Defective glucose-dependent cytosolic Ca2+ handling in islets of GK and nSTZ rat models of type 2 diabetes

We examined to what extent the abnormal glucose-dependent insulin secretion observed in NIDDM (non-insulin-dependent diabetes mellitus) is related to alterations in the handling of cytosolic Ca2+ of islets of Langerhans. Using two recognized rat models of NIDDM, the GK (Goto-Kakizaki) spontaneous model and the nSTZ (neonatal streptozotocin) induced model, we could detect several common alterations in the glucose-induced [Ca2+]i cytosolic responses. First, the initial reduction of [Ca2+]i following high glucose (16.7 mM) observed routinely in islets obtained from non-diabetic Wistar rats could not be detected in GK and nSTZ islets. Second, a delayed response for glucose to induce a subsequent 3% increase of [Ca2+]i over basal level was observed in both GK (321+/-40 s, n=11) and nSTZ (326+/-38 s, n=13) islets as compared with Wistar islets (198+/-20 s, n=11), values representing means+/-s.e.m. Third, the rate of increase in [Ca2+]i in response to a high glucose challenge was 25% and 40% lower in GK and nSTZ respectively, as compared with Wistar islets. Fourth, the maximal [Ca2+](i) level reached after 10 min of perifusion with 16.7 mM glucose was lower with GK and nSTZ islets and represented respectively 60% and 90% of that of Wistar islets. Further, thapsigargin, a blocker of Ca2+/ATPases (SERCA), abolished the initial reduction in [Ca2+]i observed in response to high glucose and induced fast [Ca2+]i oscillations with high amplitude in Wistar islets. The latter effect was not seen in GK and nSTZ islets. In these two NIDDM models, several common alterations in glucose-induced Ca2+ handling were revealed which may contribute to their poor glucose-induced insulin secretion.     

Measurement of cytosolic free calcium concentration in relation to insulin release in normal rat pancreatic islets

The effects of glucose (16.7 mM), potassium (50 mM), forskolin (20 microM), dibutyryl cyclic AMP (1 mM) and arachidonic acid (82 microM) upon insulin release and cytosolic free calcium concentrations ([Ca2+]i) were investigated in normal rat pancreatic islets. Potassium, forskolin and dibutyryl cyclic AMP significantly raised [Ca2+]i, but elicited only minimal insulin release. In the absence of extracellular Ca2+, glucose evoked insulin release, but failed to augment [Ca2+]i. Arachidonic acid increased [Ca2+]i both in Ca-depleted and -repleted medium, but promoted insulin release only in Ca2+-repleted environment. These new observations clearly demonstrate by direct measurements that although [Ca2+]i is an important factor in exocytotic insulin release, its effect is subject to amplification or antagonism.     

Glyburide and tolbutamide induce desensitization of insulin release in rat pancreatic islets by different mechanisms.

Insulin secretion was studied in rat pancreatic islets after 24-h exposure to various glyburide or tolbutamide concentrations. Glucose-induced insulin release was significantly (P < 0.05) reduced in islets cultured with 0.1 microM glyburide or 100 microM tolbutamide (2098 +/- 187, 832 +/- 93, and 989 +/- 88 pg/islet.h in control, glyburide-exposed, and tolbutamide-exposed islets, respectively). When glyburide-treated islets were stimulated with glyburide or tolbutamide, insulin release was also impaired compared to that in control islets (P < 0.05). In contrast, tolbutamide-exposed islets showed an impaired response to tolbutamide, but a normal response to glyburide. To investigate the mechanism of the sulfonylurea-induced impairment of insulin secretion, we measured insulin release and Rb+ efflux (a marker of the K+ channel activity) in a perifusion system and islet Ca2+ uptake under static conditions. Insulin release in response to 16.7 mM glucose increased in control islets from 9.4 +/- 1.1 to 131 +/- 19 pg/islet.min (first phase secretion peak). Simultaneously, the fractional 86Rb+ efflux declined from 0.015 +/- 0.002% to 0.006 +/- 0.001% (change in decrement, -63.5%). Glucose-induced insulin release in glyburide- and tolbutamide-treated islets was significantly reduced (first phase peak, 22.1 +/- 5 and 39.7 +/- 8 pg/islet.min, respectively; P < 0.05), and the fractional 86Rb+ efflux decrement was -21 +/- 6% for glyburide (P < 0.005 vs. control islets) and -65 +/- 4% (not different from control) for tolbutamide. When glyburide- or tolbutamide-exposed islets were stimulated with the corresponding sulfonylurea, insulin release was impaired compared to that in control islets (P < 0.05), but, again, 86Rb+ efflux was impaired (P < 0.05) only in glyburide-exposed islets. When 45Ca2+ uptake was studied, the increase in glucose concentration from 2.8 to 16.7 mM increased calcium uptake in control islets from 1.76 +/- 0.58 to 7.27 +/- 1.36 pmol/islet.2 min (n = 4). Preexposure to 0.1 microM glyburide did not change calcium uptake at a glucose concentration of 2.8 mM (1.44 +/- 0.45 pmol/islet.2 min) but significantly reduced calcium uptake stimulated by 16.7 mM glucose (3.21 +/- 0.35 pmol/islet.2 min; n = 4; P < 0.005 compared to control islets). In contrast, preexposure to 100 microM tolbutamide did not change either basal or glucose-stimulated calcium uptake (1.44 +/- 0.45 and 6.90 +/- 0.81 pmol/islet.2 min, respectively; n = 4). These data show that in vitro chronic exposure of pancreatic islets to the sulfonylureas glyburide and tolbutamide impairs their ability to respond to a subsequent glucose or sulfonylurea stimulation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Islet NADPH oxidase activity is modulated unevenly by different secretagogues

NADPH oxidase expression and activity have been measured in pancreatic islets under normal conditions, but its potential modulatory role upon insulin secretion remains unclear. We have currently studied NADPH oxidase activity in islets isolated from normal rats as well as the effect of its inhibition upon insulin secretion stimulated by different secretagogues. Glucose, arginine, fatty acids and KCl increased islet NADPH oxidase activity in a stimulus-dependent manner. DPI inhibited such increase in different proportions and affected unevenly insulin secretion, namely, it decreased the effect of high glucose, increased that of oleic acid and KCl, without changing the one induced by palmitic acid. Our data provide evidence that the contribution of NADPH activity to reactive oxygen species production in normal rat islets as well as its effect upon insulin secretion is uneven and highly stimulus-dependent.     

Regulation of calcium fluxes in pancreatic islets: the role of membrane depolarization

The effect of K+-induced depolarization on calcium fluxes and insulin release from isolated islets were investigated in order to elucidate the mechanism by which glucose initially reduces and later increases 45Ca efflux from prelabeled and perifused rat pancreatic islets. Raising the extracellular K+ concentration from 5.0 to 20.0 mM produced a 2- to 3-fold increase in 45Ca net uptake and efflux from isolated islets. The latter effect was dependent on the presence of extracellular Ca2+, suggesting that it resulted from the entry of calcium into the islet cells. In the presence of 20 mM K+, 16.7 mM glucose failed to stimulate 45Ca efflux, while 20 mM K+ further enhanced 45Ca efflux from islets perifused in the presence of the high concentration of glucose. These findings suggest that the effect of glucose to stimulate 45Ca efflux from perifused islets depends mainly on the glucose-induced depolarization of the cell membrane. In the absence of extracellular calcium, 20 mM K+ failed to mimick the effect of glucose to reduce 45Ca efflux. Glucose (16.7 mM) decreased 45Ca efflux from islets perifused in the presence of 20 mM K+ and antagonized the effect of 20 mM K+ to stimulate 45Ca efflux from perifused islets. It is concluded that K+-induced plasma membrane depolarization reproduces the effect of glucose to stimulate but not to inhibit 45Ca efflux from perifused islets.     

Glucose regulation of insulin secretion independent of the opening or closure of adenosine triphosphate-sensitive K+ channels in beta cells

Two major pathways are implicated in the stimulation of insulin secretion by glucose. The K+-ATP channel-dependent pathway involves closure of these channels, depolarization of the beta-cell membrane, acceleration of Ca2+ influx, and a rise in cytosolic free Ca2+ ([Ca2+]i). The K+-ATP channel-independent pathway potentiates the stimulation of exocytosis by high [Ca2+]i. To determine whether this second pathway is influenced by the configuration of the channel, we compared the effects of glucose on [Ca2+]i and insulin secretion in mouse islets under three conditions. First, in the presence of 20, 25, and 30 mM K+, i.e. without pharmacological action on K+-ATP channels, [Ca2+]i and insulin secretion were already elevated at 3 mM glucose. High glucose (20 mM) caused a transient decrease in [Ca2+]i followed by an ascent to slightly above control levels, and rapidly stimulated insulin secretion. Second, opening of K+-ATP channels with diazoxide did not influence [Ca2+]i and insulin secretion at 3 mM glucose and high K+. However, high glucose now caused a sustained lowering of [Ca2+]i accompanied by a slow increase in secretion that augmented with the K+ concentration. Third, when K+-ATP channels were blocked and beta-cells depolarized by high concentrations of tolbutamide or glibenclamide, [Ca2+]i and insulin secretion were elevated even in low glucose. High glucose transiently lowered [Ca2+]i, which then increased to or slightly above control levels, while insulin secretion was rapidly stimulated. Under all conditions the correlation between [Ca2+]i and insulin secretion was excellent at low and high glucose levels, and high glucose increased release at all [Ca2+]i. The potentiation of Ca2+-induced exocytosis by glucose is thus independent of the closed or open state of K+-ATP channels. It is only when the channels are opened by diazoxide that the increase in release is a strict amplification of the action of Ca2+. When the channels are closed (sulfonylureas) or still closable (high K+ alone), the effect of glucose on secretion also comprises a slight increase in [Ca2+]i and, in the latter case, is not strictly K+-ATP channel independent.     

Verapamil corrects abnormal metabolism of pancreatic islets and insulin secretion in phosphate depletion.

Phosphate depletion (PD) causes impaired insulin secretion and metabolic derangements in pancreatic islets. We studied PD, pair-weighed (PW), and PD and PW rats treated with verapamil (PD-V and PW-V) to examine the mechanisms of these derangements. Cytosolic calcium ([Ca2+]i) in PD islets was higher than that in PW, PD-V, and PW-V islets, and the values in the latter three groups were not different. Both basal and stimulated ATP in PD islets were lower than those in PW, PW-V, or PD-V islets. The maximum velocity (Vmax) of Ca(2+)-ATPase and the Km and Vmax of Na+,K(+)-ATPase were reduced in PD islets. In both PD-V and PW-V, the Vmax of Ca(2+)-ATPase was higher than that in PD, but lower than that in PW. Both initial and second phases of insulin secretion by PD islets were lower than those by PW and PW-V islets. In PD-V rats, insulin secretion was greater than that in PD rats, but only the second phase was significantly higher. The data are consistent with either of the following possibilities: 1) PD causes a change in the permeability of islets, allowing increased entry of Ca2+ into them and a fall in ATP of islets; the latter would impair the activity of both ATPases, leading to reduced Ca2+ extrusion from islets and, hence, an elevation in their [Ca2+]i; or 2) the primary defect in PD is a reduction in the activities of ATPases of islets due to the fall in ATP secondary to phosphorus deficiency. The decreased Ca2+ extrusion that ensues, even in the face of normal Ca2+ entry, will result in high [Ca2+]i. In either of these scenarios the rise in [Ca2+]i would inhibit mitochondrial oxygen consumption and ATP production, further lowering the ATP content of the islets. The higher [Ca2+]i and low ATP of PD underlie the impaired insulin secretion. Verapamil, by blocking normal or augmented Ca2+ entry into the islets, mitigates or prevents the derangements in islet function and metabolism.     

Glucagon-like peptide-1 stimulates insulin secretion by a Ca2+-independent mechanism in Zucker diabetic fatty rat islets of Langerhans

This study investigates the mechanisms responsible for glucagon-like peptide-1 (GLP-1)-induced insulin secretion in Zucker diabetic fatty (ZDF) rats and their lean control (ZLC) littermates. Glucose, and 100 nmol/L GLP-1 (7-37 hydroxide) in the presence of stimulatory glucose concentrations, induced insulin secretion in islets from ZLC animals. In contrast, ZDF islets hypersecreted insulin at low glucose (5 mmol/L) and were poorly responsive to 15 mmol/L glucose stimulation, but increased insulin secretion following exposure to GLP-1. The insulin secretory response to 100 nmol/L GLP-1 was reduced by 88% in ZLC islets exposed to exendin 9-39. The intracellular Ca2+ concentration ([Ca2+]i) increased in fura-2-loaded ZLC islets following stimulation with 12 mmol/L glucose alone or GLP-1 in the presence of 12 mmol/L glucose. The increases in [Ca2+]i and insulin secretion in ZLC islets induced by GLP-1 were attenuated by 1 micromol/L nitrendipine. In contrast, neither glucose nor GLP-1 substantially increased [Ca2+]i in ZDF islets. Furthermore, insulin secretory responses to GLP-1 were not significantly inhibited in ZDF islets by nitrendipine. However, the insulin secretory response to GLP-1 in both ZLC and ZDF islets was ablated by cholera toxin. Our findings indicate that in ZLC islets, GLP-1 induces insulin secretion by a mechanism that depends on Ca2+ influx through voltage-dependent Ca2+ channels, whereas in ZDF islets, the action of GLP-1 is mediated by Ca2+-independent signaling pathways.     

Glucose controls cytosolic Ca2+ and insulin secretion in mouse islets lacking adenosine triphosphate-sensitive K+ channels owing to a knockout of the pore-forming subunit Kir6.2

Glucose-induced insulin secretion is classically attributed to the cooperation of an ATP-sensitive potassium (K ATP) channel-dependent Ca2+ influx with a subsequent increase of the cytosolic free Ca2+ concentration ([Ca2+]c) (triggering pathway) and a K ATP channel-independent augmentation of secretion without further increase of [Ca2+]c (amplifying pathway). Here, we characterized the effects of glucose in beta-cells lacking K ATP channels because of a knockout (KO) of the pore-forming subunit Kir6.2. Islets from 1-yr and 2-wk-old Kir6.2KO mice were used freshly after isolation and after 18 h culture to measure glucose effects on [Ca2+]c and insulin secretion. Kir6.2KO islets were insensitive to diazoxide and tolbutamide. In fresh adult Kir6.2KO islets, basal [Ca2+]c and insulin secretion were marginally elevated, and high glucose increased [Ca2+]c only transiently, so that the secretory response was minimal (10% of controls) despite a functioning amplifying pathway (evidenced in 30 mm KCl). Culture in 10 mm glucose increased basal secretion and considerably improved glucose-induced insulin secretion (200% of controls), unexpectedly because of an increase in [Ca2+]c with modulation of [Ca2+]c oscillations. Similar results were obtained in 2-wk-old Kir6.2KO islets. Under selected conditions, high glucose evoked biphasic increases in [Ca2+]c and insulin secretion, by inducing K ATP channel-independent depolarization and Ca2+ influx via voltage-dependent Ca2+ channels. In conclusion, Kir6.2KO beta-cells down-regulate insulin secretion by maintaining low [Ca2+]c, but culture reveals a glucose-responsive phenotype mainly by increasing [Ca2+]c. The results support models implicating a K ATP channel-independent amplifying pathway in glucose-induced insulin secretion, and show that K ATP channels are not the only possible transducers of metabolic effects on the triggering Ca2+ signal.     

Microfluidic glucose stimulation reveals limited coordination of intracellular Ca2+ activity oscillations in pancreatic islets

The pancreatic islet is a functional microorgan involved in maintaining normoglycemia through regulated secretion of insulin and other hormones. Extracellular glucose stimulates insulin secretion from islet beta cells through an increase in redox state, which can be measured by NAD(P)H autofluorescence. Glucose concentrations over approximately 7 mM generate synchronous oscillations in beta cell intracellular Ca2+ concentration ([Ca2+]i), which lead to pulsatile insulin secretion. Prevailing models assume that the pancreatic islet acts as a functional syncytium, and the whole islet [Ca2+]i response has been modeled in terms of islet bursting and pacemaker models. To test these models, we developed a microfluidic device capable of partially stimulating an islet, while allowing observation of the NAD(P)H and [Ca2+]i responses. We show that beta cell [Ca2+]i oscillations occur only within regions stimulated with more than approximately 6.6 mM glucose. Furthermore, we show that tolbutamide, an antagonist of the ATP-sensitive K+ channel, allows these oscillations to travel farther into the nonstimulated regions of the islet. Our approach shows that the extent of Ca2+ propagation across the islet depends on a delicate interaction between the degree of coupling and the extent of ATP-sensitive K+-channel activation and illustrates an experimental paradigm that will have utility for many other biological systems.     

Somatostatin inhibits oxidative respiration in pancreatic beta-cells

Somatostatin potently inhibits insulin secretion from pancreatic beta-cells. It does so via activation of ATP-sensitive K+-channels (KATP) and G protein-regulated inwardly rectifying K+-channels, which act to decrease voltage-gated Ca2+-influx, a process central to exocytosis. Because KATP channels, and indeed insulin secretion, is controlled by glucose oxidation, we investigated whether somatostatin inhibits insulin secretion by direct effects on glucose metabolism. Oxidative metabolism in beta-cells was monitored by measuring changes in the O2 consumption (DeltaO2) of isolated mouse islets and MIN6 cells, a murine-derived beta-cell line. In both models, glucose-stimulated DeltaO2, an effect closely associated with inhibition of KATP channel activity and induction of electrical activity (r > 0.98). At 100 nm, somatostatin abolished glucose-stimulated DeltaO2 in mouse islets (n = 5, P < 0.05) and inhibited it by 80 +/- 28% (n = 17, P < 0.01) in MIN6 cells. Removal of extracellular Ca2+, 5 mm Co2+, or 20 microm nifedipine, conditions that inhibit voltage-gated Ca2+ influx, did not mimic but either blocked or reduced the effect of the peptide on DeltaO2. The nutrient secretagogues, methylpyruvate (10 mm) and alpha-ketoisocaproate (20 mm), also stimulated DeltaO2, but this was unaffected by somatostatin. Somatostatin also reversed glucose-induced hyperpolarization of the mitochondrial membrane potential monitored using rhodamine-123. Application of somatostatin receptor selective agonists demonstrated that the peptide worked through activation of the type 5 somatostatin receptor. In conclusion, somatostatin inhibits glucose metabolism in murine beta-cells by an unidentified Ca2+-dependent mechanism. This represents a new signaling pathway by which somatostatin can inhibit cellular functions regulated by glucose metabolism.     

Effects of protein kinase C activation on inositol phosphate generation and intracellular Ca2+ mobilization in bovine parathyroid cells

Activators of protein kinase C, such as phorbol myristate acetate (PMA) and the synthetic diacylglycerol dioctanoylglycerol (diC8), either stimulate or inhibit PTH release depending on the extracellular Ca2+ concentration. By increasing PTH release at high extracellular Ca2+, these agents, in effect, block high Ca2(+)-induced inhibition of secretion. Since raising extracellular Ca2+ increases intracellular free Ca2+ ([Ca2+]i) and inositol trisphosphate (InsP3) formation in parathyroid cells, we assessed the effects of PMA pretreatment on [Ca2+]i and InsP3 to ascertain whether these second messengers might be altered by protein kinase C activation. Preincubation of parathyroid cells with PMA (10(-6) M) significantly lowered the intracellular Ca2+ response to raising extracellular Ca2+ from 0.5-2.0 mM. The peak increase in [Ca2+]i averaged 475 +/- 11 nM in PMA-treated cells compared to 703 +/- 44 nM in control cells. High extracellular Ca2(+)-induced InsP3 accumulation was also reduced after incubating the cells with PMA. To determine whether intracellular Ca2+ stores and/or transmembrane Ca2+ uptake were affected by activating protein kinase C, we examined intracellular Ca2+ responses to the Ca2+ ionophore ionomycin after PMA pretreatment. At 0.5 mM Ca2+, ionomycin (10(-6) M) increased [Ca2+]i to an initial peak of 738 +/- 49 nM followed by a sustained increase to 501 +/- 30 nM in control cells (n = 15). After exposure to PMA (greater than or equal to 20 min), however, peak and sustained increments in [Ca2+]i were significantly lower at 550 +/- 32 and 394 +/- 16 nM, respectively (P less than 0.02, n = 8). In the absence of extracellular Ca2+, basal [Ca2+]i was 197 +/- 5 and peaked at 323 +/- 15 nM with ionomycin (10(-6) M) in PMA-treated cells (n = 16). The latter value was significantly less than the peak increase in [Ca2+]i to 461 +/- 19 nM observed with ionomycin (10(-6) M) in control cells (P less than 0.001, n = 15). With respect to secretion, either of the protein kinase C agonists (i.e. PMA or diC8) or the Ca2+ ionophore ionomycin inhibited PTH release at 0.5 mM Ca2+. To determine whether the concomitant activation of protein kinase C- and Ca2(+)-dependent pathways could additively suppress PTH release, we assessed the effects of ionomycin and either PMA or diC8 on secretion. PTH release at 0.5 mM Ca2+ was reduced in an additive manner by either of these protein kinase C agonists plus ionomycin. At 2 mM Ca2+, protein kinase C agonists stimulated PTH release.(ABSTRACT TRUNCATED AT 400 WORDS)     

Glyburide increases cytosolic-free calcium concentrations in normal rat pancreatic islet cells

We have attempted to delineate the effect of glyburide on the regulation of cytosolic-free calcium concentrations, [Ca2+]i, in normal rat pancreatic islet cells. In the presence of extracellular calcium (1 mmol/L), glyburide increased [Ca2+]i from 70 nmol/L to 260 nmol/L in a dose-dependent manner. The maximal effect was seen at a concentration of 2 mumol/L with half-maximal stimulation observed at .25 mumol/L. The effect of glyburide (.25 mumol/L) was inhibited 90% by the calcium channel blocker, verapamil (30 mumol/L). At a maximally effective concentration of glyburide (2 mumol/L), the inhibitory effect of verapamil was only 17%. In the absence of extracellular calcium, glyburide increased [Ca2+]i from 55 nmol/L to 107 nmol/L, indicating its ability to mobilize intracellular calcium stores. These results correlated well with the ability of glyburide (2 mumol/L) to stimulate insulin secretion both in the presence (from 38 +/- 5 mumol/L/10 islets to 131 +/- 28 microU/10 islets) and in the absence (from 49 +/- 4 microU/10 islets to 93 +/- 7 microU/10 islets) of extracellular Ca2+. The present observations suggest that glyburide promotes calcium influx via voltage-dependent calcium channels and may mobilize intracellular calcium stores.     

The role of Ca(2+)-related events in glucose-stimulated desensitization of insulin secretion

The spontaneous decline of insulin secretion (third phase) that occurs under a variety of secretory conditions is well documented and suggests a general impairment or desensitization of the secretory process. We have examined several aspects of Ca2+ flux as well as regulators of Ca-linked second messenger events in freshly isolated rat islets chronically stimulated with glucose over 24 h, a period that encompasses initial (hour 1), peak (hour 3), and subsequent impaired or desensitized (hour 20-22) secretion. In islets incubated for these periods in HB104 medium with 22 mM glucose, 45Ca2+ uptake did not vary (12.6 +/- 1.6 vs. 10.2 +/- 1.7 vs. 13.2 +/- 3.4 pmol Ca2+/islet.10 min at 1, 3, and 22 h, respectively). Chronic incubation in 2 mM glucose reduced total Ca2+ uptake at each of the time periods, but, again, uptake did not change with desensitization (9.8 +/- 1.4 vs. 6.6 +/- 2.1 vs. 7.8 +/- 2.3 pmol Ca2+/islet.10 min). In 11 mM glucose, the Ca channel antagonist verapamil (1-10 microM) reduced insulin secretion by 55-80% in a dose-dependent manner over 1-3 h; islets continuously exposed to verapamil escaped inhibition by 20 h even at the highest concentration. However, in islets first exposed to 10 microM verapamil only during 20-22 h, hourly insulin secretion was suppressed 25%, 45%, and 33% at 20, 21, and 22 h, respectively, indicating that glucose-desensitized islets were still sensitive to further inhibition of Ca channels. Staurosporine (1 microM), an inhibitor of protein kinase-C activity, progressively inhibited glucose-stimulated insulin secretion from 48% at 1 h to more than 80% by 3 h; again, this inhibitory effect was lost by 20 h of chronic staurosporine. When staurosporine was first administered at 20 h, insulin secretion was modestly suppressed and returned to control values in the next hour. With continuous glucose, the islet response to positive stimulation of endogenous C-kinase activity by carbachol was maintained. The Ca/calmodulin inhibitor trifluoroperazine also inhibited insulin secretion by 75-80% during 1-3 h and continued to exert inhibitory effects through 23 h of continuous administration. We conclude that even though insulin secretion has desensitized to glucose, 1) Ca2+ entry is unchanged and is still regulated by glucose, 2) voltage-dependent Ca channels are still sensitive to blockade by acute verapamil, but can desensitize to chronic verapamil; 3) stimulus-enhanced C-kinase activity may be especially labile during glucose-induced desensitization, while 4) possible Ca/calmodulin potentiation of secretion persists through the three secretory phases.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ca2+-induced Ca2+ release in pancreatic islet beta-cells: critical evaluation of the use of endoplasmic reticulum-targeted "cameleons"

Elevated glucose concentrations cause Ca2+ influx and the exocytotic release of insulin from pancreatic islet beta-cells. Whether increases in cytosolic free Ca2+ concentration also mobilize Ca2+ from intracellular stores (Ca2+-induced Ca2+ release) is unresolved. Endoplasmic reticulum-targeted cameleons have previously been used to explore the involvement of endoplasmic reticulum (ER) Ca2+ release in these cells, albeit with differing conclusions. Cameleons comprise two spectrally shifted green fluorescent proteins, enhanced cyan and yellow fluorescent protein, whose orientation is affected by Ca2+, changing intramolecular fluorescence resonance energy transfer. By measuring pH in the cytosol and ER lumen, we demonstrate that high K+ concentrations (>20 mm) acidify both compartments in clonal MIN6 beta-cells when external bicarbonate concentrations are low (<5 mm), interfering with measurements using Ycam-2 and Ycam-4ER. However, when intracellular pH is strongly buffered (24 mm HCO3-), glucose or cell depolarization increases ER [Ca2+] monitored with Ycam-4ER. KCl-induced increases in ER [Ca2+] were diminished when intracellular stores were sensitized with 1 mm caffeine and inhibited by pretreatment with ryanodine. Furthermore, preincubation with ryanodine tended to slow the falling phase of the ER Ca2+ transient after cell depolarization with KCl and reduced the peak cytosolic [Ca2+]. By contrast, stimulation with glucose increased ER [Ca2+] both in the absence and presence of caffeine or ryanodine. These observations suggest that Ca2+-induced ER Ca2+ release can occur in beta-cells under some conditions but may not be essential for glucose-stimulated insulin secretion.     

Diphenylhydantoin suppresses glucose-induced insulin release by decreasing cytoplasmic H+ concentration in pancreatic islets

Diphenylhydantoin (DPH), which is clinically used in the treatment of epilepsy, inhibits glucose-induced insulin release from pancreatic islets by a mechanism that remains unknown. In the present study, DPH is shown to suppress glucose-induced insulin release concentration-dependently. In dynamic experiments, 20 microm DPH suppressed 16.7 mm glucose-induced biphasic insulin release. DPH also suppressed insulin release in the presence of 16.7 mm glucose, 200 microm diazoxide, and 30 mm K+ without affecting the intracellular Ca2+ concentration. DPH suppressed ATP content and mitochondrial membrane hyperpolarization in the presence of 16.7 mm glucose without affecting glucose utilization, glucose oxidation, and reduced nicotinamide adenine dinucleotide phosphate fluorescence. DPH increased cytoplasmic pH in the presence of high glucose, but the increase was abolished under Na+ -deprived conditions and HCO3- -deprived conditions, suggesting that Na+ and HCO3- transport across the plasma membrane are involved in the increase in cytoplasmic pH by DPH. Alkalization by adding NH4+ to the extracellular medium also suppressed insulin release, ATP content, and mitochondrial membrane hyperpolarization. Because ATP production from the mitochondrial fraction in the presence of substrates was decreased by increased pH in the medium, DPH suppresses mitochondrial ATP production by reducing the H+ gradient across mitochondrial membrane. Using permeabilized islets, the increase in pH was shown to decrease Ca2+ efficacy at a clamped concentration of ATP in the exocytotic system. Taken together, DPH inhibits glucose-induced insulin secretion not only by inhibiting mitochondrial ATP production, but also by reducing Ca2+ efficacy in the exocytotic system through its alkalizing effect on cytoplasm.     

Time-dependent potentiation of the beta-cell is a Ca2+-independent phenomenon

When isolated rat pancreatic islets are treated with 16.7 mM glucose, a time-dependent potentiation (TDP) of insulin release occurs that can be detected by subsequent treatment with 50 mM KCl. It has been thought that TDP by glucose is a Ca2+-dependent phenomenon and only occurs when exposure to glucose is carried out in the presence of Ca2+. In contrast to this, we now demonstrate TDP under stringent Ca2+-free conditions (Ca2+-free buffer containing 1 mM EGTA). In fact, under these Ca2+-free conditions glucose caused an even stronger TDP than in the presence of Ca2+. TDP induced by glucose in the absence of extracellular Ca2+ was unaffected by inhibitors of protein kinase C (PKC). However, cerulenin or tunicamycin, two inhibitors of protein acylation, eradicated TDP without affecting glucose metabolism. The TDP by glucose was not associated with an increase in the cytosolic free Ca2+ concentration ([Ca2+]i) during subsequent treatment with high K+. Exposure of islets to forskolin under Ca(2+)-free conditions did not cause TDP despite a large increase in the cellular cAMP levels. In conclusion, glucose alone induces TDP under stringent Ca2+-free conditions when [Ca2+]i was significantly lowered. Protein acylation is implicated in the underlying mechanism of TDP.     

Immediate and time-dependent effects of glucose on insulin release: differential calcium requirements

Glucose regulates insulin release in a complex manner; apart from its acute secretory action it induces time-dependent effects which modulate subsequent islet responses. The Ca2+ sensitivities of the diverse secretory events generated by glucose were investigated in the perfused rat pancreas. First- and second-phase insulin responses to 16.7 mmol/l glucose were obliterated in the presence of 5 mmol/l EgTA; threshold Ca2+ concentrations for significant responses were 0.25 mmol/l for second-phase, and 'O' (no Ca2+ added, approx 20 mumol/l) for first-phase release (both around 10% of control). The apparent Km of the Ca2+ dependencies were 0.6 mmol/l for first-phase, and 1.25 mmol/l for second-phase release. Time-dependent potentiation was demonstrated by subjecting the pancreas to two 40-min 16.7 mmol/l glucose stimuli separated by a 30-min rest period; this amplified the first-phase response to the second stimulus 2.5 +/- 0.9-fold. Also the generation of potentiation was Ca2+ dependent, with characteristics similar to those of the acute second-phase insulin response (apparent Km approximately 1.0 mmol/l Ca2+). In contrast, the amplified first-phase response to the second glucose pulse retained its high sensitivity to Ca2+, thus resembling the unprimed first-phase. The inhibitory message of glucose was demonstrated by applying two sequential 5-min pulses of 8.3 mmol/l glucose: the insulin response to the second stimulus was reduced by 43 +/- 9%. Addition of EgTA to the first glucose pulse had no effect on the inhibition of the second insulin response. Thus: 1. Despite its high sensitivity to Ca2+, also first-phase release is fully dependent on extracellular Ca2+.2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Free cytosolic calcium and secretagogue-stimulated initial pancreatic exocrine secretion

In order to establish the role of secretagogue-induced changes in free cytosolic Ca2+ ([Ca2+]i) in pancreatic enzyme secretion, we measured the effects of carbachol, cholecystokinin-octapeptide (CCK-OP), bombesin, substance P, and bromo-A23187 on amylase release and [Ca2+]i in guinea pig pancreatic acini loaded with the Ca2+-selective fluorescent indicator, fura-2. Evaluation of time courses and dose-response curves indicated that carbachol, CCK-OP, bombesin, and substance P cause extracellular Ca2+-independent transient increases in [Ca2+]i and transient bursts in amylase release (initial secretion). The potencies for the secretagogues to increase [Ca2+]i and initial amylase release were similar. Bromo-A23187 also caused an extracellular Ca2+-independent transient increase in [Ca2+]i and amylase release. In the absence of extracellular Ca2+, sequential additions of substance P followed by carbachol caused transient increases in [Ca2+]i correlating with transient bursts in amylase release. In contrast, in acini first treated with carbachol, the ability of substance P to increase [Ca2+]i and amylase release was blocked. Sustained secretion caused by the secretagogues was dependent on extracellular Ca2+ but occurred at basal [Ca2+]i. Increasing [Ca2+]i during the sustained phase of stimulation by increasing the extracellular Ca2+ concentration or with bromo-A23187 did not increase the rate of sustained secretion.     

Glucose stimulation of insulin release in the absence of extracellular Ca2+ and in the absence of any increase in intracellular Ca2+ in rat pancreatic islets.

Insulin secretion has been studied in isolated rat pancreatic islets under stringent Ca(2+)-depleted, Ca(2+)-free conditions. Under these conditions, the effect of 16.7 mM glucose to stimulate insulin release was abolished. Forskolin, which activates adenylyl cyclase, also failed to stimulate release in the presence of either low or high glucose concentrations. A phorbol ester (phorbol 12-myristate 13-acetate; PMA) increased the release rate slightly and this was further increased by 16.7 mM glucose. Remarkably, in the presence of both forskolin and PMA, 16.7 mM glucose strongly augmented insulin release. The augmentation was concentration dependent and monophasic and had a temporal profile similar to the "second phase" of glucose-stimulated insulin release, which is seen under normal conditions when Ca2+ is present. Metabolism is required for the effect because mannoheptulose abolished the glucose response. Other nutrient secretagogues, alpha-ketoisocaproate, and the combination of leucine and glutamine augmented release under the same conditions. Norepinephrine, a physiological inhibitor of insulin secretion, totally blocked the stimulation of release by forskolin and PMA and the augmentation of release by glucose. Thus, under the stringent Ca(2+)-free conditions imposed, the stimulation of insulin release by forskolin and PMA, as well as the augmentation of release by glucose, is under normal physiological control. As no increase in intracellular [Ca2+] was observed, the results demonstrate that glucose can increase the rate of exocytosis and insulin release by pancreatic islets in a Ca(2+)-independent manner. This interesting pathway of stimulus-secretion coupling for glucose appears to exert its effect at a site beyond the usual elevation of intracellular [Ca2+] and is not due to an activation by glucose of protein kinase A or C.