Department of Environmental, Occupational, and Geospatial Health Sciences, CUNY Graduate School of Public Health and Health Policy, City University of New York, New York, NY 10027, USA.
Advanced Science Research Center (ASRC), Structural Biology Initiative, Graduate Center, City University of New York, New York, NY 10031, USA.
Nutrients. 2022 Jul 22;14(15):3022. doi: 10.3390/nu14153022.
Background: Pancreatic beta cells regulate bioenergetics efficiency and secret insulin in response to glucose and nutrient availability. The mechanistic Target of Rapamycin (mTOR) network orchestrates pancreatic progenitor cell growth and metabolism by nucleating two complexes, mTORC1 and mTORC2. Objective: To determine the impact of mTORC1/mTORC2 inhibition on amino acid metabolism in mouse pancreatic beta cells (Beta-TC-6 cells, ATCC-CRL-11506) using high-resolution metabolomics (HRM) and live-mitochondrial functions. Methods: Pancreatic beta TC-6 cells were incubated for 24 h with either: RapaLink-1 (RL); Torin-2 (T); rapamycin (R); metformin (M); a combination of RapaLink-1 and metformin (RLM); Torin-2 and metformin (TM); compared to the control. We applied high-resolution mass spectrometry (HRMS) LC-MS/MS untargeted metabolomics to compare the twenty natural amino acid profiles to the control. In addition, we quantified the bioenergetics dynamics and cellular metabolism by live-cell imaging and the MitoStress Test XF24 (Agilent, Seahorse). The real-time, live-cell approach simultaneously measures the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) to determine cellular respiration and metabolism. Statistical significance was assessed using ANOVA on Ranks and post-hoc Welch t-Tests. Results: RapaLink-1, Torin-2, and rapamycin decreased L-aspartate levels compared to the control (p = 0.006). Metformin alone did not affect L-aspartate levels. However, L-asparagine levels decreased with all treatment groups compared to the control (p = 0.03). On the contrary, L-glutamate and glycine levels were reduced only by mTORC1/mTORC2 inhibitors RapaLink-1 and Torin-2, but not by rapamycin or metformin. The metabolic activity network model predicted that L-aspartate and AMP interact within the same activity network. Live-cell bioenergetics revealed that ATP production was significantly reduced in RapaLink-1 (122.23 ± 33.19), Torin-2 (72.37 ± 17.33) treated cells, compared to rapamycin (250.45 ± 9.41) and the vehicle control (274.23 ± 38.17), p < 0.01. However, non-mitochondrial oxygen consumption was not statistically different between RapaLink-1 (67.17 ± 3.52), Torin-2 (55.93 ± 8.76), or rapamycin (80.01 ± 4.36, p = 0.006). Conclusions: Dual mTORC1/mTORC2 inhibition by RapaLink-1 and Torin-2 differentially altered the amino acid profile and decreased mitochondrial respiration compared to rapamycin treatment which only blocks the FRB domain on mTOR. Third-generation mTOR inhibitors may alter the mitochondrial dynamics and reveal a bioenergetics profile that could be targeted to reduce mitochondrial stress.
胰腺β细胞通过核形成两个复合物 mTORC1 和 mTORC2 来调节生物能量效率并响应葡萄糖和营养物质的可用性来分泌胰岛素。雷帕霉素（mTOR）机制靶标网络通过核形成两个复合物 mTORC1 和 mTORC2 来协调胰腺祖细胞的生长和代谢。目的：使用高分辨率代谢组学（HRM）和活线粒体功能，确定 mTORC1/mTORC2 抑制对小鼠胰腺β细胞（Beta-TC-6 细胞，ATCC-CRL-11506）中氨基酸代谢的影响。方法：将胰腺β TC-6 细胞用以下物质孵育 24 小时：RapaLink-1（RL）；Torin-2（T）；雷帕霉素（R）；二甲双胍（M）；RapaLink-1 和二甲双胍的组合（RLM）；Torin-2 和二甲双胍（TM）；与对照相比。我们应用高分辨率质谱（HRMS）LC-MS/MS 非靶向代谢组学来比较二十种天然氨基酸图谱与对照。此外，我们通过活细胞成像和 MitoStress Test XF24（Agilent， Seahorse）来量化生物能动力学和细胞代谢。实时活细胞方法同时测量耗氧率（OCR）和细胞外酸化率（ECAR），以确定细胞呼吸和代谢。使用 Ranks ANOVA 和事后 Welch t-检验评估统计显着性。结果：与对照相比，RapaLink-1、Torin-2 和雷帕霉素降低了 L-天冬氨酸水平（p = 0.006）。二甲双胍单独使用不会影响 L-天冬氨酸水平。然而，与对照相比，所有治疗组的 L-天冬酰胺水平均降低（p = 0.03）。相反，只有 mTORC1/mTORC2 抑制剂 RapaLink-1 和 Torin-2 降低了 L-谷氨酸和甘氨酸水平，而雷帕霉素或二甲双胍则没有。代谢活性网络模型预测 L-天冬氨酸和 AMP 在同一活性网络中相互作用。活细胞生物能学显示，与雷帕霉素（250.45 ± 9.41）和载体对照（274.23 ± 38.17）相比，RapaLink-1（122.23 ± 33.19）和 Torin-2（72.37 ± 17.33）处理的细胞中 ATP 产生显着减少，p < 0.01。然而，RapaLink-1（67.17 ± 3.52）、Torin-2（55.93 ± 8.76）或雷帕霉素（80.01 ± 4.36，p = 0.006）之间的非线粒体耗氧量没有统计学差异。结论：与仅阻断 mTOR 的 FRB 结构域的雷帕霉素相比，RapaLink-1 和 Torin-2 对 mTORC1/mTORC2 的双重抑制作用改变了氨基酸谱并降低了线粒体呼吸。第三代 mTOR 抑制剂可能会改变线粒体动力学并揭示可能有助于减少线粒体应激的生物能谱。
