Prasad Konkal-Matt R, Thimmalapura Pushpa-Rekha R, Woode Eunice A A, Nadler Jerry L
Division of Endocrinology and Metabolism, University of Virginia Health Science Center, Charlottesville 22908-2980, USA.
Biochem Biophys Res Commun. 2003 Aug 29;308(3):427-32. doi: 10.1016/s0006-291x(03)01418-9.
Leukocyte type 12-lipoxygenase (12-LO) is an enzyme specifically expressed in the beta cells of the pancreas. 12-LO oxidizes fatty acids such as arachidonic acid and linoleic acids to their respective hydroperoxides. Increased concentration of lipid hydroperoxides causes oxidative stress and this could lead to cellular dysfunction. Increased expression of 12-LO in beta cells has been observed with use of inflammatory cytokines and during the prediabetic phase of beta cell dysfunction in the Zucker diabetic fatty rat model. Also mice deficient in 12-LO expression show a decreased incidence of immune-mediated diabetes. To further understand the role of 12-LO in beta cell metabolism, we over-expressed mouse leukocyte type 12-LO in INS-1 cells (transformed rat beta cell line) using an adeno-associated virus (AAV) vector system. In 12-LO over-expressing cells, cell-associated 12-HETE levels increased approximately 5- and approximately 3-fold in the culture supernatant. In the cells over-expressing 12-LO, glucose-stimulated insulin secretion (GSIS) decreased by 25-30% one hour after exposure to high glucose (15mM). By 2h, GSIS decreased by 50-54% at high glucose levels. These data suggest that increased 12-LO products can reduce the synthesis, processing or secretion of insulin in beta cells. We next examined the effect of 12-LO over-expression on mitogen-activated protein kinases (MAPK) by Western blot analyses using antibodies specific for different phospho-MAP kinases. Over-expression of 12-LO led to an activation of c-Jun N-terminal kinase while it markedly reduced Erk1 and 2 phosphorylation (4-fold). Further, over-expression of 12-LO led to induction of apoptosis in beta cells as determined by DNA ladder assay. These results suggest that increased 12-LO plays a key role in altering beta cell metabolism. Thus, increased 12-LO expression can have a detrimental effect on pancreatic beta cell function and viability, suggesting that blockade of 12-LO activity or expression could provide a novel way to protect beta cells from inflammatory injury.
白细胞12 - 脂氧合酶（12 - LO）是一种在胰腺β细胞中特异性表达的酶。12 - LO将脂肪酸（如花生四烯酸和亚油酸）氧化为各自的氢过氧化物。脂质氢过氧化物浓度升高会导致氧化应激，进而可能导致细胞功能障碍。在使用炎性细胞因子时以及在Zucker糖尿病脂肪大鼠模型中β细胞功能障碍的糖尿病前期阶段，已观察到β细胞中12 - LO的表达增加。此外，12 - LO表达缺陷的小鼠免疫介导性糖尿病的发病率降低。为了进一步了解12 - LO在β细胞代谢中的作用，我们使用腺相关病毒（AAV）载体系统在INS - 1细胞（转化的大鼠β细胞系）中过表达小鼠白细胞12 - LO。在过表达12 - LO的细胞中，细胞相关的12 - HETE水平在培养上清液中增加了约5倍和约3倍。在过表达12 - LO的细胞中，暴露于高葡萄糖（15mM）1小时后，葡萄糖刺激的胰岛素分泌（GSIS）降低了25 - 30%。到2小时时，在高葡萄糖水平下GSIS降低了50 - 54%。这些数据表明，12 - LO产物增加可降低β细胞中胰岛素的合成、加工或分泌。接下来，我们通过使用针对不同磷酸化MAP激酶的特异性抗体进行蛋白质印迹分析，研究了12 - LO过表达对丝裂原活化蛋白激酶（MAPK）的影响。12 - LO的过表达导致c - Jun N端激酶的激活，同时显著降低了Erk1和2的磷酸化（4倍）。此外，通过DNA梯状分析确定，12 - LO的过表达导致β细胞凋亡的诱导。这些结果表明，12 - LO增加在改变β细胞代谢中起关键作用。因此，12 - LO表达增加可能对胰腺β细胞功能和活力产生有害影响，这表明阻断12 - LO活性或表达可能为保护β细胞免受炎性损伤提供一种新方法。
