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)     

Effects of protein kinase C inhibitors on insulin secretory responses from rodent pancreatic islets

The contribution of protein kinase C (PKC) to the regulation of insulin release from perifused islets was explored using staurosporine or G? 6976 to inhibit the enzyme. Phorbol 12-myristate 13-acetate (PMA, 500 nM) addition to rat islets resulted in a slowly rising insulin secretory response. While minimally effective alone, the addition of 500 nM forskolin together with PMA resulted in a synergistic secretory response. The conventional protein-kinase-C isoform inhibitor G? 6976 (1 microM) completely abolished PMA-induced secretion. However, the combination of forskolin plus PMA significantly enhanced secretion from G? 6976-treated islets. Similar to previous findings made with staurosporine, G? 6976 (1 microM) enhanced the first phase and reduced the second phase of 20 mM glucose-induced secretion from rat islets. Additional studies were conducted comparing the secretory responses of perifused rat or mouse islets to glucose. Dramatic species differences to the hexose were observed. For example, 35-40 min after the onset of stimulation with 8, 10 or 20 mM glucose insulin release rates from mouse islets averaged 32+/-6, 84+/-27 or 131+/-17 pg/islet per minute, respectively. The responses from rat islets averaged 115+/-28, 561+/-112 or 800+/-46 pg/islet per minute at this time point. Islet insulin stores were comparable in both species. The addition of 5 microM carbachol, 500 nM forskolin or 20 mM KCl to mouse islets together with 20 mM glucose resulted in a dramatic augmentation of insulin output. The responses to carbachol or forskolin, but not KCl, were inhibited by 50 nM staurosporine. However, staurosporine (50 nM) reduced insulin secretion from rat islets stimulated with KCl plus 20 mM glucose. G? 6976 potentiated 20 mM glucose-induced secretion from mouse islets. These studies demonstrate that 1 microM G? 6976 completely abolishes PMA-induced release from rat islets and has a modest inhibitory effect on 20 mM glucose-induced secretion. G? 6976 (1 microM) had no inhibitory effect on 20 mM glucose-induced release from mouse islets. These studies also confirm that staurosporine inhibits both PKC- and PKA-mediated events in islets and this lack of specificity may account for its more pronounced inhibition of release when compared to G? 6976. Finally, significant species differences to PKC inhibitors exist between mouse and rat islets.     

ACTH and alpha-MSH inhibit leptin expression and secretion in 3T3-L1 adipocytes: model for a central-peripheral melanocortin-leptin pathway

Leptin is the 167 amino-acid protein product of the Lep (obese) gene that is released predominantly from adipose tissue and circulates at levels related to the amount of fat. Leptin expression is hormonally regulated: insulin and glucocorticoids are stimulators, while inhibitors include beta-adrenergic agonists and testosterone. Recently, adenylate cyclase-coupled melanocortin receptors have been identified in murine adipose tissue, the 3T3-L1 adipocyte cell line, and in human fat tissue. These studies prompted us to evaluate the effects of pro-opiomelanocortin (POMC)-derived peptides on leptin production and expression in 3T3-L1 adipocytes in culture. 3T3-L1 pre-adipocytes differentiated by the insulin/indomethacin (I/I) method produced leptin at levels that were two times higher than those obtained in cells differentiated by the more traditional insulin/dexamethasone/isobutylmethylxanthine (I/D/M) method. By RT-PCR studies, 3T3-L1 cells expressed both the melanocortin 2 receptors (MC2-R) and melanocortin 5 receptors (MC5-R) isoforms of the melanocortin receptor at an early stage of differentiation. When I/I differentiated 3T3-L1 adipocytes were incubated with different concentrations of dibutyryl cAMP (db-cAMP) or POMC-derived peptides (ACTH and alpha-MSH), ACTH and alpha-MSH stimulated cAMP production after 30 min (2-fold increase) associated with a dose-dependent inhibition of leptin secretion (ACTHz.Gt;alpha-MSH; IC(50)=3.2+/-0.4 SE and 36+/-5 nM, respectively), maximal after 3 h of incubation (30% inhibition). In addition, 100 nM ACTH and alpha-MSH induced a 60% reduction in leptin expression by RT-PCR. Incubation of cells with 0.5 mM db-cAMP led to a more prominent inhibition of leptin expression and secretion (up to 80% at 1 and 24 h, respectively). The ACTH and alpha-MSH inhibitory effects on leptin secretion were mediated by activation of the MC2-R and MC5-R and were reversed by the MC-R antagonists ACTH(11-24) and ACTH(7-38). In summary, we have shown that POMC-peptides are potent inhibitors of leptin expression and production in 3T3-L1 adipocytes. The finding of ACTH/alpha-MSH receptor-induced inhibition of leptin production and expression in adipocytes support the possibility that there is a control mechanism for modulation of adipose tissue function via a melanocortin-leptin axis.     

Glucose-induced insulin secretion from islets of fasted rats: modulation by alternate fuel and neurohumoral agonists

Islets from fed and 24-h-fasted rats were studied immediately after collagenase isolation. (1) After a 24-h fast, the insulin secretory responses to 8 mM glucose measured during perifusion were reduced by more than 90% from islets of fasted donors. (2) Increasing glucose to 11 or 27.5 mM resulted in enhanced insulin secretion from islets of fasted animals. (3) Fasting did not reduce islet insulin content. (4) Responses to 8 or 27.5 mM glucose were not affected if fatty acid-free albumin was used during the perifusion. (5) Inclusion of alpha-ketoisocaproate (5 mM), monomethyl succinate (10 mM) or carbachol (10 microM) significantly amplified insulin release from fasted islets in the simultaneous presence of 8 mM glucose. (6) Phospholipase C activation by glucose, carbachol or their combination was not adversely affected by fasting. (7) The response to the protein kinase C activator, phorbol 12-myristate 13-acetate (500 nM), was reduced by about 60% after fasting. (8) Extending the fast to 48 h resulted in a severe decline in response to 11 mM glucose; however, the further addition of 10 microM carbachol still enhanced release from these islets. The results confirm that caloric restriction impairs islet sensitivity to glucose stimulation and that protein kinase C may be involved in the reduction of glucose-induced insulin release from these islets. The activation of phospholipase C by cholinergic stimulation may contribute to the maintenance of insulin secretion from calorically restricted animals. These results also demonstrate that free fatty acids are not essential for glucose to evoke secretion from isolated islets of fasted donors.     

The possible mechanisms by which phanoside stimulates insulin secretion from rat islets

We recently showed that phanoside, a gypenoside isolated from the plant Gynostemma pentaphyllum, stimulates insulin secretion from rat pancreatic islets. To study the mechanisms by which phanoside stimulates insulin secretion. Isolated pancreatic islets of normal Wistar (W) rats and spontaneously diabetic Goto-Kakizaki (GK) rats were batch incubated or perifused. At both 3 x 3 and 16 x 7 mM glucose, phanoside stimulated insulin secretion several fold in both W and diabetic GK rat islets. In perifusion of W islets, phanoside (75 and 150 microM) dose dependently increased insulin secretion that returned to basal levels when phanoside was omitted. When W rat islets were incubated at 3 x 3 mM glucose with 150 muM phanoside and 0 x 25 mM diazoxide to keep K-ATP channels open, insulin secretion was similar to that in islets incubated in 150 microM phanoside alone. At 16 x 7 mM glucose, phanoside-stimulated insulin secretion was reduced in the presence of 0 x 25 mM diazoxide (P<0 x 01). In W islets depolarized by 50 mM KCl and with diazoxide, phanoside stimulated insulin release twofold at 3 x 3 mM glucose but did not further increase the release at 16 x 7 mM glucose. When using nimodipine to block L-type Ca2+ channels in B-cells, phanoside-induced insulin secretion was unaffected at 3 x 3 mM glucose but decreased at 16 x 7 mM glucose (P<0 x 01). Pretreatment of islets with pertussis toxin to inhibit exocytotic Ge-protein did not affect insulin response to 150 microM phanoside. Phanoside stimulated insulin secretion from Wand GK rat islets. This effect seems to be exerted distal to K-ATP channels and L-type Ca2+ channels, which is on the exocytotic machinery of the B-cells.     

Biochemical mechanisms involved in monomethyl succinate-induced insulin secretion.

Esters of succinic acid stimulate insulin secretion from pancreatic beta-cells. Using collagenase-isolated rat islets, the transduction mechanisms involved were investigated. In freshly isolated perifused islets, monomethyl succinate (MMSucc), in the presence of basal (2.75 mM) glucose, stimulated insulin release in a biphasic pattern. This secretory response was dependent on extracellular calcium movement into the beta-cell, since the calcium channel blocker nitrendipine (5 microM) abolished it. The glucokinase inhibitor mannoheptulose (20 mM) had no effect on its secretory action, while the protein kinase-C inhibitor staurosporine (20 nM) reduced secretion to MMSucc. In addition, while ineffective alone, the diacylglycerol kinase inhibitor monooleoylglycerol (25 microM) potentiated MMSucc-induced insulin release. A similarly amplified response occurred in the presence of forskolin (0.25 microM), a compound that elevates islet cAMP levels. The sodium salt of succinic acid (20 mM) had no effect on insulin release in the presence or absence of forskolin. Prior treatment with MMSucc in the presence of 2.75 mM glucose sensitized islets to the usually weak insulin secretory effect of 7.5 mM glucose. Other groups of islets were incubated for 2 h with myo-[2-3H]inositol to label their phosphoinositide pools. These islets were subsequently stimulated, and the kinetics of [3H]inositol efflux and insulin secretion were measured. MMSucc induced a rapid and sustained dose-dependent increase in [3H]inositol efflux rates. In batch-incubated islets, MMSucc increased inositol phosphate levels. Finally, MMSucc (20 mM), in the presence of 8 mM glucose, did not influence the detritiation of [5-3H]glucose, but reduced the oxidation of [U-14C] glucose. These results support the following conclusions. First, MMSucc is a potent activator of islet phosphoinositide hydrolysis. Second, the activation of protein kinase-C appears to contribute to the acute insulin secretory effect of MMSucc. Third, MMSucc-induced increases in phosphoinositide hydrolysis contribute at least in part to its ability to acutely stimulate insulin release and prime the beta-cell to subsequent stimulation. Finally, mitochondrial events associated with the oxidative metabolism of MMSucc may underlie its insulinotropic action.     

Involvement of sodium channels and two types of calcium channels in the regulation of adrenocorticotropin release

Recent electrophysiological studies from this laboratory demonstrated that anterior lobe corticotropes exhibited a tetrodotoxin-sensitive sodium current and two types of voltage-dependent calcium currents, consisting of low threshold (transient) and high threshold (long lasting) components. The present report describes cytophysiological and cytochemical studies that used specific blockers of each of these currents to assess their role in the regulation of CRF binding and ACTH secretion and storage. Two dihydropyridines, nimodipine and the pure antagonist enantiomer (-)R202-791, which block high threshold Ca2+ channels, decreased 1 h basal release by 54-74% and CRF-mediated (5 min or 3 h) release completely. Percentages of CRF-bound cells were reduced as much as 74%; however, the inhibitory effect on percentages of CRF-bound cells could be reversed by adding 10 nM Bay K 8644, (a pure dihydropyridine agonist) with the antagonists. CdCl2, which blocks both high and low threshold calcium currents, inhibited basal and CRF-stimulated ACTH release, but only the highest concentration (0.1 mM) reduced percentages of CRF-bound cells. Involvement of the low threshold Ca2+ channels could not be proved by adding dihydropyridine antagonists with 0.1 mM CdCl2. Basal and CRF-mediated ACTH release were blocked by the potent sodium channel blocker tetrodotoxin, and the highest concentration (3 microM) reduced percentages of CRF-bound cells. Basal (1 h) and CRF-stimulated (5 min) ACTH release were also inhibited in medium containing 1 mM EGTA and no Ca2+; however, percentages of CRF-bound cells were within the normal range. Densitometric analysis of stains for ACTH showed an increase in the concentration of stain per cell after a 1-h exposure to the highest concentrations of the inhibitors or to no Ca2+ and 1 mM EGTA coupled with a significant (10%) decrease in corticotrope cell area. Finally, in the last series of tests, the Bay K 8644 agonist or arginine vasopressin were used to study mechanisms of augmentation of basal or CRF-mediated ACTH release. Bay K 8644 augmented basal release in a concentration of 1 microM and CRF-mediated release in a concentration of 100 nM or 1 microM. After pretreatment with either Bay K 8644 or arginine vasopressin (10 nM) there was a significant (30%) increase in the percentage of CRF-bound cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

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)     

CaV1.2 rather than CaV1.3 is coupled to glucose-stimulated insulin secretion in INS-1 832/13 cells

In clonal beta-cell lines and islets from different species, a variety of calcium channels are coupled to glucose-stimulated insulin secretion. The aim of this study was to identify the voltage-gated calcium channels that control insulin secretion in insulinoma (INS)-1 832/13 cells. The mRNA level of Ca(V)1.2 exceeded that of Ca(V)1.3 and Ca(V)2.3 two-fold. Insulin secretion, which rose tenfold in response to 16.7 mM glucose, was completely abolished by 5 microM isradipine that blocks Ca(V)1.2 and Ca(V)1.3. Similarly, the increase in intracellular calcium in response to 15 mM glucose was decreased in the presence of 5 microM isradipine, and the frequency of calcium spikes was decreased to the level seen at 2.8 mM glucose. By contrast, inhibition of Ca(V)2.3 with 100 nM SNX-482 did not significantly affect insulin secretion or intracellular calcium. Using RNA interference, Ca(V)1.2 mRNA and protein levels were knocked down by approximately 65% and approximately 34% respectively, which reduced insulin secretion in response to 16.7 mM glucose by 50%. Similar reductions in calcium currents and cell capacitance were seen in standard whole-cell patch-clamp experiments. The remaining secretion of insulin could be reduced to the basal level by 5 microM isradipine. Calcium influx underlying this residual insulin secretion could result from persisting Ca(V)1.2 expression in transfected cells since knock-down of Ca(V)1.3 did not affect glucose-stimulated insulin secretion. In summary, our results suggest that Ca(V)1.2 is critical for insulin secretion in INS-1 832/13 cells.     

Inhibition of cholesterol biosynthesis impairs insulin secretion and voltage-gated calcium channel function in pancreatic beta-cells

Insulin secretion from pancreatic beta-cells is mediated by the opening of voltage-gated Ca2+ channels (CaV) and exocytosis of insulin dense core vesicles facilitated by the secretory soluble N-ethylmaleimide-sensitive factor attachment protein receptor protein machinery. We previously observed that beta-cell exocytosis is sensitive to the acute removal of membrane cholesterol. However, less is known about the chronic changes in endogenous cholesterol and its biosynthesis in regulating beta-cell stimulus-secretion coupling. We examined the effects of inhibiting endogenous beta-cell cholesterol biosynthesis by using the squalene epoxidase inhibitor, NB598. The expression of squalene epoxidase in primary and clonal beta-cells was confirmed by RT-PCR. Cholesterol reduction of 36-52% was observed in MIN6 cells, mouse and human pancreatic islets after a 48-h incubation with 10 mum NB598. A similar reduction in cholesterol was observed in the subcellular compartments of MIN6 cells. We found NB598 significantly inhibited both basal and glucose-stimulated insulin secretion from mouse pancreatic islets. CaV channels were markedly inhibited by NB598. Rapid photolytic release of intracellular caged Ca2+ and simultaneous measurements of the changes in membrane capacitance revealed that NB598 also inhibited exocytosis independently from CaV channels. These effects were reversed by cholesterol repletion. Our results indicate that endogenous cholesterol in pancreatic beta-cells plays a critical role in regulating insulin secretion. Moreover, chronic inhibition of cholesterol biosynthesis regulates the functional activity of CaV channels and insulin secretory granule mobilization and membrane fusion. Dysregulation of cellular cholesterol may cause impairment of beta-cell function, a possible pathogenesis leading to the development of type 2 diabetes.     

Dual effect of cell-cell contact disruption on cytosolic calcium and insulin secretion

Cell-to-cell interactions play an important role in insulin secretion. Compared with intact islets, dispersed pancreatic beta-cells show increased basal and decreased glucose-stimulated insulin secretion. In this study, we used mouse MIN6B1 cells to investigate the mechanisms that control insulin secretion when cells are in contact with each other or not. RNAi-mediated silencing of the adhesion molecule E-cadherin in confluent cells reduced glucose-stimulated secretion to the levels observed in isolated cells but had no impact on basal secretion. Dispersed cells presented high cytosolic Ca(2+) activity, depolymerized cytoskeleton and ERK1/2 activation in low glucose conditions. Both the increased basal secretion and the spontaneous Ca(2+) activity were corrected by transient removal of Ca(2+) or prolonged incubation of cells in low glucose, a procedure that restored the ability of dispersed cells to respond to glucose (11-fold stimulation). In conclusion, we show that dispersed pancreatic beta-cells can respond robustly to glucose once their elevated basal secretion has been corrected. The increased basal insulin secretion of dispersed cells is due to spontaneous Ca(2+) transients that activate downstream Ca(2+) effectors, whereas engagement of cell adhesion molecules including E-cadherin contributes to the greater secretory response to glucose seen in cells with normal intercellular contacts.     

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.     

Forskolin-induced desensitization of pancreatic beta-cell insulin secretory responsiveness: possible involvement of impaired information flow in the inositol-lipid cycle.

Isolated rat islets of Langerhans were incubated for 2 h in a myo-[2-3H]inositol-containing solution to label their phosphoinositides. Also included during this labeling period was forskolin (0.1-5 microM), a compound established to elevate islet cAMP levels. These islets were subsequently perifused, and their insulin secretory responses to 20 mM glucose or 1 microM of the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA) were assessed. Determined in parallel with secretion were [3H] inositol efflux patterns and, at the termination of the perifusion, labeled inositol phosphate accumulation. The following major observations were made. 1) Forskolin had no deleterious effect on the total amount of [3H]inositol incorporated by the islets during the labeling period. 2) However, labeling in forskolin resulted in subsequent dose-dependent decreases in 20 mM glucose-induced insulin secretion, [3H]inositol efflux and inositol phosphate accumulation. 3) Inclusion of the diacylglycerol (DAG) kinase inhibitor monooleoylglycerol (50 microM) restored to a significant degree glucose-induced release from forskolin-desensitized islets. 4) Pretreatment with 5 microM forskolin had no deleterious effect on TPA-induced insulin release. 5) Prior exposure to forskolin also impaired phosphoinositide hydrolysis in response to cholecystokinin stimulation. 6) Similar to forskolin, labeling in isobutylmethylxanthine (1 mM) reduced in a parallel fashion islet [3H]inositol efflux and insulin secretion in response to 20 mM glucose stimulation. These findings demonstrate that prior chronic elevation of islet cAMP levels suppresses the activation of phospholipase-C in response to subsequent stimulation. Defective insulin secretory responsiveness of these islets appears to be the result of impaired generation of phosphoinositide-derived second messenger molecules, particularly DAG. By substituting for DAG, however, TPA circumvents this biochemical lesion and evokes a normal insulin secretory response from forskolin-pretreated islets.     

Mobilization of Ca2+ from intracellular stores of pancreatic beta-cells by the calcium store blocker TMB-8

TMB-8 has been used experimentally in many cell types, including endocrine cells, because of its ability to block the efflux of Ca2+ from intracellular stores without affecting influx. Unexpectedly, TMB-8 potentiates stimulated insulin release from pancreatic islets, a process believed to be dependent on the level of cytosolic Ca2+. In the present study, while having no effect on basal insulin release (in the presence of 2.8 mM glucose), TMB-8 (10, 30, and 100 microM) caused a concentration-dependent increase in 45Ca2+ efflux from 45Ca2+-preloaded islets. TMB-8 (100 microM) stimulated 45Ca2+ efflux even in the absence of extracellular Ca2+. In the presence of 5.6 mM glucose, TMB-8 (30 and 100 microM) potentiated insulin release and again increased 45Ca2+ efflux in a concentration-dependent manner. Similarly, insulin release stimulated by isobutylmethylxanthine (IBMX) was potentiated significantly, and IBMX-stimulated 45Ca2+ efflux was increased by the simultaneous introduction of 30 microM TMB-8. Thus, in pancreatic islets, TMB-8 appears to mobilize Ca2+ from intracellular stores, rather than inhibit the efflux as has been commonly accepted. In further studies, using insulin-secreting beta-cells of the RINm5F cell line, TMB-8 was shown to increase the cytosolic Ca2+ concentration in the presence and absence of extracellular Ca2+. This confirmed that mobilization of intracellular Ca2+ was occurring in the pancreatic beta-cell in response to TMB-8. Furthermore, a rise in cytosolic Ca2+ of not more than 10 nM (as induced with KCl) was found to mimick the effect of TMB-8 in conjunction with IBMX. No additional effect of TMB-8 to alter Ca2+ handling at the plasma membrane was found when 45Ca2+ uptake experiments were performed. Therefore, the paradoxical mobilization of beta-cell Ca2+ by TMB-8 appears to be a sufficient explanation for its potentiating effect on the rate of insulin secretion.     

Pancreastatin, a presumed product of chromogranin-A (secretory protein-I) processing, inhibits secretion from porcine parathyroid cells in culture

Chromogranin-A, also referred to as secretory protein-I, is a 50K protein found in and secreted by endocrine cells, in which it is costored with the native hormone. Porcine chromogranin-A contains a sequence identical to pancreastatin, a 49-amino acid, C-terminally amidated peptide that has been isolated from porcine pancreas, suggesting that chromogranin-A is the precursor of pancreastatin. Pancreastatin has been found to be a potent inhibitor of glucose-stimulated insulin release. As it is possible that pancreastatin inhibits secretion from other chromogranin-A-containing tissues in which it may be formed, we tested its action on dispersed porcine parathyroid cells in culture. Secretion of chromogranin-A and PTH was up to 6-fold greater at 0.5 mM Ca2+ than at 3.0 mM Ca2+. Pancreastatin (1 nM) reduced the secretion of both chromogranin-A and PTH at 0.5 mM Ca2+ to approximately the levels found at 3.0 mM Ca2+, but did not affect secretion at 3.0 mM Ca2+. Pancreastatin (0.01-1.0 nM) inhibited secretion of chromogranin-A in a dose-dependent fashion. Preincubation of the cells with pancreastatin was not required for inhibition. Transfer of inhibited cells to medium without pancreastatin led to restoration of secretion within 90 min. Phorbol myristate acetate (1.6 microM) stimulated secretion of PTH and chromogranin-A at 3.0 mM Ca2+, but not at 0.5 mM Ca2+. Pancreastatin reversed this stimulation, demonstrating that its inhibition was independent of Ca2+ concentration. These results are consonant with pancreastatin playing a physiological role in modulation of secretion by the parathyroid and, by extension, other endocrine tissues.     

Influence of staurosporine, nitrendipine and monooleoylglycerol on interleukin-1-induced insulin secretion and phosphoinositide hydrolysis.

The monokine interleukin-1 alpha (IL-1) induces a glucose-dependent increase in insulin secretion, an effect tentatively attributed to its ability to increase beta cell phosphoinositide (PI) hydrolysis. In the present experiments, the effects of the protein kinase C inhibitor staurosporine (20 nM), the calcium channel antagonist nitrendipine (5 microM), and the diacylglycerol kinase inhibitor monooleoylglycerol (MOG, 25 microM) on 40 nM IL-1-induced increments in insulin release from perifused islets and inositol phosphate levels in [3H]inositol prelabeled islets were assessed. In perifused islets, insulin secretion in response to IL-1 in the presence of 7 mM glucose averaged 313 +/- 43 pg/islet/min 35-40 min after the onset of stimulation. Release from control islets perifused in the presence of 7 mM glucose alone averaged 56 +/- 6 pg/islet/min at this time point. The addition of staurosporine together with IL-1 reduced insulin secretion at this time point to 88 +/- 21 pg/islet/min. This level of IL-1 caused significant increases in inositol phosphate accumulation in the presence of 7 mM glucose but not 2.75 mM glucose. Staurosporine was without a significant effect on inositol phosphate accumulation in response to the monokine. In contrast, nitrendipine (5 microM) inhibited insulin release and inositol phosphate accumulation in a parallel fashion. Finally, MOG significantly amplified release to the monokine without significantly affecting its impact on inositol phosphate accumulation. Nitrendipine or staurosporine blocked this amplifying effect of MOG on secretion. These results emphasize the role of PI hydrolysis in IL-1-induced insulin secretion and suggest further that calcium influx is essential for IL-1 to fully activate both PI hydrolysis and insulin secretion.     

Phosphoinositide hydrolysis and insulin secretion in response to glucose stimulation are impaired in isolated rat islets by prolonged exposure to the sulfonylurea tolbutamide

Isolated rat islets of Langerhans were incubated for 2 h in a [3H]inositol-containing medium supplemented with 7 mM glucose and the sulfonylurea tolbutamide (50-200 microM). After labeling, the ability of these islets to respond during a subsequent perifusion to 20 mM glucose or 15 mM alpha-ketoisocaproate (KIC) was assessed. The following major observations were made. Prior exposure to tolbutamide inhibited [3H]inositol efflux, inositol phosphate accumulation, and the insulin secretory responses of subsequently perifused islets to 20 mM glucose stimulation. When present during the 2-h labeling period, the calcium channel blocker nitrendipine (500 nM), a compound that abolishes tolbutamide-induced increases in PI hydrolysis, blocked these inhibitory effects of tolbutamide. In addition, the diacylglycerol kinase inhibitor monooleoylglycerol (50 microM) restored the impaired second phase insulin secretory response noted after a 2-h tolbutamide exposure. Prior exposure to tolbutamide (200 microM) also desensitized the islet, in terms of [3H] inositol phosphate accumulation, [3H]inositol efflux, and insulin secretory responses, to 15 mM KIC. The inclusion of monooleoylglycerol during the stimulatory period with KIC restored second phase insulin secretion. The results support the conclusion that chronic tolbutamide-induced increases in PI hydrolysis render the beta-cell insensitive to a subsequent 20-mM glucose or 15-mM KIC stimulus. Blocking tolbutamide-induced increases in PI hydrolysis during the labeling period eliminates the adverse effects of the sulfonylurea. The ineffectiveness of glucose and KIC to maintain insulin secretory responses from prior tolbutamide-exposed islets appears to be the result of the inability of these agonists to appropriately activate PI hydrolysis.     

The desensitization of normal B-cells to glucose in vitro is transient and not related to high glucose levels

To examine the postulated phenomenon of glucotoxicity toward the B-cell, islets isolated from normal adult rats were cultured for 1-6 days in RPMI medium at various glucose concentrations. Insulin release and (pro)insulin biosynthesis by these islets were then measured in short term incubations. The 1-day cultured islets (at 9.7 mM glucose) displayed a deficient glucose-stimulated insulin release which was partially restored in the presence of forskolin (5 microM). By contrast they exhibited a consistent insulin release in response to ketoisocaproate (10 mM), 12-O-tetradecanoylphorbol-13-acetate (2 microM), or the combination of Ba2+ (2 mM) and theophylline (1.4 mM) in the absence of extracellular Ca2+. Desensitization of their B-cells was not specific for glucose, since glyceraldehyde (10 mM) or leucine (10 mM) also failed to stimulate insulin release. Desensitization was not related to glucose concentration (5.6, 9.7, or 16.7 mM) in the medium during the 1-day culture period and was restricted to the secretory function, with no impairment of the biosynthesis process. The desensitization to glucose was transient, and high glucose levels (9.7 and 16.7 mM) in the culture medium favored restoration of the subsequent secretory response to the hexose. Under conditions of recovery of B-cell sensitivity to glucose in vitro (5 days at 9.7 mM glucose), the secretory response to acute glucose was in fact significantly enhanced after an additional exposure (1 day) to very high glucose levels (22 or 55 mM). The present results argue against 1) the possibility that islets suffer from some unspecific decreased viability after a 1-day culture period; and 2) the hypothesis that glucose insensitivity in the 1-day cultured islets is primarily caused by a direct deleterious effect of high glucose concentrations on the B-cells. They, rather, reinforce the view that high glucose levels are actually crucial in the preservation of the insulin secretory response to glucose of islets maintained in tissue culture.     

A role for kisspeptin in islet function

Aims/hypothesis We investigated the production of kisspeptin (KISS1) and the KISS1 receptor, GPR54, in pancreatic islets and determined the effects of exogenous kisspeptin on insulin secretion.Methods RT-PCR and immunohistochemistry were used to detect expression of KISS1 and GPR54 mRNAs and the production of KISS1 and GPR54 in human and mouse islets and in beta (MIN6) and alpha- (alphaTC1) cell lines. The effects of KISS1 on basal and glucose-induced insulin secretion from mouse and human islets were measured in a perifusion system.Results KISS1 and GPR54 mRNAs were both detected in human and mouse islets, and GPR54 mRNA expression was also found in the MIN6 and alphaTC1 endocrine cell lines. In sections of mouse pancreas, KISS1 and GPR54 immunoreactivities were co-localised in both beta and alpha cells within islets, but were not detected in the exocrine pancreas. Exposure of mouse and human islets to KISS1 caused a stimulation of glucose-induced (20 mmol/l) insulin secretion, but had no effect on the basal rate of secretion at a sub-stimulatory concentration of glucose (2 mmol/l). In contrast, KISS1 inhibited insulin secretion from MIN6 cells at both 2 and 20 mmol/l glucose. KISS1 had no significant effect on glucagon secretion from mouse islets.Conclusions/interpretation This is the first report to show that the GPR54/KISS1 system is expressed in the endocrine pancreas, where it influences beta cell secretory function. These observations suggest an important role for this system in the normal regulation of islet function.     

Structure, function and transforming potential of the epidermal growth factor receptor

We have examined the pharmacology of the voltage-sensitive Ca2+ channels (VSCCs) that mediate gonadotropin secretion from primary cultures of rat pituitary cells, stimulated by either cell depolarization or by binding of gonadotropin-releasing hormone (GnRH). We also measured single-cell [Ca2+]i transients using fura-2 in gonadotropes identified by a reverse hemolytic plaque assay employing an antiserum to luteinizing hormone (LH). Cell depolarization evoked by either 50 mM K+ or 30 microM veratridine induced 2- to 6-fold increases in gonadotropin secretion over basal levels. GnRH caused 6- to 20-fold increases in follicle-stimulating hormone (FSH) and LH secretion, respectively, with maximal stimulation at 100 nM GnRH. K(+)- or GnRH-induced FSH release was largely prevented by co-incubation with 1 mM CdCl. Tetrodotoxin (TTX, 5 microM) prevented the veratridine-, but not the K(+)- or GnRH-induced, stimulation of FSH secretion. Nitrendipine (Ntd, 1 microM) produced 35-50% inhibition (NS) of both FSH and LH release stimulated by either 50 mM K+ or 100 nM GnRH. Ntd also inhibited the K(+)-induced [Ca2+]i rise (greater than 90%), as well as the secondary, plateau phase of the GnRH-induced elevation of [Ca2+]i (100% inhibition). Omega-conotoxin (omega-CgTx, 100 nM) partially suppressed FSH and LH release (NS) due to both K+ (33% each) and GnRH (44% and 18%, respectively). omega-CgTx showed variable effects on [Ca2+]i transients evoked by K+ or GnRH ranging from clear inhibition to no effect. We conclude that influx of extracellular Ca2+ is one of several fundamental events underlying the depolarization- or receptor-activated release of LH and FSH, and that this influx can be inhibited by dihydropyridine-sensitive ('L') Ca2+ channels. Two classes of L-channels may exist in gonadotropes, that differ in their sensitivity to omega-CgTx.