Karolinska Institutet, Department of Clinical Science and Education, Unit for Diabetes Research, Södersjukhuset, SE-118 83 Stockholm, Sweden.
Biochem Biophys Res Commun. 2012 Oct 12;427(1):91-5. doi: 10.1016/j.bbrc.2012.09.010. Epub 2012 Sep 11.
Evidence is emerging that elevated serum free fatty acids (hyperlipidemia) contribute to the pathogenesis of type-2-diabetes, and lipotoxicity is observed in many cell types. We recently published data indicating lipotoxic effects of simulated hyperlipidemia also in GLP-1-secreting cells, where the antidiabetic drug metformin conferred protection from lipoapoptosis. The aim of the present study was to identify mechanisms involved in mediating lipotoxicity and metformin lipoprotection in GLP-1 secreting cells. These signaling events triggered by simulated hyperlipidemia may underlie reduced GLP-1 secretion in diabetic subjects, and metformin lipoprotection by metformin could explain elevated plasma GLP-1 levels in diabetic patients on chronic metformin therapy. The present study may thus identify potential molecular targets for increasing endogenous GLP-1 secretion through enhanced viability of GLP-1 secreting cells in diabetic hyperlipidemia and obesity.
We have studied molecular mechanisms mediating lipotoxicity and metformin-induced lipoprotection in GLP-1-secreting L-cells in vitro, using the murine GLUTag cell line as a model. Diabetic hyperlipidemia was simulated in this cell system by addition of the fatty acid palmitate. Caspase-3 activity was used as a measure of GLUTag cell apoptosis. ROS production was determined using a fluorescent probe, and the activation of intracellular signaling pathways was assessed by Western blotting.
Palmitate increased ROS production in GLP-1 secreting cells, and the lipotoxic effects of palmitate were abolished in the presence of the antioxidant Trolox. Further, palmitate phosphorylated p38 and inhibition of p38 using the p38 inhibitor SB203580 significantly reduced palmitate-induced caspase-3 activity. Pre-incubation of palmitate with metformin further increased palmitate induced ROS production, while significantly reducing the expression of p38.
This study demonstrates that palmitate induces ROS production and that the palmitate induced lipotoxicity is the result of increased ROS production, where the ROS sensitive MKK3/6-p38 pathway mediates lipoapoptosis of GLP-1-secreting cells. Further, in the presence of simulated hyperlipidemia, metformin increases ROS production. However, metformin significantly decreases the expression of p38, indicating that metformin mediated lipoprotection involves reduced activity of the p38 signaling pathway.
有证据表明，血清游离脂肪酸升高（高血脂）会导致 2 型糖尿病的发病机制，并且许多细胞类型都存在脂毒性。我们最近发表的数据表明，模拟高血脂也会对 GLP-1 分泌细胞产生脂毒性作用，而抗糖尿病药物二甲双胍可防止脂肪细胞凋亡。本研究的目的是确定介导 GLP-1 分泌细胞脂毒性和二甲双胍脂蛋白保护的机制。这些由模拟高脂血症引发的信号事件可能是糖尿病患者 GLP-1 分泌减少的基础，而二甲双胍在糖尿病患者慢性二甲双胍治疗中升高血浆 GLP-1 水平可能是通过增强 GLP-1 分泌细胞的活力来解释的。因此，本研究可能会确定通过增强糖尿病高脂血症和肥胖症中 GLP-1 分泌细胞的活力来增加内源性 GLP-1 分泌的潜在分子靶标。
我们使用鼠 GLUTag 细胞系作为模型，在体外研究了介导 GLP-1 分泌细胞脂毒性和二甲双胍诱导的脂蛋白保护的分子机制。在该细胞系统中，通过添加脂肪酸棕榈酸来模拟糖尿病高脂血症。用 caspase-3 活性作为 GLUTag 细胞凋亡的测定。用荧光探针测定 ROS 产生，用 Western blot 测定细胞内信号通路的激活。
棕榈酸增加了 GLP-1 分泌细胞中的 ROS 产生，而抗氧化剂 Trolox 的存在则消除了棕榈酸的脂毒性作用。此外，棕榈酸磷酸化 p38，用 p38 抑制剂 SB203580 抑制 p38 可显著降低棕榈酸诱导的 caspase-3 活性。用二甲双胍预孵育棕榈酸进一步增加了棕榈酸诱导的 ROS 产生，同时显著降低了 p38 的表达。
本研究表明，棕榈酸诱导 ROS 产生，而棕榈酸诱导的脂毒性是 ROS 产生增加的结果，其中 ROS 敏感的 MKK3/6-p38 途径介导 GLP-1 分泌细胞的脂肪细胞凋亡。此外，在模拟高脂血症的情况下，二甲双胍增加了 ROS 的产生。然而，二甲双胍显著降低了 p38 的表达，表明二甲双胍介导的脂蛋白保护涉及 p38 信号通路活性的降低。
