Preconditioning with Associated Blocking of Ca2+ Inflow Alleviates Hypoxia-Induced Damage to Pancreatic β-Cells

ObjectiveBeta cells of pancreatic islets are susceptible to functional deficits and damage by hypoxia. Here we aimed to characterize such effects and to test for and pharmacological means to alleviate a negative impact of hypoxia.Methods and DesignRat and human pancreatic islets were subjected to 5.5 h of hypoxia after which functional and viability parameters were measured subsequent to the hypoxic period and/or following a 22 h re-oxygenation period. Preconditioning with diazoxide or other agents was usually done during a 22 h period prior to hypoxia.ResultsInsulin contents decreased by 23% after 5.5 h of hypoxia and by 61% after a re-oxygenation period. Preconditioning with diazoxide time-dependently alleviated these hypoxia effects in rat and human islets. Hypoxia reduced proinsulin biosynthesis (3H-leucine incorporation into proinsulin) by 35%. Preconditioning counteracted this decrease by 91%. Preconditioning reduced hypoxia-induced necrosis by 40%, attenuated lowering of proteins of mitochondrial complexes I–IV and enhanced stimulation of HIF-1-alpha and phosphorylated AMPK proteins. Preconditioning by diazoxide was abolished by co-exposure to tolbutamide or elevated potassium (i.e. conditions which increase Ca2+ inflow). Preconditioning with nifedipine, a calcium channel blocker, partly reproduced effects of diazoxide. Both diazoxide and nifedipine moderately reduced basal glucose oxidation whereas glucose-induced oxygen consumption (tested with diazoxide) was unaffected. Preconditioning with diaxoxide enhanced insulin contents in transplants of rat islets to non-diabetic rats and lowered hyperglycemia vs. non-preconditioned islets in streptozotocin-diabetic rats. Preconditioning of human islet transplants lowered hyperglycemia in streptozotocin-diabetic nude mice.Conclusions1) Prior blocking of Ca2+ inflow associates with lesser hypoxia-induced damage, 2) preconditioning affects basal mitochondrial metabolism and accelerates activation of hypoxia-reactive and potentially protective factors, 3) results indicate that preconditioning by K+-ATP-channel openers has therapeutic potential for islet transplantations.

**研究目的**：胰岛β细胞（pancreatic islet beta cells）易受缺氧诱导的功能缺损与损伤。本研究旨在明确此类缺氧效应的特征，并筛选可减轻缺氧负面影响的药理学干预手段。
**方法与实验设计**：将大鼠及人源胰岛暴露于5.5小时的缺氧环境中，分别于缺氧结束后、以及22小时复氧周期结束后检测其功能与存活相关参数。采用二氮嗪（diazoxide）或其他试剂进行预处理的操作，通常于缺氧前22小时完成。
**实验结果**：缺氧5.5小时后，胰岛胰岛素含量下降23%；经22小时复氧后，该含量较对照组仅余61%。二氮嗪预处理可呈时间依赖性地减轻大鼠及人源胰岛的上述缺氧损伤。缺氧可使胰岛素原生物合成过程（以3H-亮氨酸掺入胰岛素原的量为检测指标）降低35%，而二氮嗪预处理可抵消91%的该降幅。此外，缺氧诱导的胰岛坏死率可被预处理降低40%，预处理还可缓解线粒体复合物I~IV蛋白水平的下调，并增强缺氧诱导因子1α（HIF-1-alpha）与磷酸化腺苷酸活化蛋白激酶（AMPK）的表达激活。联合应用甲苯磺丁脲（tolbutamide）或高钾环境（即可增加钙离子内流的条件）可完全阻断二氮嗪的预处理保护效应。采用钙通道阻滞剂硝苯地平（nifedipine）进行预处理，可部分复刻二氮嗪的保护作用。二氮嗪与硝苯地平均可适度降低基础状态下的葡萄糖氧化速率，但经二氮嗪测试的葡萄糖诱导性氧耗未受显著影响。在大鼠胰岛移植实验中，二氮嗪预处理可提升移植至非糖尿病大鼠体内的胰岛胰岛素含量；在链脲佐菌素诱导的糖尿病大鼠模型中，预处理组胰岛可较未预处理组更有效地降低宿主高血糖症状。此外，人源胰岛移植预处理可减轻链脲佐菌素诱导的糖尿病裸小鼠的高血糖状态。
**研究结论**：1）预先阻断钙离子内流可减轻缺氧诱导的胰岛损伤；2）预处理可调控基础线粒体代谢，并加速缺氧应答性潜在保护因子的激活；3）本研究结果表明，ATP敏感性钾通道开放剂的预处理方案，在胰岛移植领域具备潜在治疗价值。