Fig 1.
ATC induces insulin secretion via cADPR/NAADP-dependent Ca2+ signals in pancreatic β cell.
(A) Representative tracing of the Ca2+ response to ATC (400 μM), Arginine (400 μM) (AG), Thiazolidine-2-carboxylic acid (400 μM) (T2C) and T2C + Arg treatments. (B) Representative tracing of the Ca2+ response to ATC (400 μM) and OTC (1 mM) treatments. (C) Comparisons of NAADP formation among ATC, OTC, Arg, T2C and T2C + AG treatment. (D) Comparisons of cADPR formation among ATC, OTC, Arg, T2C and T2C + Arg treatment. (E) Comparisons of insulin secretion among ATC, OTC, Arg, T2C and T2C + AG treatment. (F) Blood glucose levels in vehicle (closed circle, n = 5)- and ATC (5 mg/kg; open circle, n = 5, 10 mg/kg; closed triangle, n = 5, 20 mg/kg; open triangle, n = 5)-treated db/db mice following intraperitoneal injection of glucose after overnight fasting. (G) Plasma insulin levels during intraperitoneal glucose tolerance testing in vehicle (closed circle, n = 5)- and ATC (5 mg/kg; open circle, n = 5, 10 mg/kg; closed triangle, n = 5, 20 mg/kg; open triangle, n = 5)-treated db/db mice. *, P<0.05 versus CON level. #, P<0.05 versus ATC treated level. All data are expressed as the Mean ± SEM.
Fig 2.
ATC-induced NAADP and cADPR formation and involvement of SOCE in ATC-induced Ca2+ signaling in pancreatic β cell.
(A) Time course of NAADP and cADPR production following ATC treatment (B) Effect of Ca2+ second messenger inhibitors on ATC-induced Ca2+ signals. XesC (2 μM), Ned19 (100 μM) and 8-Br-cADPR (100 μM) were used. (C) Representative tracings of the Ca2+ response to ATC in the absence and presence of extracellular Ca2+. (D) Representative tracings of the Ca2+ response to ATC in the presence of SKF 96365 (10 μM) (E and F) Effect of Ca2+ second messenger inhibitors on ATC-induced cADPR and NAADP formation. (G) Effect of Ca2+ second messenger inhibitors on ATC-induced insulin secretion. *, P<0.05 versus CON level. #, P<0.05 versus ATC treated level. All data are expressed as the Mean ± SEM.
Fig 3.
ATC-induced NAADP and cADPR formation in a cAMP-dependent manner in pancreatic β cell.
(A) Effect of H89 (10 μM) on ATC-induced Ca2+ signals. (B) Effect of Ca2+ second messenger inhibitors on ATC-induced cAMP formation. (C and D) Effect of H89 and Rp-cAMP (100 μM) on ATC-induced cADPR and NAADP formation. (E) Effect of Ca2+ second messenger inhibitors and cAMP antagonist on ATC-induced GSH formation. (F-H) Effect of GSH inhibitor, Diehtyl Maleate (DEM) (50 μM) on ATC-induced formation of cAMP, cADPR and NAADP. (I) Inhibitory effect of cAMP antagonists or DEM on ATC-induced insulin secretion. *, P<0.05 versus CON level. #, P<0.05 versus ATC treated level. All data are expressed as the Mean ± SEM.
Fig 4.
NOS involves in ATC-induced cADPR formation but not in NAADP formation in pancreatic β cell.
(A) Effect of L-NG-Nitroarginine Methyl Ester (L-NAME) (5 mM) on ATC-induced Ca2+ signal. (B) Effect of L-NAME and Ca2+ second messenger inhibitors on ATC-induced nitrite formation. (C) Effect of cGMP antagonist, (Rp)-8-pCPT-cGMPS (20 μM) on ATC-induced NO formation. (D and E) Effect of L-NAME and cGMP antagonist on ATC-induced cADPR and NAADP formation. (F) Effect of L-NAME and cGMP antagonists on ATC-induced insulin secretion. *, P<0.05 versus CON level. #, P<0.05 versus ATC treated level. All data are expressed as the Mean ± SEM.
Fig 5.
nNOS plays a major role in ATC-induced Ca2+ signaling and insulin secretion in pancreatic β cell.
(A) Effect of nNOS inhibitor, ARL17477 (30 μM) and iNOS inhibitor, 1400W (100 μM) on ATC-induced Ca2+ signals. (B-D) Effect of nNOS and iNOS inhibitors on ATC-induced NO, cADPR, NAADP formation. (E) Effect of nNOS knock down (KD) on ATC-induced NO formation. (inset) Representative immunoblots for quantifications of nNOS protein expression in pancreatic β cell after infection with lentiviral particles expressing scrambled or nNOS-specific short hairpin (shRNA). (F) Effect of nNOS KD on ATC-induced Ca2+ signal. (G and H) Effects of nNOS KD on ATC-induced cADPR and NAADP formation. (I) Effect of nNOS inhibitors and nNOS KD on ATC-induced insulin secretion. *, P<0.05 versus CON level. #, P<0.05 versus ATC treated level. All data are expressed as the Mean ± SEM.
Fig 6.
ATC-induced Ca2+ signals, cADPR and NAADP production, and insulin secretion in pancreatic β cell from wild-type (WT) and CD38 knock-out (KO) mice.
(A) Representative tracings of the Ca2+ response to ATC in pancreatic β cell prepared from WT and CD38 KO mice. (B and C) ATC-stimulated NAADP and cADPR formation in WT and CD38 KO mice. (D and E) ATC-stimulated cAMP and NO formation in WT and CD38 KO mice. (F) ATC-stimulated insulin secretion in WT and CD38 KO mice. *, P<0.05 versus CON level. #, P<0.05 versus ATC treated level. All data are expressed as the Mean ± SEM.
Fig 7.
Schematic representation of ATC-induced insulin secretion via cADPR and NAADP production as well as role of NO in pancreatic β cell.
Arginine Thiazolidine Carboxylate (ATC) enters and is divided into TC and arginine. TC contributes for Glutathione (GSH) formation, which stimulates adenylyl cyclase, resulting in the production of cAMP. cAMP/PKA activates NSE to produce NAADP, releasing Ca2+ from lysosome-related acidic organelles. NAADP-mediated increase of intracellular Ca2+ levels results in the activation of NOS. At this moment, arginine is provided as a substrate for Nitric Oxide syntase (NOS). Resulting Nitric Oxide (NO) synthesis activate guanylyl cyclase (GC)/protein kinase G (PKG). PKG activates CD38 to produce cADPR. cADPR-mediated Ca2+ release from the ER Ca2+ stores. cADPR-mediated Ca2+ release regulates the Ca2+ influx through store-operated Ca2+ entry (SOCE), resulting in insulin secretion in pancreatic β cells. GLP-1, an insulin secretion inducing hormone, also uses similar Ca2+ signalling pathway for insulin secretion in pancreatic β cells.