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345 Time-dependent potentiation of the -cell is a Ca2+-independent phenomenon S Yamada, M Komatsu, T Aizawa, Y Sato, H Yajima, T Yada1, S Hashiguchi2, K Yamauchi and K Hashizume Department of Aging Medicine and Geriatrics, Shinshu University School of Medicine, Matsumoto 390–8621, Japan 1Department of Physiology, Jichi Medical School, Minamikawachi, Kawachi, Tochigi 329–0498, Japan 2Department of 2nd Internal Medicine, Faculty of Medicine, Kagoshima University, Kagoshima 890–8520, Japan (Requests for offprints should be addressed to M Komatsu; Email: [email protected]) Abstract When isolated rat pancreatic islets are treated with However, cerulenin or tunicamycin, two inhibitors of 16·7 mM glucose, a time-dependent potentiation (TDP) protein acylation, eradicated TDP without affecting glu- of insulin release occurs that can be detected by subse- cose metabolism. The TDP by glucose was not associated quent treatment with 50 mM KCl. It has been thought with an increase in the cytosolic free Ca2+ concentration that TDP by glucose is a Ca2+-dependent phenomenon ([Ca2+]i) during subsequent treatment with high K+. and only occurs when exposure to glucose is carried out in Exposure of islets to forskolin under Ca2+-free conditions the presence of Ca2+. In contrast to this, we now did not cause TDP despite a large increase in the cellular demonstrate TDP under stringent Ca2+-free conditions cAMP levels. In conclusion, glucose alone induces TDP (Ca2+-free buffer containing 1 mM EGTA). In fact, under under stringent Ca2+-free conditions when [Ca2+]i was these Ca2+-free conditions glucose caused an even significantly lowered. Protein acylation is implicated in the stronger TDP than in the presence of Ca2+. TDP induced underlying mechanism of TDP. 2+ by glucose in the absence of extracellular Ca was Journal of Endocrinology (2002) 172, 345–354 unaffected by inhibitors of protein kinase C (PKC). Introduction kinases C (PKC) and A (PKA), by phorbol 12-myristate 13-acetate (PMA) and forskolin respectively, under strin- The pancreatic -cell is an excitable endocrine cell gent Ca2+-free conditions in the presence of EGTA that displays a variety of electrophysiological and ionic (Komatsu et al. 1995). Stimulation of insulin release by a responses upon stimulation with nutrients (Mears & combination of a nutrient and 1-oleoyl-2-acetyl-glycerol, Atwater 2000). Since the pioneering work of Grodsky and another activator of PKC, under the stringent Ca2+-free Bennett (1966), it has been accepted that extracellular conditions was also reported by others (Malaisse et al. Ca2+ is absolutely required for glucose stimulation of the 1985). This glucose stimulation of the -cell was potent -cell (Wollheim & Sharp 1981). Thus, Ca2+ influx and occurred under significantly lowered [Ca2+]i through L-type voltage-dependent Ca2+ channels and (Komatsu et al. 1995, Straub et al. 2001). Yet, pharmaco- consequent elevation of cytosolic free Ca2+ concentration logical activation of kinases, especially PKC, was a pre- ([Ca2+]i) has been thought to play an indispensable role in requisite for the Ca2+-independent glucose stimulation of -cell stimulus-secretion coupling (Wollheim & Sharp the -cell to be observed. Partly due to such requirements 1981, Komatsu et al. 1989). Moreover, glucose requires for pharmacological manoeuvres, the physiological rel- extracellular Ca2+ to increase cAMP production in the evance of the Ca2+-independent glucose action has been pancreatic -cell (Charles et al. 1975). Glucose-induced questioned (Sato et al. 1998). ionic events have now been proven to originate, for the As an extension of our efforts to establish the glucose most part, from the closure of ATP-sensitive K+ (KATP) action ‘beyond ionic events’ in the -cell (Komatsu et al. channels and the resultant membrane depolarization, 1997, Aizawa et al. 1998), identification of a novel which is due to elevation of ATP or the ATP/ADP ratio Ca2+-independent glucose action was made in the present produced by glucose metabolism (Seino et al. 2000). study. We report here that time-dependent potentiation In 1995, it was shown that glucose augments insulin (TDP) of the islet -cell by glucose is a Ca2+-independent release triggered by pharmacological activation of protein phenomenon. It had been reported previously that glucose Journal of Endocrinology (2002) 172, 345–354 Online version via http://www.endocrinology.org 0022–0795/02/0172–345 2002 Society for Endocrinology Printed in Great Britain Downloaded from Bioscientifica.com at 09/28/2021 06:00:02AM via free access + 346 S YAMADA and others · Glucose primes -cells in a Ca2 -independent manner failed to exhibit TDP under Ca2+-free conditions in the presence of EGTA (Zawalich et al. 1988, Chalmers & Sharp 1989, Taguchi et al. 1995). However, TDP occurred when the priming exposure to glucose was carried out in the presence of diazoxide (Taguchi et al. 1995), a KATP channel activator, or in the nominally Ca2+-free buffer without EGTA (Grill et al. 1978, 1979, Malaisse & Sener 1987, Taguchi et al. 1995). Furthermore, simple elevation of [Ca2+]i by high K+ or sulfonylurea does not cause TDP (Taguchi et al. 1995). Taken together, it Figure 1 Schematic presentation of the protocol used for has been considered that 1) a glucose-induced rise in evaluation of Ca2+-independent priming of insulin release. The 2+ [Ca ]i is not required for TDP, and that 2) extracellular islets were initially preincubated in Ca2+-free KRB/EGTA buffer Ca2+ concentrations ([Ca2+]o) in the micromolar range containing test substances (see text) at 37 C for 60 min and thus normal, resting [Ca2+]i are critically required for (Preincubation=priming period). Then they were stimulated with glucose-induced TDP. Contrary to these assumptions, we 50 mM KCl in regular KRB buffer containing 2·8 mM glucose and 2·5 mM CaCl2 with 250 M diazoxide (Test incubation). At the found that glucose alone, without any other stimulating end of the Test incubation, the buffer was collected for agents, time-dependently potentiated insulin release under radioimmunoassay of insulin. stringent Ca2+-free conditions. Accordingly, a detailed characterization of this novel aspect of stimulus-secretion coupling in the -cell was performed. 250 µM diazoxide, the rack(s) was placed into a 37 C water bath again and additional incubation was carried out Materials and Methods for 30 min (Test incubation). Thus, the medium used for the Test incubation was the same for all experiments, and Isolation of pancreatic islets did not contain test substances. The media change usually took 5 to 10 min. At the end of the Test incubation, the Male Wistar rats weighing 250–450 g were killed by CO2 medium was collected and kept at20 C until assayed asphyxiation. Immediately after death, the pancreases were for insulin. surgically removed, and the islets were isolated by colla- In one series of experiments, buffer with various con- genase dispersion (Lacy & Kostianovsky 1967). Krebs– 2+ ff centrations of Ca was used during Preincubation to Ringer bicarbonate (KRB) bu er containing 129 mM examine the dependency of the glucose effect on extra- NaCl, 5 mM NaHCO3, 4·8 mM KCl, 1·2 mM KH2PO4, cellular Ca2+ concentrations ([Ca2+]o). In another series of 1·2 mM MgSO4, 2·5 mM CaCl2, 5·6 mM glucose, 0·1% experiments, 8·3 mM glucose was used in place of 50 mM BSA, and 10 mM HEPES at pH 7·4 was used for isolation K+ as a secretagog during the Test incubation without and pooling of the islets. diazoxide. When the effects of cerulenin or tunicamycin, inhibi- tors of protein acylation (Schlesinger & Malfer 1982, Measurements of insulin release Patterson & Skene 1994, Jochen et al. 1995, Hurley Insulin release was measured in static incubation and et al. 2000), were examined, islets were first incubated perifusion experiments. In static incubation, batches of 5 in regular KRB buffer (2·8 mM glucose and 2·5 mM size-matched islets per tube were used. The typical CaCl2) containing various concentrations of cerulenin or experimental condition for static incubation is illustrated in tunicamycin for 30 min at 37 C. Then, after washing Fig. 1. The islets were first incubated for 60 min at 37 C with 1 ml Ca2+-free KRB/EGTA buffer containing in 1 ml Ca2+-free KRB buffer containing 129 mM NaCl, 2·8 mM glucose, incubation experiments were carried 5 mM NaHCO3, 4·8 mM KCl, 1·2 mM KH2PO4, out as described above. Cerulenin or tunicamycin was 1·2 mM MgSO4, 1 mM EGTA, and 0·2% BSA and included throughout the experiment. Knowing that pro- 10 mM HEPES at pH 7·4 (Ca2+-free KRB/EGTA longed (>3 h) exposure of the cell to tunicamycin also buffer) including the indicated concentrations of glucose causes inhibition of N-linked glycosylation of proteins and/or test substances (Preincubation). At the end of the (Kadowaki et al. 2000, Marmorstein et al. 2000), exposure preincubation, all the tubes in a rack or racks were placed to tunicamycin was limited to a maximum of 1 h. into an ice-cold water bath to stop glucose metabolism at Although cerulenin inhibits fatty acid synthase (FAS) once. Then the islets were washed with 1 ml ice-cold (Saitoh et al. 1996), little FAS activity was dected in the KRB buffer containing 2·5 mM Ca2+, 2·8 mM glucose -cell (Brun et al. 1996). Thus, cerulenin inhibition of and 250 µM diazoxide. After the introduction of 1 ml fresh FAS, if any, is not expected to significantly affect -cell KRB buffer containing a depolarizing concentration function. In the experiments with palmitate, the final (50 mM) of KCl, 2·5 mM Ca2+, 2·8 mM glucose and concentration of palmitate was 0·6 mM in the presence of Journal of Endocrinology (2002) 172, 345–354 www.endocrinology.org Downloaded from Bioscientifica.com at 09/28/2021 06:00:02AM via free access + Glucose primes -cells in a Ca2 -independent manner · S YAMADA and others 347 100 µM (0·68%) free fatty acid (FFA)-free BSA, which single cells by treatment with Ca2+-free KRB buffer with approximates to 10 µM of the unbound form of palmitate 0·1 mM EGTA.

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