Mossakowski Medical Research Centre Polish Academy of Sciences, Laboratory of Molecular and Cellular Nephrology, Gdansk, Poland.
Mossakowski Medical Research Centre Polish Academy of Sciences, Laboratory of Molecular and Cellular Nephrology, Gdansk, Poland.
Biochim Biophys Acta Mol Basis Dis. 2018 Jan;1864(1):115-125. doi: 10.1016/j.bbadis.2017.10.014. Epub 2017 Oct 13.
Podocyte insulin sensitivity is critical for glomerular function, and the loss of appropriate insulin signaling leads to alterations and disorders featuring diabetic nephropathy. Energy-sensing pathways, such as AMP-dependent protein kinase (AMPK) and protein deacetylase SIRT1, have been shown to play an important role in insulin resistance. The absence of a stimulating effect of insulin on glucose uptake into podocytes after exposure to hyperglycemic conditions has been demonstrated to be related to a decreased level and activity of SIRT1 protein, leading to reduced AMPK phosphorylation. The present work was undertaken to investigate metformin's ability to restore the insulin responsiveness of podocytes by regulating SIRT1 and AMPK activities. Primary rat podocytes cultured with standard or high glucose concentrations for 5days were transfected with siRNAs targeting SIRT1, AMPKα1, or AMPKα2. SIRT1 activity was measured by a fluorometric method. Insulin-stimulated changes in glucose uptake were used to detect insulin resistance. Podocyte permeability was measured by a transmembrane albumin flux assay to examine podocytes functioning. Our results demonstrated that metformin activated SIRT1 and AMPK, prevented hyperglycemia-induced reduction of SIRT1 protein levels, ameliorated glucose uptake into podocytes, and decreased glomerular filtration barrier permeability. Furthermore, metformin activated AMPK in a SIRT1-independent manner, as the increase in AMPK phosphorylation after metformin treatment was not affected by SIRT1 downregulation. Therefore, the potentiating effect of metformin on insulin-resistant podocytes seemed to be dependent on AMPK, as well as SIRT1 activity, establishing multilateral effects of metformin action.
足细胞的胰岛素敏感性对肾小球功能至关重要，适当的胰岛素信号丢失会导致糖尿病肾病的改变和紊乱。能量感应途径，如 AMP 依赖的蛋白激酶（AMPK）和蛋白去乙酰化酶 SIRT1，已被证明在胰岛素抵抗中发挥重要作用。在高糖条件下暴露后，胰岛素对足细胞葡萄糖摄取的刺激作用丧失与 SIRT1 蛋白水平和活性降低有关，导致 AMPK 磷酸化减少。本研究旨在探讨二甲双胍通过调节 SIRT1 和 AMPK 活性恢复足细胞胰岛素反应的能力。用标准或高葡萄糖浓度培养 5 天的原代大鼠足细胞用靶向 SIRT1、AMPKα1 或 AMPKα2 的 siRNA 转染。通过荧光法测量 SIRT1 活性。通过检测葡萄糖摄取来检测胰岛素抵抗。通过跨膜白蛋白通量测定法测量足细胞通透性，以检查足细胞功能。我们的结果表明，二甲双胍激活了 SIRT1 和 AMPK，阻止了高血糖诱导的 SIRT1 蛋白水平降低，改善了葡萄糖摄取到足细胞中，并降低了肾小球滤过屏障的通透性。此外，二甲双胍以 SIRT1 非依赖性方式激活 AMPK，因为二甲双胍处理后 AMPK 磷酸化的增加不受 SIRT1 下调的影响。因此，二甲双胍对胰岛素抵抗足细胞的增强作用似乎依赖于 AMPK 以及 SIRT1 活性，从而确立了二甲双胍作用的多方面影响。
