Bone marrow fat infiltration is one of the hallmarks of aging and osteoporotic bones. Marrow adipocytes produce substantial amounts of palmitic acid (PA). PA is toxic to bone-forming osteoblasts in vitro, affecting their differentiation, function, and survival. Since rapamycin (RAP)-induced inhibition of target of rapamycin complex 1 (mTORC1) activates autophagy and prevents apoptosis, we hypothesized that RAP may preserve osteoblast viability and reduce PA-induced lipotoxicity. Normal human osteoblasts were incubated with RAP in the presence of a lipotoxic concentration of PA or vehicle for 24 and 48 hours. Expression of LC3 protein levels and the phosphorylation of the direct mTORC1 target p70S6K1-T389 were quantified by Western blot. Lysosomes and autophagosomes were studied using confocal fluorescence imaging, lysotracker, and live-cell imaging. RAP reduced PA-induced apoptosis. In addition, PA-induced autophagosome formation increased substantially over the time-course, an effect that was significantly regulated by the presence of RAP in the media. In addition, LC3I/II ratios were higher in PA-induced cells with RAP whereas p70S6K1-T389 were lower in PA and RAP together. In summary, this study highlights the role of the RAP-sensitive mTORC1 pathway in normal human osteoblasts under lipotoxic conditions. RAP-associated therapies could, potentially, be targeted for specific roles in osteoporosis and aging bone.

中文翻译：

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雷帕霉素通过调节细胞凋亡和自噬作用，影响棕榈酸酯诱导的成骨细胞脂毒性。

骨髓脂肪浸润是衰老和骨质疏松骨骼的标志之一。骨髓脂肪细胞产生大量的棕榈酸（PA）。PA在体外对成骨成骨细胞有毒，影响其分化，功能和存活。由于雷帕霉素（RAP）诱导的雷帕霉素复合物1（mTORC1）靶标的抑制作用会激活自噬并阻止细胞凋亡，因此我们假设RAP可以保留成骨细胞的活力并降低PA诱导的脂毒性。在脂毒性浓度的PA或赋形剂存在下，将正常人成骨细胞与RAP孵育24和48小时。通过Western印迹定量LC3蛋白的表达和直接mTORC1靶标p70S6K1-T389的磷酸化。使用共聚焦荧光成像，lysotracker，和活细胞成像。RAP减少了PA诱导的细胞凋亡。此外，PA诱导的自噬体的形成在整个时间过程中都显着增加，该效应由培养基中RAP的存在显着调节。此外，PA诱导的RAP细胞中LC3I / II比更高，而PA和RAP的p70S6K1-T389则更低。总而言之，这项研究突出了RAP敏感的mTORC1途径在脂毒性条件下在正常人成骨细胞中的作用。RAP相关疗法可能有针对性地在骨质疏松症和骨骼老化中发挥特定作用。在PA诱导的RAP细胞中，LC3I / II比值较高，而在PA和RAP中，p70S6K1-T389的LC3I / II比值较低。总而言之，这项研究突出了RAP敏感的mTORC1途径在脂毒性条件下在正常人成骨细胞中的作用。RAP相关疗法可能有针对性地在骨质疏松症和骨骼老化中发挥特定作用。在PA诱导的RAP细胞中，LC3I / II比值较高，而在PA和RAP中，p70S6K1-T389的LC3I / II比值较低。总而言之，这项研究突出了RAP敏感的mTORC1途径在脂毒性条件下在正常人成骨细胞中的作用。RAP相关疗法可能有针对性地在骨质疏松症和骨骼老化中发挥特定作用。