Potential role of the CD38/cADPR signaling pathway as an underlying mechanism of the effects of medetomidine on insulin and glucose homeostasis

Transcription

1 Veternary Anaesthesa and Analgesa, 2013, 40, do: /vaa SHORT COMMUNICATION Potental role of the CD38/cADPR sgnalng pathway as an underlyng mechansm of the effects of medetomdne on nsuln and glucose homeostass Alonso GP Guedes*, Elane P Rude* & Mathur S Kannan *Veternary Clncal Scences Department, College of Veternary Medcne, Unversty of Mnnesota, St. Paul, MN, USA Department of Veternary and Bomedcal Scences, College of Veternary Medcne, Unversty of Mnnesota, St. Paul, MN, USA Correspondence: Alonso GP Guedes, Department of Surgcal & Radologcal Scences, School of Veternary Medcne, Unversty of Calforna, 2112 Tupper Hall, One Shelds Avenue, Davs, CA 95616, USA. E-mal: aguedes@ucdavs.edu Abstract Objectve To nvestgate the CD38/cADPR sgnalng pathway as possble underlyng mechansm of the effects of medetomdne on nsuln and glucose homeostass. Anmals Thrty-two C57BL/6 mce of both sexes. Methods Wld-type (WT) and CD38-knockout (CD38 / ) mce receved medetomdne (50 lg kg 1 ) or a smlar volume of 0.9% NaCl (control) by ntrapertoneal (IP) njecton (each group n = 8). The mce were euthanzed 45 mnutes later wth sodum pentobarbtal IP and blood was sampled va cardac puncture. Insuln and glucose concentratons were measured by radommunoassay and by the oxygen rate method, respectvely. Data were analyzed wth ANOVA and Bonferron post hoc (5% sgnfcance) and are shown as mean SD. Results Plasma nsuln and glucose concentratons were smlar between WT and CD38 / mce under control condtons. As compared to controls, medetomdne admnstraton produced a statstcally sgnfcant decrease n plasma nsuln concentratons n the WT mce whereas the decrease n the CD38 / mce was not statstcally sgnfcant. Correspondngly, medetomdne caused a sgnfcantly greater ncrease n plasma glucose concentratons n the WT than n the CD38 / mce. Concluson The CD38/cADPR sgnalng pathway may be one underlyng mechansm of the glucose and nsuln effects of the alpha-2 adrenergc receptor agonst medetomdne and lkely other drugs of ts class. Keywords alpha-2 agonsts, dabetes melltus, hyperglycema, nsulnoma, pancreas. Introducton Under physologc condtons, nsuln release from pancreatc b-cells s ntated by ncreases n ntracellular ATP levels as glucose s metabolzed (Fg. 1). Ths leads to ncreases n ntracellular calcum concentratons ([ ] ) va two separate pathways. One nvolves extracellular nflux through voltage-dependent channels that are actvated by changes n plasma membrane potental due to nhbton of ATP-senstve K + channels (Ashcroft et al. 1988). The other nvolves ntracellular release va ryanodne receptors (RyR) that are actvated by cyclc ADP-rbose (cadpr) as t accumulates ntracellularly secondary to ncreasng ATP levels. In the pancreas, cadpr s both syntheszed and degraded by CD38, a 45-kDa type II transmembrane glycoproten. Intracellular accumulaton of 512

2 Mechansm of medetomdne on nsuln secreton AGP Guedes et al. ADPR NAD + Medetomdne CD38 CNT TRP ATP AC Cx43 K ATP VOCC camp PKA CICR ADP ATP PKC NAD + ER CaM Knase II -dependent nsuln secreton Stmulates Antagonzes Fgure 1 Model detalng the two pathways nvolved n glucose-nduced nsuln release n b-cells and possble subcellular mechansm of the nhbtory effect of medetomdne on nsuln secreton. Both pathways begn wth ncreases n ntracellular ATP concentratons followng glucose metabolsm. In one pathway, depcted to the left, ATP-senstve K + channels (K ATP ) are nhbted thus ncreasng membrane potental wth subsequent actvaton of voltage-dependent channels (VOCC) and extracellular nflux whch leads to nsuln secreton. The other pathway, depcted to the rght, requres cyclc ADPrbose (cadpr), a calcum-sgnalng molecule syntheszed from b-ncotnamde adenne dnucleotde (b-nad) by the ADPrbosyl cyclase CD38 (cyclase actvty), and degraded to ADP-rbose (ADPR) by the same enzyme through ts cadpr hydrolase actvty. Here, ATP produced by glucose metabolsm prevents cadpr degradaton by compettvely nhbtng the hydrolase actvty of CD38. Ths leads to extracellular accumulaton of cadpr, whch reaches the ntracellular compartment va concentratve nucleosde transporters (CNT) or va CD38 tself to subsequently actvate ryanodne receptors (RyR) n the endoplasmc retculum (ER) and produce release. Ths then trggers further release va calcum-nduced calcum release (CICR) mechansms that, n concert wth extracellular nflux va cadpr-actvated transent receptor potental melastatn-2 (TRPM2) channels, ultmately culmnates n nsuln secreton. Crtcal n ths process s the phosphorylaton and senstzaton of RyR by /calmoduln-dependent proten knase II (CaM knase II) and cyclc AMP-dependent proten knase (PKA). The CD38/cADPR pathway s termnated when calcum-dependent proten knase (PKC) phosphorylates conexn 43 (Cx43) hemchannels preventng the substrate b-nad from reachng the extracellular catalytc doman of CD38. A possble crosstalk mechansm between medetomdne and the CD38/cADPR system may nvolve downregulaton of PKA as stmulaton of alpha-2-ar actvates nhbtory G-protens (Ga), whch nhbts adenylyl cyclase (AC) actvty, camp producton and, consequently, actvaton of proten PKA. Hence, medetomdne could effectvely prevent PKA-medated phosphorylaton of ryanodne receptors (RyR), thereby precludng the crtcally mportant senstzaton of the RyR to the cadpr sgnal. ATP nhbts the degradaton pathway resultng n accumulaton of cadpr (Takasawa et al. 1998; Okamoto & Takasawa 2002). Medetomdne s a hghly selectve alpha-2-adrenergc receptor (AR) agonst wth strong sedatve, analgesc and neurohormonal propertes. The antagonsm of nsuln secreton and elevaton n blood glucose levels caused by alpha-2-ar agonsts are well recognzed but the underlyng mechansms have not been fully elucdated. Inhbton of nsuln release 2013 The Authors. Veternary Anaesthesa and Analgesa 2013 Assocaton of Veternary Anaesthetsts and the Amercan College of Veternary Anesthesa and Analgesa, 40,

3 Mechansm of medetomdne on nsuln secreton AGP Guedes et al. followng actvaton of alpha-2 A and alpha-2 C AR subtypes occurs va dstnct sgnalng pathways. Stmulaton of alpha-2 A AR subtypes, whch are coupled to nhbtory G-protens (Ga), cause reducton n extracellular nflux due to plasma membrane hyperpolarzaton caused by decreases n camp producton (Peterhoff et al. 2003). The downstream mechansm followng actvaton of the alpha- 2 C AR subtype remans uncertan but occurs through a pathway not nvolvng adenylyl cyclase or changes n membrane potental (Peterhoff et al. 2003). Ths mechansm may nvolve the CD38/cADPR sgnalng pathway gven ts promnent role n nsuln secreton (Kato et al. 1995, 1999) and ts crosstalk wth G- proten-coupled receptors n many cell types, ncludng pancreatc b-cells (Bruzzone et al. 2008). The goal of ths study was to explore the possblty that the CD38/cADPR sgnalng system may represent a mechansm downstream from alpha-2-ar underlyng the effects of medetomdne n nsuln and glucose homeostass. Materal and methods The study protocol and expermental procedures were revewed and approved by the Insttutonal Anmal Care and Use Commttee of the Unversty of Mnnesota. Thrty-two specfc pathogen-free, week-old, male and female C57BL/6J wld-type (WT) and CD38-knockout [CD38 / ] mce (backcrossed 12 generatons to C57BL/6J) obtaned through n-house breedng were used n ths study. The orgnal breedng pars were purchased from Jackson Laboratores (ME, USA). They were housed n a 12-hour lght-dark schedule wth food and water avalable ad lbtum. Wld type and CD38 / mce receved medetomdne (50 lg kg 1 ; Pfzer Anmal Health, PA, USA; each group n = 8) or a smlar volume of 0.9% NaCl (each group n = 8) by ntrapertoneal (IP) njecton, and were euthanzed 45 mnutes later wth an IP njecton of sodum pentobarbtal (180 mg kg 1 ; Butler, OH, USA). Food and water were not wthheld pror to the experments. Blood (approxmately 1.0 ml) was collected mmedately after euthanasa through cardac puncture and placed nto tubes contanng EDTA. The blood was spun mmedately and the plasma harvested and stored at 80 C for later nsuln and glucose measurements. Insuln was measured va radommunoassay (LINCO Research, MO, USA) by the Cornell Unversty Anmal Health Dagnostc Center accordng to the laboratory standard operatng procedures. The senstvty of the assay s 0.1 ng ml 1. Glucose was measured by the oxygen rate method usng a glucose oxygen electrode (Beckman Coulter, Inc., CA, USA) by the Veternary Clncal Pathology Laboratory of the Veternary Medcal Center, Unversty of Mnnesota, accordng to standard laboratory procedures. Statstcal analyses were performed usng GRAPHPAD Prsm verson 5.0c for MAC OS (GraphPad Software, CA, USA). One-way ANOVA was used to analyze the data between the groups. When sgnfcant dfferences were detected, the data were submtted to the Bonferron post-test. Sgnfcance level was set at p < 0.05 and, unless otherwse noted, the data are presented as mean SD. Results After admnstraton of 0.9% NaCl (control), plasma nsuln concentratons were ng ml 1 n the WT and ng ml 1 n the CD38 / mce. These values were not sgnfcantly dfferent. After admnstraton of medetomdne, plasma nsuln concentratons were ng ml 1 n the WT and ng ml 1 n the CD38 / mce. On average, plasma nsuln was 67% and 43% lower n the WT and CD38 / mce, respectvely. Compared to controls, medetomdne admnstraton sgnfcantly decreased plasma nsuln concentratons n the WT mce. The decrease n the CD38 / mce was not enough to reach statstcal sgnfcance. After admnstraton of 0.9% NaCl, plasma glucose concentratons were mg dl 1 n the WT and mg dl 1 n the CD38 / mce. These values were not sgnfcantly dfferent. After admnstraton of medetomdne, plasma glucose concentratons were mg dl 1 n the WT and mg dl 1 n the CD38 / mce. In the mce treated wth medetomdne, plasma glucose concentratons were sgnfcantly hgher n the WT mce (mean 47% ncrease) compared to the CD38 / mce (mean 19% ncrease). Compared to controls, plasma glucose concentraton was sgnfcantly hgher after medetomdne admnstraton n the WT mce whereas t ncreased slghtly, statstcally not sgnfcantly, n the CD38 / mce. Dscusson The goal of ths study was to nvestgate a possble role of the CD38/cADPR sgnalng system as a The Authors. Veternary Anaesthesa and Analgesa 2013 Assocaton of Veternary Anaesthetsts and the Amercan College of Veternary Anesthesa and Analgesa, 40,

4 Mechansm of medetomdne on nsuln secreton AGP Guedes et al. subcellular mechansm underlyng the effects of medetomdne on nsuln and glucose homeostass. The physologcal role of the CD38/cADPR sgnalng n nsuln secreton and glucose homeostass has been shown n studes usng CD38 / and CD38- overexpressed mce (Kato et al. 1995, 1999). Plasma nsuln and glucose concentratons were smlar n CD38 / and WT mce when measured after salne admnstraton, and were largely smlar to values reported prevously n mce of the same genotype and genetc background (Kato et al. 1999). Some varaton n the plasma concentratons of nsuln and glucose was evdent probably because of stress assocated wth handlng and IP njectons. The presence of an ntact CD38/cADPR sgnalng system resulted n greater effect of medetomdne upon nsuln secreton, suggestng a functonal crosstalk between ths sgnalng pathway and the alpha-2-ar. The specfc pathway lnkng the two sgnalng systems s not known but proten knase A (PKA) s one lkely canddate gven ts promnent role n senstzng the RyR to cadpr (Okamoto & Takasawa 2002) and ts downregulaton by alpha- 2-AR agonsts (Correa-Sales et al. 1992). It may also ental ndrect reducton n the CD38 cyclase actvty, thus decreasng cadpr formaton, va a PKAdependent mechansm (Bruzzone et al. 2008). Whle the reducton n plasma nsuln concentratons n the CD38 / mce was not large enough to reach statstcal sgnfcance, t ndcates the presence of addtonal operatve mechansms for ths endocrne acton of medetomdne. Elucdaton of such mechansm was beyond the scope of the present nvestgaton, but may nvolve nhbton of medators of ntracellular release (e.g., PKA, /calmoduln-dependent proten knase II) or extracellular nflux (ATP-senstve potassum channels or K ATP channels, transent receptor potental melastatn 2 channels or TRPM2 channels) snce both mechansms are mportant for nsuln secreton, although they have also been drectly or ndrectly lnked to cadpr (Ashcroft et al. 1988; Okamoto & Takasawa 2002; Togash et al. 2006; Bruzzone et al. 2008). Interacton wth CD157, another enzyme wth homology to CD38 expressed n pancreatc slet cells, s less lkely snce t possesses cyclase and hydrolase actvtes n acdc but not at physologc ph and thus s mprobable to partcpate n nsuln secreton n vvo (Furuya et al. 1995). The fact that cadpr loss s not complete n CD38 / mce mght suggest that CD157 and/or yet undentfed members of the famly compensate for the absence of CD38. However, n pancreatc b-cells, loss of cadpr-medated nsuln secreton s functonally replaced by the nostol trsphosphate (IP 3 ) system (Okamoto & Takasawa 2002). A model depctng the role of CD38/cADPR n nsuln release and possble crosstalk mechansms between medetomdne and to nhbt nsuln release n b-cells s present n Fg. 1. A moderate but sgnfcant ncrease n plasma glucose concentratons followng medetomdne admnstraton was observed n WT mce whereas only a mld, non-sgnfcant ncreased occurred n CD38 / mce. The dstnctve effects of medetomdne on plasma glucose concentratons between WT and CD38 / mce s lkely due to ts effects on nsuln secreton rather than an effect on the perpheral actons of nsuln (.e., glucose uptake). In the CD38 / mce, glucose ntolerance results from attenuaton of glucose-nduced nsuln release rather than alteraton n perpheral nsuln resstance, and ths phenotype can be rescued by targeted expresson of CD38 cdna n b-cells (Kato et al. 1995, 1999). Functonally, the results of the present study provde ndrect evdence that the CD38/ cadpr sgnalng system may have a role on the actons of medetomdne upon nsuln and glucose homeostass. In concluson, the results presented here suggest that the CD38/cADPR sgnalng pathway may be an underlyng mechansm for the effect of medetomdne on nsuln secreton and glucose homeostass. Acknowledgements Ths study was funded by the Small Companon Anmal Research Grants of the Veternary Research and Graduate Program, College of Veternary Medcne, Unversty of Mnnesota. References Ashcroft FM, Ashcroft SJ, Harrson DE (1988) Propertes of sngle potassum channels modulated by glucose n rat pancreatc beta-cells. J Physol 400, Bruzzone S, Bodrato N, Usa C et al. (2008) Abscsc acd s an endogenous stmulator of nsuln release from human pancreatc slets wth cyclc ADP rbose as second messenger. J Bol Chem 283, Correa-Sales C, Nacf-Coelho C, Red K et al. (1992) Inhbton of adenylate cyclase n the locus coeruleus medates the hypnotc response to an alpha 2 agonst n the rat. J Pharmacol Exp Ther 263, Furuya Y, Takasawa S, Yonekura H et al. (1995) Clonng of a cdna encodng rat bone marrow stromal cell 2013 The Authors. Veternary Anaesthesa and Analgesa 2013 Assocaton of Veternary Anaesthetsts and the Amercan College of Veternary Anesthesa and Analgesa, 40,

5 Mechansm of medetomdne on nsuln secreton AGP Guedes et al. antgen 1 (BST-1) from the slets of Langerhans. Gene 165, Kato I, Takasawa S, Akabane A et al. (1995) Regulatory role of CD38 (ADP-rbosyl cyclase/cyclc ADP-rbose hydrolase) n nsuln secreton by glucose n pancreatc beta cells. Enhanced nsuln secreton n CD38-expressng transgenc mce. J Bol Chem 270, Kato I, Yamamoto Y, Fujmura M et al. (1999) CD38 dsrupton mpars glucose-nduced ncreases n cyclc ADP-rbose, [Ca2+], and nsuln secreton. J Bol Chem 274, Okamoto H, Takasawa S (2002) Recent advances n the Okamoto model: the CD38-cyclc ADP-rbose sgnal system and the regeneratng gene proten (Reg)-Reg receptor system n beta-cells. Dabetes 51(Suppl 3), S462 S473. Peterhoff M, Seg A, Brede M et al. (2003) Inhbton of nsuln secreton va dstnct sgnalng pathways n alpha2-adrenoceptor knockout mce. Eur J Endocrnol 149, Takasawa S, Akyama T, Nata K et al. (1998) Cyclc ADPrbose and nostol 1,4,5-trsphosphate as alternate second messengers for ntracellular Ca2 + moblzaton n normal and dabetc beta-cells. J Bol Chem 273, Togash K, Hara Y, Tomnaga T et al. (2006) TRPM2 actvaton by cyclc ADP-rbose at body temperature s nvolved n nsuln secreton. EMBO J 25, Receved 4 June 2012; accepted 22 August The Authors. Veternary Anaesthesa and Analgesa 2013 Assocaton of Veternary Anaesthetsts and the Amercan College of Veternary Anesthesa and Analgesa, 40,