Moffitt J H, Fielding B A, Evershed R, Berstan R, Currie J M, Clark A
Oxford Centre for Diabetes, Endocrinology and Metabolism, Churchill Hospital, Oxford, OX3 7LJ, UK.
Diabetologia. 2005 Sep;48(9):1819-29. doi: 10.1007/s00125-005-1861-9. Epub 2005 Aug 11.
AIMS/HYPOTHESIS: Long-term exposure of beta cells to lipids, particularly saturated fatty acids in vitro, results in cellular dysfunction and apoptosis (lipotoxicity); this could contribute to obesity-related diabetes. Our aims were to relate cell death to intracellular triglyceride concentration, composition and localisation following incubation of INS1 cells in saturated and unsaturated NEFA in high and low glucose concentrations.
Insulin-producing INS1 cells were cultured (24 h; 3 and 20 mmol/l glucose) with palmitic, oleic or linoleic acids and the resulting intracellular lipids were analysed by gas chromatography and microscopy. Cell death was determined by quantitative microscopy and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and glucose-stimulated insulin secretion by ELISA.
All NEFA (0.5 mmol/l, 0.5% albumin) inhibited glucose-stimulated (20 mmol/l) insulin secretion. Cytotoxicity was evident only with palmitic acid (p<0.05), in which case intracellular triglyceride consisted largely of tripalmitin in angular-shaped dilated endoplasmic reticulum. Cytotoxicity and morphological disruption were reduced by addition of unsaturated NEFA. Triglyceride content (control cells; 14.5 ng/mug protein) increased up to 10-fold following incubation in NEFA (oleic acid 153.2 ng/mug protein; p<0.05) and triglyceride and phospholipid fractions were both enriched with the specific fatty acid added to the medium (p<0.05).
CONCLUSIONS/INTERPRETATION: In INS1 cells, palmitic acid is converted in the endoplasmic reticulum to solid tripalmitin (melting point >65 degrees C), which could induce endoplasmic reticulum stress proteins and signal apoptosis; lipid-induced apoptosis would therefore be a consequence of the physicochemical properties of these triglycerides. Since cellular triglycerides composed of single species of fatty acid are not likely to occur in vivo, destruction of beta cells by saturated fatty acids could be predominantly an in vitro scenario.
目的/假设：β细胞长期暴露于脂质中，尤其是体外的饱和脂肪酸，会导致细胞功能障碍和凋亡（脂毒性）；这可能与肥胖相关的糖尿病有关。我们的目的是在高糖和低糖浓度下，将INS1细胞与饱和及不饱和游离脂肪酸（NEFA）孵育后，研究细胞死亡与细胞内甘油三酯浓度、组成及定位之间的关系。
将产生胰岛素的INS1细胞（培养24小时；葡萄糖浓度为3和20 mmol/L）与棕榈酸、油酸或亚油酸一起培养，然后通过气相色谱法和显微镜分析所得的细胞内脂质。通过定量显微镜和3-（4,5-二甲基噻唑-2-基）-2,5-二苯基四氮唑溴盐（MTT）法测定细胞死亡情况，并用酶联免疫吸附测定法（ELISA）检测葡萄糖刺激的胰岛素分泌。
所有NEFA（0.5 mmol/L，0.5%白蛋白）均抑制葡萄糖刺激（20 mmol/L）的胰岛素分泌。仅棕榈酸具有明显的细胞毒性（p<0.05），在这种情况下，细胞内甘油三酯主要由位于角形扩张内质网中的三棕榈精组成。添加不饱和NEFA可降低细胞毒性和形态破坏。在NEFA中孵育后，甘油三酯含量（对照细胞；14.5 ng/μg蛋白质）增加至10倍（油酸为153.2 ng/μg蛋白质；p<0.05），并且甘油三酯和磷脂组分均富含添加到培养基中的特定脂肪酸（p<0.05）。
结论/解读：在INS1细胞中，棕榈酸在内质网中转化为固态三棕榈精（熔点>65℃），这可能诱导内质网应激蛋白并引发凋亡信号；因此，脂质诱导的凋亡是这些甘油三酯物理化学性质的结果。由于由单一脂肪酸种类组成的细胞内甘油三酯在体内不太可能出现，饱和脂肪酸对β细胞的破坏可能主要是体外情况。
