GPCR; islets of Langerhans; GPR17
Settore BIO/09 - Fisiologia
Physiological society
Expression and function of the atypical purinergic receptor GPR17 in the endocrine pancreas / E. Di Cairano, V. Meraviglia, A. Ulivi, P. Rosa, S. Moretti, F. Daniele, F. Bertuzzi, S. La Rosa, V. Sacchi, C. Perego. ((Intervento presentato al convegno Physiology tenutosi a London nel 2014.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/2434/252496
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