Insulin secretion is stimulated by ethanol extract of Anemarrhena asphodeloides in isolated islet of healthy Wistar and diabetic Goto-Kakizaki Rats.

BACKGROUND: The hypoglycemic effect of extract of Anemarrhena asphodeloides has been accounted for by the substance mangiferin which increases insulin sensitivity. The present study aimed to investigate whether an ethanol extract of Anemarrhena asphodeloides would stimulate insulin secretion and if so, further elucidate the mechanism behind this effect. METHODS: Isolated pancreatic islets of normal Wistar rats and spontaneously diabetic Goto-Kakizaki (GK) rats were batch incubated or perifused to study effect of Anemarrhena asphodeloides extract (TH2) on insulin release. RESULTS: At 3.3 mM glucose, 2, 4, and 8 mg/ml TH2 increased the insulin release of Wistar rat islets 2.5-, 4.1-, and 5.7-fold, respectively (p < 0.05) and of GK rat islets 1.7-, 3.0-, and 6.3-fold, respectively (p < 0.01). Similarly at 16.7 mM glucose, 2, 4 and 8 mg/ml TH2 increased insulin release of Wistar rat islets 1.5-, 2.2-, and 3.8-fold, respectively (p < 0.05) and of GK rat 2.5-, 4.2-, and 11.9-fold, respectively (p < 0.01). In perifusions of islets, TH2 also increased insulin secretion that returned to basal levels when TH2 was omitted from the perifusate. Mangiferin had no effect on insulin secretion of islets. In islets depolarized by 30 mM KCl and B-cell K-ATP channels kept open by 0.25 mM diazoxide, TH2 (8 mg/ml) further enhanced insulin secretion at 3.3 but not at 16.7 mM glucose. Pertussis toxin suppressed the insulin stimulating effect of 2 and 8 mg/ml TH2 by 35 % and 47 % (p < 0.05 and p < 0.001, respectively). CONCLUSIONS: Ethanol extract of the roots of Anemarrhena asphodeloides contains a substance, TH2, that stimulates insulin secretion both at 3.3 and 16.7 mM glucose in islets of normal Wistar and diabetic GK rats. The mechanism behind TH2-stimulated insulin secretion involves an effect on the exocytotic machinery of the B-cell, mediated via pertussis toxin-sensitive Gi- (or Ge-) proteins.     

Glucose stimulates glucagon release in single rat alpha-cells by mechanisms that mirror the stimulus-secretion coupling in beta-cells

In isolated rat pancreatic alpha-cells, glucose, arginine, and the sulfonylurea tolbutamide stimulated glucagon release. The effect of glucose was abolished by the KATP-channel opener diazoxide as well as by mannoheptulose and azide, inhibitors of glycolysis and mitochondrial metabolism. Glucose inhibited KATP-channel activity by 30% (P<0.05; n=5) and doubled the free cytoplasmic Ca2+ concentration. In cell-attached recordings, azide opened KATP channels. The N-type Ca2+-channel blocker omega-conotoxin and the Na+-channel blocker tetrodotoxin inhibited glucose-induced glucagon release whereas tetraethylammonium, a blocker of delayed rectifying K+ channels, increased secretion. Glucagon release increased monotonically with increasing K+ concentrations. omega-Conotoxin suppressed glucagon release to 15 mM K+, whereas a combination of omega-conotoxin and an L-type Ca2+-channel inhibitor was required to abrogate secretion in 50 mM K+. Recordings of cell capacitance revealed that glucose increased the exocytotic response evoked by membrane depolarization 3-fold. This correlated with a doubling of glucagon secretion by glucose in intact rat islets exposed to diazoxide and high K+. In whole-cell experiments, exocytosis was stimulated by reducing the cytoplasmic ADP concentration, whereas changes of the ATP concentration in the physiological range had little effect. We conclude that glucose stimulates glucagon release from isolated rat alpha-cells by KATP-channel closure and stimulation of Ca2+ influx through N-type Ca2+ channels. Glucose also stimulated exocytosis by an amplifying mechanism, probably involving changes in adenine nucleotides. The stimulatory action of glucose in isolated alpha-cells contrasts with the suppressive effect of the sugar in intact islets and highlights the primary importance of islet paracrine signaling in the regulation of glucagon release.     

Morphological and functional characteristics of islets neoformed during tissue culture of fetal rat pancreas

Cell suspensions prepared by collagenase digestion of the pancreas of rat fetuses (21.5 days) were cultured for 7-9 days in RPMI medium containing 10 mM glucose. Exocrine cells disappeared rapidly, whereas fibroblasts and endocrine cells proliferated. These latter were first arranged in monolayers but progressively reorganized in neoformed islets essentially composed of B-cells. Total insulin content of the culture dishes increased until day 9, and fractional insulin release was about 20% per day. After 1 week, islets incubated in glucose-free medium released less than 1% of their insulin content over 2 h. Glucose (16.7 mM) caused a slower and weaker (3-fold) stimulation than 10 mM leucine or arginine (3-5-fold). The effects of the three secretagogues were potentiated by theophylline, but only those of glucose and leucine were inhibited by diazoxide. These neoformed islets thus retain a fetal character (relatively low responsiveness to glucose), but the stimulus-specificity of the inhibition by diazoxide is the same as in adult islets. This technique may be useful for studying the mechanisms which govern the organization of pancreatic endocrine cells in islets, and which underlie their functional maturation during the perinatal period.     

Adenosine triphosphate-sensitive K+ channels may not be the sole regulators of glucose-induced electrical activity in pancreatic B-cells.

Stimulation of insulin release by glucose requires Ca2+ influx in pancreatic B-cells. This influx occurs during phases of electrical activity (slow waves of membrane potential with superimposed spikes) that can be monitored with intracellular microelectrodes. It has been suggested that closure of ATP-sensitive K+ channels contributes to the increase in electrical activity (and, hence, in Ca2+ influx and insulin release) produced by suprathreshold (greater than 7 mM) concentrations of glucose. If this is the sole mechanism of control, the decrease in electrical activity that follows a decrease in glucose concentration should be mimicked by opening these ATP-sensitive K+ channels. This was achieved by diazoxide, which selectively and directly acts at the channel level (without decreasing B-cell metabolism), and azide, which indirectly opens the channels by inhibiting mitochondrial ATP production. Stepwise lowering of the glucose concentration from 15 to 8 mM progressively decreased electrical activity in B-cells. This decrease was characterized by a shortening of the slow waves and a lengthening of the intervals between the slow waves, with little change in slow wave frequency. Similar changes followed the addition of azide (250-750 microM) to a medium containing 15 mM glucose. In contrast, in the presence of 15 mM glucose, diazoxide (5-20 microM) considerably increased the interval duration, but did not shorten the slow waves, thus causing a marked fall in slow wave frequency. In B-cells persistently depolarized by 30 mM glucose, diazoxide restored slow waves and intervals that were much longer than those recorded when the same cells were stimulated by 15 mM glucose. In conclusion, decreasing mitochondrial ATP production with azide is more able to reproduce the effects of a decrease in glucose concentration on electrical activity in B-cells than a direct pharmacological opening of ATP-sensitive K+ channels with diazoxide. This suggests that ionic channels other than ATP-sensitive K+ channels are under metabolic control and may contribute to the regulation of electrical activity by glucose.     

Abnormal expression of pancreatic islet exocytotic soluble N-ethylmaleimide-sensitive factor attachment protein receptors in Goto-Kakizaki rats is partially restored by phlorizin treatment and accentuated by high glucose treatment

The role of glucotoxicity in dysregulation of islet exocytotic soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex proteins and insulin response was explored in the hyperglycemic Goto-Kakizaki (GK) rat. Syntaxin-1A and vesicle-associated membrane protein isoform 2, which drive insulin granule exocytotic fusion, and the associated nSec1, which modulates the SNARE complex assembly, were diminished in GK pancreatic islets to approximately 40% of the levels in control Wistar rat islets. Phlorizin treatment (12 d) induced normoglycemic control in GK rats, resulting in partial restoration of the insulin response to glucose. Furthermore, islet SNARE complex and nSec1 proteins increased by about 40%. Phlorizin treatment did not affect levels of islet SNARE proteins in controls or on the same SNARE complex proteins in GK rat brain. To examine the role of hyperglycemia per se, GK and control rat islets were exposed for 5 d in culture to 5.5 and 16.7 mM glucose. High glucose treatment greatly increased the levels of synaptosomal-associated membrane protein of 25 kDa and, less markedly, the levels of syntaxin-1A and nSec1 in control islets more than in GK rat islets, whereas levels were reduced in both. This was accompanied by sustained impairment of the insulin response to glucose in GK islets and a normal response in control islets. Thus, GK islets demonstrate dysregulation of SNARE protein expression, and their compensatory increase by high glucose exposure is abrogated. Conversely, normoglycemic control results in partial replenishment of these critical components of the insulin exocytotic machinery and improvement in the insulin response. We propose that dysregulation of SNARE proteins is an important mechanism behind glucotoxicity-mediated impairment of the insulin response to glucose.     

Diazoxide attenuates glucose-induced defects in first-phase insulin release and pulsatile insulin secretion in human islets

Humans with type-2 diabetes mellitus (TTDM) have hyperglycemia ( approximately 11 mM) and impaired glucose-mediated insulin secretion characterized by impaired first-phase insulin release (FPIR) and pulsatile insulin release. Culture of islets from nondiabetic humans in very high glucose concentrations ( approximately 20-30 mM) for 96 h causes impaired FPIR. We sought to determine 1). whether human islets cultured at a glucose concentration of approximately 11 mM (comparable to TTDM) recapitulates impaired insulin secretion in TTDM, specifically impaired FPIR and insulin pulse mass with an increased proinsulin/insulin (PI/I) secretion ratio; and 2). whether these changes can be attenuated by addition of diazoxide to islets cultured with 11 mM glucose. Islets cultured with 11 mM glucose for 96 h had 75% depleted insulin stores (P < 0.05), decreased FPIR and insulin pulse mass (P < 0.05), and an approximately 3-fold increase in the ratio of PI/I islet content and in secretion ratio (P < 0.05). Addition of diazoxide to islets cultured with 11 mM glucose decreased insulin secretion during static incubation, leading to relative preservation of insulin stores and enhanced insulin secretion during subsequent perifusion; FPIR increased by 162% (P < 0.05) and insulin pulse mass by 150% (P < 0.05) vs. no diazoxide. The mean islet PI/I content and islet PI/I secretion ratio were also decreased by approximately 70% (P < 0.05) by prior addition of diazoxide to islets during culture with 11 mM glucose. FPIR and insulin pulse mass were related to islet insulin stores (P < 0.001 for FPIR and P < 0.001 for pulse amplitude). In conclusion, the pattern of defects of insulin secretion present in TTDM (impaired FPIR and pulsatile insulin secretion, increased PI/I ratio) can be recapitulated in human islets cultured with 11 mM glucose for 96 h. These defects can be at least partially offset by concurrent inhibition of insulin secretion by diazoxide, which also preserves insulin stores. Defective insulin secretion in TTDM may be, at least in part, due to depletion of available insulin stores secondary to chronic increased demand (insulin resistance and hyperglycemia) in the setting of a decreased beta-cell mass.     

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.     

胰岛素分泌的第二细胞调控点作用机制

为探讨胰岛素分泌时β细胞内第二调控位点的作用机制，以小鼠胰岛细胞为材料，用二氮嗪（ｄｉａｚｏｘｉｄｅ）阻断β细胞膜上Ｋ＋－ＡＴＰ通道作用后，研究胰岛素分泌时的ＡＴＰ、二磷酸腺苷（ＡＤＰ）、三磷酸鸟苷（ＧＴＰ）和三磷酸尿苷（ＵＴＰ）代谢变化。在此条件下，胰岛素分泌量依然随葡萄糖浓度增加而增加；与此同时，ＡＴＰ随之增高，ＡＤＰ随之减少，而ＡＴＰ＋ＡＤＰ值则持续上升，ＡＴＰ／ＡＤＰ值与胰岛素分泌量呈高度的线性正相关；此外，ＧＴＰ和ＵＴＰ水平亦随葡萄糖浓度升高而升高。提示β细胞分泌胰岛素时存在着第二细胞调控点，ＡＴＰ／ＡＤＰ值在其作用机制中起重要作用，ＧＴＰ和ＵＴＰ也参与了胰岛素分泌的调控。     

Glucose enhances adenylyl cyclase responses in normal but not diabetic GK rat islets

In the GK rat model of type 2 diabetes, adenylyl cyclase (AC) expression and stimulation are increased. Whether the prevalent glucose level has any effects on AC responses is, however, unclear. We have studied concurrent insulin release and cyclic adenosine monophosphate (cAMP) generation in response to 5 microM forskolin in islets cultured for 48 hours in 5.5 or 11 mM glucose. Insulin release was impaired in GK rat islets, irrespective of culture condition, in response to 3.3 and 16.7 mM glucose and was fully restored by forskolin through exaggerated insulin responses. Stimulation of normal islets with 5 microM forskolin elicited different islet cAMP responses, which were dependent on the dose of glucose in the culture medium. Thus in normal islets cultured in 11 mM glucose, forskolin increased cAMP levels fivefold to sixfold at 3.3 and 16.7 mM glucose, whereas forskolin increased cAMP levels only twofold in islets cultured at 5.5 mM glucose. In GK islets, forskolin induced a consistently exaggerated approximately eightfold increase in cAMP generation irrespective of glucose concentration in the culture medium. In conclusion, culturing normal islets at hyperglycemic glucose levels (11 mM) primed and markedly enhanced cAMP generation in response to forskolin.     

Abnormal insulin secretion and glucose metabolism in pancreatic islets from the spontaneously diabetic GK rat

Summary Insulin secretion and islet glucose metabolism were compared in pancreatic islets isolated from GK/Wistar (GK) rats with spontaneous Type 2 (non-insulin-dependent) diabetes mellitus and control Wistar rats. Islet insulin content was 24.5±3.1 U/ng islet DNA in GK rats and 28.8±2.5 U/ng islet DNA in control rats, with a mean (±SEM) islet DNA content of 17.3±1.7 and 26.5±3.4 ng (p < 0.05), respectively. Basal insulin secretion at 3.3 mmol/l glucose was 0.19±0.03 · ng islet DNA^–1· h^–1 in GK rat islets and 0.40±0.07 in control islets. Glucose (16.7 mmol/l) stimulated insulin release in GK rat islets only two-fold while in control islets five-fold. Glucose utilization at 16.7 mmol/l glucose, as measured by the formation of ³H₂O from [5-³ H]glucose, was 2.4 times higher in GK rat islets (3.1±0.7 pmol · ng islet DNA^–1 · h^–1) than in control islets (1.3±0.1 pmol · ng islet DNA^–1 · h^–1; p<0.05). In contrast, glucose oxidation, estimated as the production of ¹⁴CO₂ from [U-¹⁴C]glucose, was similar in both types of islets and corresponded to 15±2 and 30±3 % (p<0.001) of total glucose phosphorylated in GK and control islets, respectively. Glucose cycling, i. e. the rate of dephosphorylation of the total amount of glucose phosphorylated, (determined as production of labelled glucose from islets incubated with ³H₂O) was 16.4±3.4% in GK rat and 6.4±1.0% in control islets, respectively (p<0.01). We conclude that insulin secretion stimulated by glucose is markedly impaired in GK rat islets. Glucose metabolism is also altered in GK rat islets, with diminished ratio between oxidation and utilization of glucose, and increased glucose cycling, suggesting links between impaired glucose-induced insulin release and abnormal glucose metabolism.     

Carbachol restores insulin release in diabetic GK rat islets by mechanisms largely involving hydrolysis of diacylglycerol and direct interaction with the exocytotic machinery

In several models of insulin resistance, cholinergically induced insulin secretion is augmented. We studied here whether this also is present in the spontaneously diabetic GK (Goto-Kakizaki) rat pancreas. Using carbachol (50 micromol/L), enhanced insulin release was elicited in perfused pancreas under normal or depolarized conditions in GK compared with control rats at 3.3 mmol/L glucose (p < 0.03). Carbachol fully normalized insulin secretion in GK rats at 16.7 mmol/L glucose through an effect abolished by atropine. Similarly, direct stimulation of protein kinase C (PKC) with the DAG-permeable compound 1-oleoyl-2-acetyl-sn-glycerol (OAG, 300 micromol/L) induced more pronounced insulin release in GK islets than in control islets. The diacylglycerol (DAG) lipase inhibitor RHC-80267 (35 micromol/L) significantly reduced carbachol effects in control and GK islets, but had no effect on OAG-induced insulin release. The enhanced insulinotropic effects of carbachol in GK islets was not accompanied by increased cyclic adenosine monophosphate (cAMP) or arachidonic acid (AA) formation in GK when compared with control islets. In conclusion, cholinergic stimulation induced enhanced insulin release in diabetic GK islets. This is largely mediated through mechanisms involving hydrolysis of DAG to AA and interaction with exocytotic steps of insulin release.     

Cholinergic agonists prime the beta-cell to glucose stimulation

The ability of the cholinergic agonists carbachol or acetylcholine to stimulate insulin release, activate phosphoinositide hydrolysis, and prime the beta-cell to the insulin stimulatory effect of 7.5 mM glucose was assessed. In the presence of 7 mM glucose, but not 2.75 mM glucose, 1 mM carbachol evoked a sustained insulin secretory response. At both glucose levels, carbachol stimulated phosphoinositide hydrolysis, an event monitored in myo-[2-3H]inositol-prelabeled islets by increases in [3H]inositol efflux and labeled inositol phosphate accumulation. Prior exposure to carbachol (0.1-1 mM) resulted in a dose-dependent increase in the subsequent insulin secretory response to 7.5 mM glucose. This sensitization developed within 2 min and lasted for at least 45 min after carbachol removal from the perifusion medium. Carbachol pretreatment also sensitized the islet to either 200 microM tolbutamide or 10 mM arginine. Prior exposure to 1 mM acetylcholine induced a similar proemial sensitization to a subsequent challenge with glucose. These results demonstrate that even though cholinergic stimulation increases phosphoinositide hydrolysis, this event is insufficient to initiate sustained insulin secretion from islets exposed to a low (2.75 mM) glucose concentration. However, this increase in phosphoinositide hydrolysis sensitizes islets to a subsequent challenge with one of several different stimuli, including glucose. Hence, this sensitization of islets to physiologically relevant glucose concentrations may represent the major contribution of vagal stimulation to the regulation of insulin secretion.     

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.     

L型和P／Q型钙通道对大鼠胰岛β细胞胰岛素双相分泌的调控

目的通过胰腺原位灌注实验结合钙通道阻断剂的应用探讨L型和P／Q型钙通道在调控大鼠胰岛B细胞胰岛素分泌中的作用。方法健康雄性sD大鼠24只,采用随机数字表法分为对照组（n=8）、L型通道阻断组（n=8）和P／Q型通道阻断组（n=8）。用戊巴比妥钠对大鼠进行腹腔注射麻醉,沿腹正中线做一切口,将胰腺与脾、胃、结肠等组织器官分离,分别在腹主动脉和门静脉上适当的位置插入硅胶导管。各组大鼠均先用37℃改良三羧酸循环4一羟乙基哌嗪乙磺酸缓冲液（modifiedKrebs—RingerHEPES）通过腹主动脉插管以1ml／min流速预灌注40min,随后开始正式灌注实验并收集门静脉插管的流出液。对照组先用含3．3mmol／L葡萄糖的ModifiedKrebs—RingerHEPES低糖缓冲液对完整的大鼠胰腺灌注10min,再用含16．7mmol／L葡萄糖的ModifiedKrebs—Ringer HEPES高糖缓冲液灌注25min;L型通道阻断组先用低糖灌注液灌注10min,再用50mnol／L依拉地平一高糖灌注液灌注25rain;P／Q型通道阻断组用低糖灌注液灌注10min,再用10nmol／L漏斗网蛛毒素一高糖灌注液灌注25min。各组均从门静脉收集每分钟的流m液,用放射免疫分析法检测其胰岛素水平。以第1～10分钟为低糖灌注液灌注期,第11～15分钟为第1时相,第16～35分钟为第2时相,计算胰岛素分泌率。多组数据比较采用单因素方差分析,进一步两两比较采用SNK．q检验。结果对照组、L型通道阻断组和P／Q型通道阻断组大鼠的体重、空腹血糖值比较差异均无统计学意义（F值分别为1．224、0．377,均P〉0．05）。大鼠基础胰岛素分泌率各组间比较差异均无统计学意义（F=0．095,P〉0．05）。高糖刺激下,L型通道阻断组的胰岛素1相和2相分泌率及分泌峰值均显著低于对照组和P／Q型通道阻断组[分别为（402±24）、（744±32）、（728±42）~U／min,F=163．879,P〈0．01;（323±29）、（568±40）、（563±19）~U／min,F=95．043,P〈0．0l;（521±43）、（1134±146）、（1083±199）p＋U／min,F：27．713,P〈0．01];P／Q型通道阻断组的胰岛素1相和2相分泌率及分泌峰值与对照组比较差异均无统计学意义（t值分别为163．879、95．043、27．713,均P〉0．05）。结论阻断L型钙通道能显著抑制高糖刺激的大鼠胰岛B细胞胰岛素双相分泌,阻断P／Q型钙通道对大鼠胰岛p细胞胰岛素的分泌未见显著影响,表明正常大鼠胰岛p细胞L型钙通道在调控胰岛素双相分泌中起主要作用,而P／Q型钙通道对胰岛素双相分泌的调控并无显著作用。     

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)     

Perifusion of monolayer-cultured B cells of the infant rat: a comparative study of the effects of 2-deoxy-2-fluoroglucose and iodoacetic acid

Cell culture techniques for monolayer islets of 3-week-old rat pancreases and the responsiveness of B cells are described. In this procedure, whole pancreatic tissues from 3-week-old rats were enzymatically dispersed and then cultured in a medium with 5.5 mM glucose plus 1 mM 2-deoxy-2-fluoroglucose or with 5.5 mM glucose following a 3-day exposure to a medium with 5.5mM glucose plus 5 microM iodoacetic acid. The use of 2-deoxy-2-fluoroglucose or iodoacetic acid allowed a selective deletion of fibroblasts, yielding large clusters that consisted mostly of islet cells. The immunocytochemical evaluation of the islet cells in these cultures showed that approximately 70% are B-cells, 20% A-cells, and 10% D-cells. On day 0, the response to 16.7 mM glucose included only a small rise in insulin secreted during the first and the second phase, and the response to 10 mM of leucine or 2-ketoisocaproate was monophasic. After being cultured for 7 days, all three secretagogues markedly stimulated insulin secretion by B cells cultured in both media, resulting in an enhancement of the biphasic pattern. However, quantitative relationships differed. Thus, the total response from B cells in 2-deoxy-2-fluoroglucose during a 30-min stimulation with glucose and leucine was significantly higher (1.6- and 1.9-fold respectively) than that from B cells in 5.5 mM glucose, although there was no significant difference in insulin secretion evoked by 2-ketoisocaproate. Furthermore, in the former B cells, the amount of insulin secreted during the second phase was 84-94% of the total insulin secretion, and in the latter it was 66-76%. Addition of 1 mM 3-isobutyl-1-methylxanthine and 10 microM forskolin resulted in a significant increase in insulin secretion by B cells in 2-deoxy-2-fluoroglucose, whereas there was no difference in the increase of insulin secretion induced by 16.7 mM glucose and 200 nM 12-o-tetradecanoyl phorbol-13-acetate. In monolayer cultures that had been maintained in both media for 15 days, the second phase of insulin secretion due to the secretagogues was slightly decreased, but the biphasicity in the response was well preserved. In conclusion, the present results suggest that B cells of 3-week-old rats may be still immature, and that the medium with 2-deoxy-2-fluoroglucose is beneficial to the continued maturation of the B-cell function in vitro.     

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.     

Interleukin-1 alpha exerts glucose-dependent stimulatory and inhibitory effects on islet cell phosphoinositide hydrolysis and insulin secretion

Isolated rat islets were incubated with myo-[2-3H]inositol to label their phosphoinositides (PI). Labeling was carried out in the presence of various glucose levels (2.75-10 mM) with or without human recombinant interleukin-1 alpha (IL-1). After the labeling period, insulin release, [3H]inositol efflux, and the accumulation of labeled inositol phosphates in perfused islets were assessed under various conditions. The following major observations were made. 1) In islets labeled for 2 h with [3H]inositol in the presence of 2.75 mM glucose, subsequent perifusion with 5.0 nM IL-1 increased insulin output, [3H]inositol efflux, and [3H]inositol phosphate accumulation in the simultaneous presence of 7 mM, but not 2.75 mM, glucose. 2) Mannoheptulose, a competitive inhibitor of islet glucokinase, blocked the stimulatory effects of IL-1 noted in the presence of 7 mM glucose. In other experiments, the conditions used during the 2-h labeling period with myo-[2-3H]inositol were varied. The following major observations were made in islets subsequently stimulated during the perifusion with 20 mM glucose. 3) Islets labeled with [3H]inositol in the presence of 2.75 mM glucose with or without 5.0 nM IL-1 responded with similar increases in PI hydrolysis and insulin output. 4) Compared to that with 2.75 mM glucose alone, labeling in the presence of 7 mM glucose alone was without any adverse effect on the subsequent PI and insulin responses of perifused islets to 20 mM glucose. 5) Labeling in the presence of 7 mM glucose plus 5.0 nM IL-1 resulted in a significant reduction in the subsequent PI and insulin responses. 6) These inhibitory effects of IL-1 were abolished if mannoheptulose was included during the 2-h incubation with 7 mM glucose plus 5.0 nM IL-1. 7) The diacylglycerol kinase inhibitor 1-monooleoylglycerol (100 microM) significantly restored insulin output after IL-1 exposure (with 7 mM glucose). 8) Similar to the results obtained with 7 mM glucose plus IL-1, incubation of islets with 8-10 mM glucose alone produced dose-dependent impairments of [3H]inositol efflux patterns and inositol phosphate accumulation. Insulin secretion was also impaired. These results demonstrate that IL-1 has glucose-dependent stimulatory and inhibitory effects on beta-cell function. Both effects appear to involve alterations in islet PI hydrolysis.     

Effects of islet amyloid polypeptide (IAPP) on insulin biosynthesis or secretion in rat islets and mouse beta TC3 cells. Biosynthesis of IAPP in mouse beta TC3 cells.

Effects of rat islet amyloid polypeptide (IAPP) on insulin biosynthesis and secretion were examined in isolated rat islets and mouse beta TC3 cells. Culture of islets or mouse beta TC3 cells for 24 h in the presence of 10(-6) M IAPP and 5.5 mM glucose had no effect on insulin mRNA levels. The rates of proinsulin biosynthesis were not altered in islets incubated in 10(-4)-10(-9) M IAPP. In beta TC3 cells, proinsulin biosynthesis was stimulated by glucose, though no effects of IAPP were shown. Addition of 10(-5) M IAPP to islets incubated in 11 mM glucose decreased the fractional insulin secretion rates; however, the secretion of insulin from beta TC3 cells was not affected by 10(-5) M IAPP. On the other hand, mouse beta TC3 cells expressed the elevated level of IAPP mRNA. Metabolic labeling of beta TC3 cells revealed the synthesis of both proIAPP and mature IAPP. In pulse chase experiments, proIAPP was processed to IAPP in a manner similar to proinsulin. These data indicate that IAPP is a possible polypeptide hormone synthesized in pancreatic beta cells though it is unlikely that IAPP is a physiologically relevant modulator of insulin biosynthesis or 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.