Isolation of INS-1-derived cell lines with robust ATP-sensitive K+ channel-dependent and -independent glucose-stimulated insulin secretion
Isolation of INS-1-derived cell lines with robust ATP-sensitive K+ channel-dependent and -independent glucose-stimulated insulin secretion
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DOI：
10.2337/diabetes.49.3.424
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发表时间：
2000-03-01
期刊：
影响因子：
7.7
通讯作者：
Newgard, CB
中科院分区：
文献类型：
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作者：
Hohmeier, HE;Mulder, H;Newgard, CB
参与胰岛素分泌调节的生化机制尚未完全了解。大鼠INS-1细胞系已被用于获得该领域的洞察力，因为它响应于生理范围内的葡萄糖浓度而分泌胰岛素。然而，反应的幅度远小于在新鲜分离的大鼠胰岛中看到的。在目前的研究中，我们已经稳定转染INS-1细胞与含有人胰岛素原基因的质粒。在抗生素选择和克隆扩增后，发现67%的所得克隆在胰岛素分泌方面对葡萄糖反应不良（与3 mmol/l葡萄糖相比，15 mmol/l葡萄糖刺激小于或等于2倍），17%的克隆中度反应（2至5倍刺激），16%的克隆强烈反应（5至13倍刺激）。反应性的差异不能归因于胰岛素含量的差异。对其中一个强反应株系（832/13）的详细分析表明，其对葡萄糖的有效反应（平均10倍）在66个群体倍增期（类似于7.5个月的组织培养）内保持稳定，在6 mmol/l葡萄糖下具有半最大刺激。此外，在存在15 mmol/l葡萄糖的情况下，100 mmol/l异丁基甲基黄嘌呤（320%）、1 mmol/l油酸/棕榈酸酯（77%）和50 nmol/l胰高血糖素样肽1（60%）可显著增强胰岛素分泌，而卡巴胆碱无影响。磺酰脲甲苯磺丁脲也能增强葡萄糖刺激的胰岛素分泌（3 mmol/l葡萄糖时为3倍，15 mmol/l葡萄糖时为50%），并被二氮嗪消除，这表明832/13细胞中ATP敏感性K+通道（K-ATP）的运作。此外，当通过在去极化K+（35 mmol/l）中孵育细胞绕过K-ATP通道时，葡萄糖在832/13细胞中比在亲本INS-1细胞中更有效地刺激胰岛素分泌，这证明存在K-ATP通道非依赖性葡萄糖感知途径。我们的结论是，INS-1细胞的克隆选择允许分离的细胞系，表现出显着增强和稳定的葡萄糖和它的几个已知的增效剂的反应。这些细胞系可能是研究β细胞功能的有吸引力的新载体。
The biochemical mechanisms involved in regulation of insulin secretion are not completely understood. The rat INS-1 cell line has been used to gain insight in this area because it secretes insulin in response to glucose concentrations in the physiological range. However, the magnitude of the response is far less than that seen in freshly isolated rat islets. In the current study, we have stably transfected INS-1 cells with a plasmid containing the human proinsulin gene. After antibiotic selection and clonal expansion, 67% of the resultant clones were found to be poorly responsive to glucose in terms of insulin secretion (less than or equal to 2-fold stimulation by 15 mmol/l compared with 3 mmol/l glucose), 17% of the clones were moderately responsive (2- to 5-fold stimulation), and 16% were strongly responsive (5- to 13-fold stimulation). The differences in responsiveness could not be ascribed to differences in insulin content. Detailed analysis of one of the strongly responsive lines (832/13) revealed that its potent response to glucose (average of 10-fold) was stable over 66 population doublings (similar to 7.5 months of tissue culture) with half-maximal stimulation at 6 mmol/l glucose. Furthermore, in the presence of 15 mmol/l glucose, insulin secretion was potentiated significantly by 100 mmol/l isobutylmethylxanthine (320%), 1 mmol/l oleate/palmitate (77%), and 50 nmol/l glucagon-like peptide 1 (60%), whereas carbachol had no effect. Glucose-stimulated insulin secretion was also potentiated by the sulfonylurea tolbutamide (threefold at 3 mmol/l glucose and 50% at 15 mmol/l glucose) and was abolished by diazoxide, which demonstrates the operation of the ATP-sensitive K+ channel (K-ATP) in 832/13 cells. Moreover, when the K-ATP channel was bypassed by incubation of cells in depolarizing K+ (35 mmol/l), insulin secretion was more effectively stimulated by glucose in 832/13 cells than in parental INS-1 cells, which demonstrates the presence of a K-ATP channel-independent pathway of glucose sensing. We conclude that clonal selection of INS-1 cells allows isolation of cell lines that exhibit markedly enhanced and stable responsiveness to glucose and several of its known potentiators. These lines may be attractive new vehicles for studies of beta-cell function.