Cersosimo Eugenio, Xu Xiaojing, Upala Sikarin, Triplitt Curtis, Musi Nicolas
Department of Medicine, Division of Diabetes and the Texas Diabetes Institute, University Health System and the University of Texas Health Science Center at San Antonio, San Antonio, Texas.
Department of Preventive and Social Medicine, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand.
Physiol Rep. 2014 Aug 19;2(8). doi: 10.14814/phy2.12123. Print 2014 Aug 1.
Differential activation/deactivation of insulin signaling, PI-3K and MAP-K pathways by high glucose and palmitate, with/out the insulin sensitizer pioglitazone (PIO), have been previously shown in vascular smooth muscle cells (VSMCs). To determine the biological impact of these molecular changes, we examined VSMC migration and proliferation ("M"&"P") patterns in similar conditions. VSMCs from healthy human coronary arteries were incubated in growth medium and "M"&"P" were analyzed after exposure to high glucose (25 mmol/L) ± palmitate (200 μmol/L) and ± PIO (8 μmol/L) for 5 h. "M"&"P" were assessed by: (1) polycarbonate membrane barrier with chemo-attractants and extended cell protrusions quantified by optical density (OD595 nm); (2) % change in radius area (2D Assay) using inverted microscopy images; and (3) cell viability assay expressed as cell absorbance (ABS) in media. "M" in 25 mmol/L glucose media increased by ~25% from baseline and % change in radius area rose from ~20% to ~30%. The addition of PIO was accompanied by a significant decrease in "M" from 0.25 ± 0.02 to 0.19 ± 0.02; a comparable decline from 0.25 ± 0.02 to 0.18 ± 0.02 was also seen with 25 mmol/L of glucose +200 μmol/L of palmitate. When PIO was coincubated with high glucose plus palmitate there was a 50% reduction in % change in radius. A ~10% increase in ABS, reflecting augmented "P" in media with 25 mmol/L glucose versus control was documented. The addition of PIO reduced ABS from 0.208 ± 0.03 to 0.183 ± 0.06. Both high glucose and palmitate showed ABS of ~0.140 ± 0.02, which decreased with PIO to ~0.120 ± 0.02, indicating "P" was reduced.
These results confirm that high glucose and palmitate stimulate VSMCs migration and proliferation in vitro, which is attenuated by coincubation with the insulin sensitizer PIO. Although, we cannot ascertain whether these functional changes are coincident with the activation/deactivation of signal molecules, our findings are consistent with the theory that differential regulation of insulin signaling pathways in VSMCs in insulin-resistant states plays an important role in inflammation, arterial wall thickening, and plaque formation during development of atherosclerosis.
先前已证实在血管平滑肌细胞（VSMC）中，高糖和棕榈酸可使胰岛素信号传导、PI-3K和MAP-K通路发生差异性激活/失活，无论有无胰岛素增敏剂吡格列酮（PIO）。为确定这些分子变化的生物学影响，我们在类似条件下检测了VSMC的迁移和增殖（“M”&“P”）模式。将来自健康人冠状动脉的VSMC在生长培养基中培养，在暴露于高糖（25 mmol/L）±棕榈酸（200 μmol/L）和±PIO（8 μmol/L）5小时后分析“M”&“P”。通过以下方法评估“M”&“P”：（1）带有化学引诱剂的聚碳酸酯膜屏障，并通过光密度（OD595 nm）对延伸的细胞突起进行定量；（2）使用倒置显微镜图像的半径面积变化百分比（二维分析）；（3）以培养基中的细胞吸光度（ABS）表示的细胞活力测定。在25 mmol/L葡萄糖培养基中的“M”较基线增加了约25%，半径面积变化百分比从约20%上升至约30%。添加PIO后，“M”显著降低，从0.25±0.02降至0.19±0.02；在25 mmol/L葡萄糖+200 μmol/L棕榈酸的情况下也观察到了类似的下降，从0.25±0.02降至0.18±0.02。当PIO与高糖加棕榈酸共同孵育时，半径变化百分比降低了50%。记录到在含有25 mmol/L葡萄糖的培养基中，ABS增加了约10%，反映出与对照相比“P”增加。添加PIO后，ABS从0.208±0.03降至0.183±0.06。高糖和棕榈酸的ABS均约为0.140±0.02，与PIO共同孵育后降至约0.120±0.02，表明“P”减少。
这些结果证实高糖和棕榈酸在体外刺激VSMC迁移和增殖，与胰岛素增敏剂PIO共同孵育可使其减弱。虽然我们无法确定这些功能变化是否与信号分子的激活/失活一致，但我们的发现与以下理论一致，即在胰岛素抵抗状态下VSMC中胰岛素信号通路的差异性调节在动脉粥样硬化发展过程中的炎症、动脉壁增厚和斑块形成中起重要作用。
