The aim of the present study was to determine the role of individual PHLPP isoforms in insulin signaling and insulin resistance in neuronal cells. PHLPP isoforms were either silenced or overexpressed individually, and the effects were observed on individual Akt isoforms, AS160 and on neuronal glucose uptake, under insulin sensitive and resistant conditions. To determine PHLPP regulation itself, we tested effect of scaffold protein, Scribble, on PHLPP isoforms and neuronal glucose uptake. We observed elevated expression of both PHLPP1 and PHLPP2 in insulin resistant neuronal cells (Neuro-2A, mouse neuroblastoma; SHSY-5Y, human neuroblastoma) as well as in the whole brain lysates of high-fat-diet mediated diabetic mice. In insulin sensitive condition, PHLPP isoforms differentially affected activation of all Akt isoforms, wherein PHLPP1 regulated serine phosphorylation of Akt2 and Akt3, while PHLPP2 regulated Akt1 and Akt3. This PHLPP mediated Akt isoform specific regulation activated AS160 affecting glucose uptake. Under insulin resistant condition, a similar trend of results were observed in Akt isoforms, AS160 and glucose uptake. Over-expressed PHLPP isoforms combined with elevated endogenous expression under insulin resistant condition drastically affected downstream signaling, reducing neuronal glucose uptake. No compensation was observed amongst PHLPP isoforms under all conditions tested, indicating independent roles and pointing towards possible scaffolding interactions behind isoform specificity. Silencing of Scribble, a scaffolding protein known to interact with PHLPP, affected cellular localization of both PHLPP1 and PHLPP2, and caused increase in glucose uptake. PHLPP isoforms play independent roles via Scribble in regulating Akt isoforms differentially, affecting AS160 and neuronal glucose uptake.

中文翻译：

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PHLPP 亚型通过 Scribble 差异调节 Akt 亚型和 AS160 影响神经元胰岛素信号和胰岛素抵抗

本研究的目的是确定单个 PHLPP 亚型在胰岛素信号传导和神经元细胞胰岛素抵抗中的作用。PHLPP 亚型单独沉默或过表达，并且在胰岛素敏感和抵抗条件下观察到对个体 Akt 亚型、AS160 和神经元葡萄糖摄取的影响。为了确定 PHLPP 调节本身，我们测试了支架蛋白 Scribble 对 PHLPP 亚型和神经元葡萄糖摄取的影响。我们观察到 PHLPP1 和 PHLPP2 在胰岛素抵抗神经元细胞（Neuro-2A，小鼠神经母细胞瘤；SHSY-5Y，人神经母细胞瘤）以及高脂肪饮食介导的糖尿病小鼠的全脑裂解物中表达升高。在胰岛素敏感条件下，PHLPP 亚型对所有 Akt 亚型的激活有不同的影响，其中PHLPP1调节Akt2和Akt3的丝氨酸磷酸化，而PHLPP2调节Akt1和Akt3。这种 PHLPP 介导的 Akt 亚型特异性调节激活了影响葡萄糖摄取的 AS160。在胰岛素抵抗条件下，在 Akt 亚型、AS160 和葡萄糖摄取中观察到类似的结果趋势。过度表达的 PHLPP 亚型与胰岛素抵抗条件下升高的内源性表达相结合，极大地影响下游信号传导，减少神经元葡萄糖摄取。在所有测试条件下，PHLPP 亚型之间没有观察到补偿，表明独立的角色并指向亚型特异性背后可能的脚手架相互作用。已知与 PHLPP 相互作用的支架蛋白 Scribble 的沉默影响 PHLPP1 和 PHLPP2 的细胞定位，并引起葡萄糖摄取增加。PHLPP 亚型通过 Scribble 在差异调节 Akt 亚型中发挥独立作用，影响 AS160 和神经元葡萄糖摄取。