Feedback inhibition of insulin on insulin secretion in isolated pancreatic islets

We have examined the effect of exogenous insulin on secretion vesicle margination and secretion vesicle lysis in isolated perifused rat pancreatic islets. Recruitment of somatostatin (SRIF) receptors to the plasma membrane was used as a marker of secretion vesicle margination, whereas insulin release reflected the process of secretion vesicle lysis. A newly designed perifusion protocol allows one to interrupt intermittently either secretion vesicle margination or secretion vesicle lysis. Islets were initially perifused with glucose (30, 100, 165, 200, or 300 mg/dl) in the presence of sodium isethionate. Sodium isethionate inhibits secretion vesicle lysis, but not the recruitment of SRIF receptors. Thus, the margination of secretion vesicles to the surface membrane continued without their lysis. Sodium isethionate was then removed, and islets were challenged with 400 microM isobutylmethylxanthine (IBMX). In the islets perifused with high glucose concentrations, IBMX lysed a greater number of vesicles and caused enhanced release of insulin. The presence of exogenous insulin during the initial phase of secretion vesicle margination did not affect subsequent IBMX-induced insulin secretion from the islets perifused with low glucose concentrations (30 or even 100 mg/dl). When the glucose concentration was increased to 165, 200, or 300 mg/dl, insulin significantly diminished IBMX-induced insulin release. In separate experiments, increasing concentrations of insulin (50, 100, and 200 microU/ml) reduced glucose-induced recruitment of SRIF receptors in a dose-dependent manner. Our observations strongly suggest the existence of a well balanced relationship between ambient glucose and insulin concentrations in terms of their positive and negative feedback actions on insulin release. Their influences seem to be exerted at the level of secretion vesicle margination at the plasma membrane.     

Pulsatile insulin secretion by human pancreatic islets.

Insulin is secreted in discrete bursts. These pulses are also present when individual or groups of islets are perifused. Interpretation of the measured frequency and magnitude of pulsatile hormone secretion requires an examination of the sensitivity and specificity of the methods for pulse detection and validation of these for the experimental apparatus and hormone assay in which they are applied. In the present study we achieve these aims for a perfusion method for measurement of pulsatile insulin release by human islets. A deconvolution technique previously developed for measurement of pulsatile hormone secretion in vivo was specifically validated for in vitro pulse detection in the present study. Deconvolution analysis reliably (>90%) detected insulin pulses with an amplitude 20% or more above baseline and recovered quantitatively the insulin secretion profile, insulin secretion rate, and insulin pulse mass from single as well as multiple perifused islets. Cluster analysis was less sensitive, but was able to detect most (>80%) pulses with an amplitude of 40% or more above baseline. With this limitation, cluster analysis is potentially useful for groups, but not single perifused human islets. Analysis of single human islets showed that enhanced insulin secretion by increased glucose concentrations in the perfusate is achieved by enhancing insulin pulse mass with no change in pulse frequency. Perfused single or groups of human islets exhibited an interpulse interval ( approximately 6-8 min) comparable to that observed in humans in vivo. Dynamic in vitro perifusion should facilitate studies of the mechanisms driving pulsatile insulin secretion.     

Beta-endorphin inhibits insulin secretion from isolated pancreatic islets

Intravenous administration of small doses of beta-endorphin causes immediate suppression of basal and glucose-stimulated insulin secretion in normal rabbits. The purpose of the present study was to determine if beta-endorphin directly inhibits glucose-stimulated insulin secretion from rabbit pancreatic islets. Islets were isolated from male New Zealand White rabbits and perifused for 1 h with medium containing 100 mg/dl glucose (M100) followed by a 1-h challenge with medium containing 300 mg/dl glucose (M300) with or without beta-endorphin and/or the specific opioid antagonist naloxone. Samples were collected every 5 min during the last 30 min of the baseline perifusion with M100 and during the 1-h challenge with the stimulatory concentration of glucose (M300). Total insulin secretion for each 1-h period was calculated by adding the areas under the curves for twice the 30-min baseline period and for the 1-h challenge period. The mean +/- SE area for the control islets during perifusion with M100 was 5.9 +/- 0.8 microU/islet.h. M300 stimulated a 4.2-fold increase in the amount of insulin secreted (24.5 +/- 3.6 microU/islet.h). The stimulated rate of insulin release was sustained throughout the 1-h test period with M300, averaging 0.42 +/- 0.02 microU insulin/islet.min. beta-Endorphin inhibited glucose-stimulated insulin secretion in a concentration-dependent manner. Maximal suppression of insulin secretion to a level well below the baseline secretion rate was produced by 300 nM beta-endorphin (1.9 +/- 0.3 microU/islet.h). The first 15 min of glucose-stimulated insulin secretion was 6 times less sensitive to the inhibitory effect of beta-endorphin than was the next 45 min. The concentrations of beta-endorphin causing 50% inhibition of glucose-stimulated insulin secretion (IC50) for the 5- to 15-, 20- to 60-, and 5- to 60-min intervals were 1.96, 0.35, and 0.57 nM, respectively. Naloxone (3 microM) had no effect on glucose-stimulated insulin secretion, but partially antagonized the inhibitory effect of 30 nM beta-endorphin (10.2 +/- 2.9 microU/islet.h naloxone plus beta-endorphin vs. 2.6 +/- 1.1 microU/islet.h beta-endorphin; P less than 0.05). These data demonstrate that beta-endorphin, at low concentrations, has a direct inhibitory effect on insulin secretion, and they support the idea that a naloxone-sensitive beta-endorphin-binding component is present in pancreatic islets.     

Glucose stimulates pulsatile insulin secretion from human pancreatic islets by increasing secretory burst mass: dose-response relationships

Insulin is secreted almost exclusively in discrete bursts, and physiological regulation is accomplished by modulation of the pulse mass. How the integrity of contiguous anatomic structures in the human pancreas (islets, splanchnic innervation, exocrine tissue, local hormones) directs the coordinated insulin secretion is not known. We posed the hypothesis that glucose stimulates insulin secretion from isolated human islets by an amplification of insulin pulse mass with no change in pulse frequency and that the glucose dose-response curve for the regulation of insulin pulse mass mirrors that recognized in vivo. Islets from five nondiabetic cadaveric donors were perifused in a recently validated perifusion system at 4 mM and subsequently at 8, 12, 16, or 24 mM glucose. The effluent was collected in 1-min intervals and used for the measurement of insulin (ELISA). Pulsatile insulin secretion was analyzed by deconvolution analysis. Total insulin secretion increased progressively (P < 0.0001). This augmentation was due to amplified pulse mass (3-fold, 24 mM vs. 4 mM glucose; P < 0.0001) with no change in pulse interval (approximately 4 min). Pulsatile insulin secretion was stimulated most effectively in a physiologic concentration range of 4-8 mM. The islet insulin content was significantly correlated to the magnitude of first and second phase insulin secretion (P < 0.0001). The quantifiable orderliness of pulsatile insulin secretion rose with escalating glucose concentration (P = 0.02). In conclusion, glucose stimulates pulsatile insulin secretion from isolated human islets by amplification of insulin pulse mass without altering pulse interval. The in vitro concentration-response relationship is comparable with that observed in vivo. These data imply that transplanted human islets should be able to reproduce glucose-regulated insulin secretion as observed in the intact human pancreas.     

Activin A stimulates insulin secretion in cultured human pancreatic islets

Activin A is a dimeric glycoprotein showing a high sequence homology with transforming growth factor-beta (TGF-beta) and playing autocrine/paracrine actions in reproductive tissues. However, since the synthesis of activin is ubiquitous it may have a role in regulating cell growth and differentiation in several tissues. Previous studies showed that activin A is expressed by insulin-positive B cells of human pancreatic islets, and women with gestational diabetes have higher serum activin A levels than healthy pregnant women at the same gestational age. The present study aimed to evaluate the effect of activin A on insulin secretion from cultured human pancreatic islets. With this purpose human pancreatic islets were incubated with varying concentrations of activin A (0.1 to 10.0 nM). In absence of glucose, activin A did not modify insulin secretion at the different concentrations used. In absence of activin A, 8.3 mM and 16.7 mM glucose significantly increased insulin secretion, with a dose-dependent pattern. In presence of a non stimulatory concentration of glucose (3.3 mM), activin A significantly increased insulin secretion starting from low concentration (0.1 nM). Furthermore, the addition of activin A to 8.3 mM and 16.7 mM glucose induced an additional effect of the dose-dependent glucose-mediated insulin secretion (p<0.001). The present data could support a role for activin A in human endocrine pancreas in modulating insulin response to different glucose concentrations.     

Potentiators of insulin secretion modulate Ca sensitivity in rat pancreatic islets

Insulin secretion stimulated by 10 mM glucose was potentiated by forskolin, an activator of adenyl cyclase, by acetyl choline which may enhance turnover of inositol phospholipids, and by tetradecanoyl phorbol acetate (TPA), an activator of protein kinase C. None of these agents initiated insulin secretion in the presence of 2 mM glucose. Glucose-stimulated insulin secretion was markedly dependent on the concentration of extracellular Ca²⁺: at or below 10 μM Ca²⁺ no insulin secretion was evoked by glucose in freshly isolated islets. The threshold Ca²⁺ requirement was increased after culture of islets for 44 h. In both fresh and cultured islets the presence of a potentiator of secretion produced both a marked increase in the maximum rate of glucose-stimulated insulin secretion and a lowering of the requirement for extracellular Ca²⁺. Thus potentiation of insulin release involves an increase in the sensitivity of the B cell to Ca²⁺.     

Prolactin regulates adenylyl cyclase and insulin secretion in rat pancreatic islets

A role for prolactin (PRL) in the regulation of adenylyl cyclase (AC), cyclic AMP (cAMP) formation and insulin secretion was studied in isolated rat pancreatic islets cultured for 4 days at 5.5 mM glucose in the absence (control) or presence of PRL (500 ng/ml). In PRL-treated islets, stimulation by glucose (8 mM), carbamylcholine chloride (CCh) and phorbol dibutyrate increased cAMP levels 40, 89, and 151%, respectively, above similarly stimulated control islets without PRL. Moreover, insulin secretion in PRL-treated islets was more than doubled in response to 8 mM glucose plus glucagon-like peptide 1 compared with control islets. PRL also increased protein kinase C (PKC) activity in cultured islets. When islets were cultured at an insulin secretion desensitizing concentration of glucose (11 mM) for 4 days, there was a decrease in forskolin-stimulated cAMP production. However, the presence of PRL with 11 mM glucose prevented the glucose-induced decrease in cAMP production. Insulin secretion in response to 17 mM glucose was also higher (P<0.02) in islets cultured with 11 mM glucose plus PRL compared with islets cultured with 11 mM glucose alone. Islet AC types -III, -V, and -VI mRNA levels increased relative to 18s rRNA following PRL treatment. In contrast, culture at 11 mM glucose decreased relative AC-III, -V and -VI mRNA levels by as much as 50%. Culture with PRL prevented the decrease in AC expression during islet culture with 11 mM glucose, and the mRNA levels remained similar to control islets cultured at 5.5 mM glucose. Thus, PRL not only increased islet AC expression and activity and insulin secretory responsiveness, but also protected islets from chronic glucose-induced inhibition of these beta-cell activation parameters.     

Ghrelin对离体大鼠胰岛分泌胰岛素的影响

目的 观察Ghrelin对离体大鼠胰岛分泌胰岛素的影响.方法 将分离纯化的大鼠胰岛置于含不同浓度葡萄糖和Ghrelin的孵育液中孵育1h,应用放射免疫分析法测定孵育液中胰岛素浓度.结果 葡萄糖浓度＞5.6 mmol/L时,浓度≥10 nmol/L的Ghrelin对胰岛素分泌有明显抑制作用(P<0.05).结论 高浓度Ghrelin可显著抑制高浓度葡萄糖诱导的胰岛素分泌.     

白藜芦醇对原代大鼠胰岛葡萄糖刺激的胰岛素分泌的影响

分离SD大鼠胰岛接种于24孔板中,用不同浓度的葡萄糖和白藜芦醇分别培养1 h或24 h,结果表明白藜芦醇孵育大鼠胰岛1 h可呈剂苗依赖地抑制大鼠高糖刺激的胰岛素分泌,1、10和100 μmol/L白藜芦醇可以分别使胰岛素的分泌降低10%、35%(P<0.05)和80%(P<0.01).显微离子成像技术爪10μmol/L的白藜芦醇可以使高糖引起的β细胞内Ca2+浓度的升高减少60%(P<0.05).白藜芦醇可使软脂酸孵育24 h大鼠胰岛的胰岛素分泌恢复到对照组的75%(P<0.01),提示白藜芦醇短期可通过调控细胞内的Ca+浓度,而抑制原代胰岛高精刺激的胰岛素分泌,长期可改善软脂酸引起的β细胞损伤.     

白藜芦醇对原代大鼠胰岛葡萄糖刺激的胰岛素分泌的影响

Isolated freshly rat islets were transferred to 24-well plates and incubated with different concentrations of glucose or resveratrol for 1 or 24 h.The results showed that resveratrol dose-dependently inhibited glucose-stimulated insulin secretion from isolated rat islets after 1 h incubation,with 10%,35%,and 80% (P<0.05 or P<0.01) decrease at the concentrations of 1,I0,and 100 μmol/L.10 μmol/L resveratrol decreased the intracellular calcium concentration by 60% (P<0.05).After incubation for 24 h,resveratrol increased palmitatesuppressed insulin secretion to 75% (P<0.01) of control.These results suggest that resveratrol acutely inhibits insulin secretion from primary pancreatic islet via regulating intracellular calcium ion concentration,and in the long run resveratrol may protect β-cells from lipotoxicity.     

白藜芦醇对原代大鼠胰岛葡萄糖刺激的胰岛素分泌的影响

Isolated freshly rat islets were transferred to 24-well plates and incubated with different concentrations of glucose or resveratrol for 1 or 24 h.The results showed that resveratrol dose-dependently inhibited glucose-stimulated insulin secretion from isolated rat islets after 1 h incubation,with 10%,35%,and 80% (P<0.05 or P<0.01) decrease at the concentrations of 1,I0,and 100 μmol/L.10 μmol/L resveratrol decreased the intracellular calcium concentration by 60% (P<0.05).After incubation for 24 h,resveratrol increased palmitatesuppressed insulin secretion to 75% (P<0.01) of control.These results suggest that resveratrol acutely inhibits insulin secretion from primary pancreatic islet via regulating intracellular calcium ion concentration,and in the long run resveratrol may protect β-cells from lipotoxicity.     

Physical exercise and pancreatic islets: Acute and chronic actions on insulin secretion

Diabetes mellitus (DM) is a great public health problem, which attacks part of the world population, being characterized by an imbalance in body glucose homeostasis. Physical exercise is pointed as a protective agent and is also recommended to people with DM. As pancreatic islets present an important role in glucose homeostasis, we aim to study the role of physical exercise (chronic adaptations and acute responses) in pancreatic islets functionality in Wistar male rats. First, animals were divided into two groups: sedentary (S) and aerobic trained (T). At the end of 8 weeks, half of them (S and T) were submitted to an acute exercise session (exercise until exhaustion), being subdivided as acute sedentary (AS) and acute trained (AT). After the experimental period, periepididymal, retroperitoneal and subcutaneous fat pads, blood, soleus muscle and pancreatic islets were collected and prepared for further analysis. From the pancreatic islets, total insulin content, insulin secretion stimulated by glucose, leucine, arginine and carbachol were analyzed. Our results pointed that body adiposity and glucose homeostasis improved with chronic physical exercise. In addition, total insulin content was reduced in group AT, insulin secretion stimulated by glucose was reduced in trained groups (T and AT) and insulin secretion stimulated by carbachol was increased in group AT. There were no significant differences in insulin secretion stimulated by arginine and leucine. We identified a possible modulating action on insulin secretion, probably related to the association of chronic adaptation with an acute response on cholinergic activity in pancreatic islets.     

Chronic exposure to glibenclamide impairs insulin secretion in isolated rat pancreatic islets

We investigated the effect of 24 h exposure to 100 nmol/l glibenclamide on insulin secretion in isolated rat pancreatic islets. The insulin content was similar in control islets and in islets preincubated with 100 nmol/l glibenclamide for 24 h. In islets preexposed to glibenclamide: 1) the subsequent response to a maximal glibenclamide stimulatory concentration (10 mumol/l, 1 h at 37 C) was greatly reduced in comparison to control islets (0.69 +/- 0.20% vs 2.16 +/- 0.41%; mean +/- SE; n = 14; p less than 0.001); 2) the response to 100 mumol/l tolbutamide stimulation was also reduced (0.55 +/- 0.15% vs 2.38 +/- 0.44%; n = 8; p less than 0.001); 3) the response to 16.7 mmo/l glucose, both in the presence or in the absence of 1 mmol/l IBMX, a phosphodiesterase inhibitor, was also diminished by about 50% (1.79 +/- 0.39% vs. 3.22 +/- 0.42%; n = 14, p less than 0.001). In glibenclamide pretreated islets, blunted responses to stimuli were confirmed also by dynamic studies using a perifusion system. The effect of glibenclamide preincubation was fully reversible: when islets cultured in the presence of glibenclamide were transferred to a glibenclamide-free medium for further 24 h, insulin release in response to glibenclamide stimulation returned to control values. We conclude that prolonged exposure of rat pancreatic islets to glibenclamide induces a reversible desensitization to a variety of metabolic stimuli. The inhibition by prolonged glibenclamide exposure of a common pathway in the mechanism of insulin release is one possible explanation for these results.     

Glutamate decarboxylase 67 contributes to compensatory insulin secretion in aged pancreatic islets

Pancreatic islets play an essential role in regulating blood glucose levels. Age-dependent development of glucose intolerance and insulin resistance results in hyperglycemia, which in turn stimulates insulin synthesis and secretion from aged islets, to fulfill the increased demand for insulin. However, the mechanism underlying enhanced insulin secretion remains unknown. Glutamic acid decarboxylase 67 (GAD67) catalyzes the conversion of glutamate into γ-aminobutyric acid (GABA) and CO₂. Both glutamate and GABA can affect islet function. Here, we investigated the role of GAD67 in insulin secretion in young (3 month old) and aged (24 month old) C57BL/6J male mice. Unlike young mice, aged mice displayed glucose-intolerance and insulin-resistance. However, aged mice secreted more insulin and showed lower fed blood glucose levels than young mice. GAD67 levels in primary islets increased with aging and in response to high glucose levels. Inhibition of GAD67 activity using a potent inhibitor of GAD, 3-mercaptopropionic acid, abrogated glucose-stimulated insulin secretion from a pancreatic β-cell line and from young and aged islets. Collectively, our results suggest that blood glucose levels regulate GAD67 expression, which contributes to β-cell responses to impaired glucose homeostasis caused by advanced aging.     

Mechanism of fat-induced attenuation of glucose-induced insulin secretion from mouse pancreatic islets

In order to investigate the mechanism behind fat-induced inhibition of glucose-induced insulin secretion a selection of enzymes that may participate in regulation of pancreatic islet glucose oxidation was studied in islets isolated from mice that had been fed on a laboratory chow diet or on a high-fat diet for 10–12 weeks. At 20 mmol/L glucose production of ¹⁴CO₂ from [U-¹⁴C]-glucose was decreased 50% in islets from fat-fed mice. At 3.3 mmol/L glucose the glucose oxidation rate was similar in the two groups. The fatinduced decrease in glucose oxidation rate was correlated with a 35% decrease in the maximal glucokinase activity. The K_m for glucose was unchanged. No differences between the diet groups were found in the activities of hexokinase, phosphofructo-1-kinase, glucose 6-phosphatase or mitochondrial glycerophosphate dehydrogenase. After preincubation with 20 mmol/L glucose the activity of cytosolic Ca²⁺-independent as well as Ca²⁺-dependent phospholipase A₂ was unchanged by fat-feeding. However, the activity of lysophospholipase was significantly increased by fat feeding, which may result in lowered concentrations of islet lysophosphatidylcholine (lysoPC). It is concluded that in fat-induced diabetic animals a decrease in islet glucokinase may contribute considerably to the decrease in islet glucose oxidation rate. Furthermore, the study raises the possibility that changes in islet lysoPC may contribute to the fat-induced attenuation of glucose-induced insulin secretion. Received: 1 February 1999 / Accepted: 19 May 1999     

Immaturity of insulin secretion by pancreatic islets isolated from one human neonate

Human β‐cells are functionally mature by the age of 1 year. The timeline and mechanisms of this maturation are unknown owing to the exceptional availability of testable tissue. Here, we report the first in vitro study of insulin secretion by islets from a 5‐day‐old newborn. Glucose was inefficient alone, but induced insulin secretion, which was concentration‐dependent, showed a biphasic time‐course and was of similar magnitude as in infant islets when β‐cell cyclic adenosine monophosphate was raised by forskolin. Tolbutamide alone was effective in low glucose, but its effect was not augmented by high glucose. Metabolic amplification by glucose was thus inoperative, in contrast to amplification by cyclic adenosine monophosphate. Newborn islets showed high basal insulin secretion that could be inhibited by diazoxide or omission of CaCl₂. Postnatal acquisition of functional maturity by human β‐cells implicates control of basal secretion and production of metabolic signals able to activate both triggering and amplifying pathways of insulin secretion.     

胰淀素对大鼠胰岛素分泌的影响及其机理的探讨

目的　为研究胰淀素（ Ａｍｙｌｉｎ） 对大鼠胰岛细胞分泌胰岛素的影响和机理。方法　用放免和荧光方法分别检测经不同浓度胰淀素温育的大鼠胰岛细胞的胰岛素分泌量、细胞内 Ｃａ 《及ｃ Ａ Ｍ Ｐ 含量。结果　在胰淀素１０μｍｏｌ／ Ｌ 组中高糖刺激下的胰岛素含量、细胞内 Ｃａ 《含量，细胞内ｃ Ａ Ｍ Ｐ 含量分别为０ ．８９ ±０ ．２１ｎｇ／ ｍｌ、６３ ±１０ｎｍｏｌ／ Ｌ、２９ ．３ ±３ ．３ｐｍｏｌ／１ ×１０６ 细胞与对照组（１ ．７５ ±０ ．４２ｎｇ／ｍｌ、１３５ ±１０ｎ ｍｏｌ／ Ｌ、３６ ．３ ±７ ．４ｐｍｏｌ／ Ｌ） 比较，其中胰岛素分泌量、细胞内 Ｃａ 《含量均明显降低， Ｐ＜ ０ ．０１ ，细胞内ｃ Ａ Ｍ Ｐ 含量有减少趋势。结论　高浓度的胰淀素抑制了胰岛细胞内 Ｃａ 《升高和／ 或细胞内ｃ Ａ Ｍ Ｐ 形成，可能是胰岛素分泌减少的原因之一。     

Regulation of glucokinase in pancreatic islets by prolactin: a mechanism for increasing glucose-stimulated insulin secretion during pregnancy

Glucokinase activity is increased in pancreatic islets during pregnancy and in vitro by prolactin (PRL). The underlying mechanisms that lead to increased glucokinase have not been resolved. Since glucose itself regulates glucokinase activity in beta-cells, it was unclear whether the lactogen effects are direct or occur through changes in glucose metabolism. To clarify the roles of glucose metabolism in this process, we examined the interactions between glucose and PRL on glucose metabolism, insulin secretion, and glucokinase expression in insulin 1 (INS-1) cells and rat islets. Although the PRL-induced changes were more pronounced after culture at higher glucose concentrations, an increase in glucose metabolism, insulin secretion, and glucokinase expression occurred even in the absence of glucose. The presence of comparable levels of insulin secretion at similar rates of glucose metabolism from both control and PRL-treated INS-1 cells suggests the PRL-induced increase in glucose metabolism is responsible for the increase in insulin secretion. Similarly, increases in other known PRL responsive genes (e.g. the PRL receptor, glucose transporter-2, and insulin) were also detected after culture without glucose. We show that the upstream glucokinase promoter contains multiple STAT5 binding sequences with increased binding in response to PRL. Corresponding increases in glucokinase mRNA and protein synthesis were also detected. This suggests the PRL-induced increase in glucokinase mRNA and its translation are sufficient to account for the elevated glucokinase activity in beta-cells with lactogens. Importantly, the increase in islet glucokinase observed with PRL is in line with that observed in islets during pregnancy.