Anello M, Rabuazzo A M, Degano C, Caltabiano V, Patanè G, Vigneri R, Purrello F
Institute of Internal Medicine, Endocrinology and Metabolism, University of Catania, Italy.
Diabetes. 1996 Apr;45(4):502-6. doi: 10.2337/diab.45.4.502.
The present study was done to achieve a better understanding of the role of ionic flux alterations in glucose-induced desensitization of pancreatic beta-cells. Moreover, we investigated the reversibility of glucose-induced desensitization after different times of exposure to high glucose to ascertain the time necessary for desensitization reversal and to determine whether it depends on the length of high glucose exposure. Glucose desensitization was obtained by incubating rat pancreatic islets for 6 h in CMRL medium containing 16.7 mmol/l glucose. At the end of this period, insulin release, 86Rb efflux, and 45Ca uptake were measured in parallel experiments. In islets cultured at 16.7 mmol/l glucose, maximal glucose-induced insulin release was reduced (848 +/- 97 pg x islet-1 x 30 min-1) in comparison to islets incubated at 5.5 mmol/l glucose (1,436 +/- 144, n = 7, P < 0.01). In contrast, insulin content was similar in the two groups, being 41.0 +/- 2.7 and 47.8 +/- 2.2 ng/islet in islets exposed to 16.7 or 5.5 mmol/l glucose, respectively (P = 0.167). The effect of glucose on both 86Rb efflux and 45Ca uptake was also significantly reduced in 16.7 mmol/l glucose-cultured islets. 86Rb efflux was inhibited only 19 +/- 4% in islets cultured at high glucose with respect to 56 +/- 7% in control islets (n = 5, P < 0.001). 45Ca uptake was 10.5 +/- 1.7 pmol/islet (mean +/- SE, n = 9) in islets cultured at high glucose with respect to 19.7 +/- 2.4 pmol/islet in control islets (P < 0.001). In contrast, the effect of glyburide (10 micromol/l) on insulin release, 86Rb efflux, and 45Ca uptake was similar in islets exposed to 5.5 or 16.7 mmol/l glucose. All the abnormalities observed in islets cultured at 16.7 mmol/l glucose were promptly and simultaneously reversible after islets were transferred in culture medium at 5.5 mmol/l glucose; both insulin secretion and glucose effects on 86Rb efflux and 45Ca uptake returned to values similar to control islets within 5 min. Also, islets exposed to high glucose for a longer period (24 h) recovered from both secretory and ionic abnormalities after 5 min of incubation in CMRL medium at 5.5 mmol/l glucose. Reversal from glucose desensitization was slower (45 - 60 min) when islets were incubated at 5.5 mmol/l glucose in Krebs-Ringer HEPES buffer instead of CMRL medium. The present data suggest that ion flux and consequent membrane-potential changes play a key role in the mechanism leading to glucose-induced desensitization of pancreatic beta-cells. Because a normal response to glyburide was observed in islets exposed to high glucose, a proximal signal defect for closure of K+ channels rather than an intrinsic defect in the channel is likely.
本研究旨在更好地理解离子通量改变在葡萄糖诱导的胰腺β细胞脱敏中的作用。此外，我们研究了在不同时间暴露于高葡萄糖后葡萄糖诱导的脱敏的可逆性，以确定脱敏逆转所需的时间，并确定其是否取决于高葡萄糖暴露的时长。通过将大鼠胰岛在含有16.7 mmol/l葡萄糖的CMRL培养基中孵育6小时来实现葡萄糖脱敏。在此时间段结束时，在平行实验中测量胰岛素释放、86Rb外流和45Ca摄取。与在5.5 mmol/l葡萄糖中孵育的胰岛相比，在16.7 mmol/l葡萄糖中培养的胰岛中，最大葡萄糖诱导的胰岛素释放减少（848±97 pg×胰岛-1×30分钟-1）（1436±144，n = 7，P < 0.01）。相反，两组的胰岛素含量相似，暴露于16.7或5.5 mmol/l葡萄糖的胰岛中胰岛素含量分别为41.0±2.7和47.8±2.2 ng/胰岛（P = 0.167）。在16.7 mmol/l葡萄糖培养的胰岛中，葡萄糖对86Rb外流和45Ca摄取的作用也显著降低。与对照胰岛中的56±7%相比，高葡萄糖培养的胰岛中86Rb外流仅被抑制19±4%（n = 5，P < 0.001）。高葡萄糖培养的胰岛中45Ca摄取为10.5±1.7 pmol/胰岛（平均值±标准误，n = 9），而对照胰岛中为19.7±2.4 pmol/胰岛（P < 0.001）。相反，在暴露于5.5或16.7 mmol/l葡萄糖的胰岛中，格列本脲（10 μmol/l）对胰岛素释放、86Rb外流和45Ca摄取的作用相似。在将胰岛转移到含有5.5 mmol/l葡萄糖的培养基中后，在16.7 mmol/l葡萄糖中培养的胰岛中观察到的所有异常迅速且同时可逆；胰岛素分泌以及葡萄糖对86Rb外流和45Ca摄取的作用在5分钟内恢复到与对照胰岛相似的值。此外，在含有5.5 mmol/l葡萄糖的CMRL培养基中孵育5分钟后，暴露于高葡萄糖更长时间（24小时）的胰岛从分泌和离子异常中恢复。当胰岛在Krebs-Ringer HEPES缓冲液而非CMRL培养基中于5.5 mmol/l葡萄糖中孵育时，从葡萄糖脱敏的逆转较慢（45 - 60分钟）。目前的数据表明，离子通量以及随之而来的膜电位变化在导致胰腺β细胞葡萄糖诱导的脱敏的机制中起关键作用。因为在暴露于高葡萄糖的胰岛中观察到对格列本脲的正常反应，所以K+通道关闭的近端信号缺陷而非通道的内在缺陷可能是原因。
