Insulin resistance in obesity and type 2 diabetes (T2D) has been shown to be associated with decreased de novo fatty acid (FA) synthesis in adipose tissue. It is known that insulin can acutely stimulate FA synthesis in adipocytes, however the mechanisms underlying this effect are unclear. The rate-limiting step in FA synthesis is catalyzed by acetyl-CoA carboxylase (ACC), known to be regulated through inhibitory phosphorylation at S79 by the AMP-activated protein kinase (AMPK). Previous results from our laboratory showed an inhibition of AMPK activity by insulin, which was accompanied by PKB-dependent phosphorylation of AMPK at S485. However, if the S485 phosphorylation is required for insulin-induced inhibition of AMPK, or if other mechanisms underlie the reduced kinase activity, is not known. In order to investigate this, primary rat adipocytes were transduced with a recombinant adenovirus encoding AMPK-WT or a non-phosphorylatable AMPK S485A mutant. AMPK activity measurements by western blotting and in vitro kinase assay revealed that WT and S485A AMPK were inhibited to a similar degree by insulin, indicating that AMPK S485 phosphorylation is not required for insulin-induced AMPK inhibition. Further analysis suggested an involvement of decreased AMP/ATP ratios in the insulin-induced inhibition of AMPK activity, whereas a possible contribution of phosphodiesterases was excluded. Furthermore, we show that insulin-induced AMPK S485 phosphorylation also occurs in human adipocytes, suggesting it to be of an importance yet to be revealed. Altogether, this study increases our understanding of how insulin regulates AMPK activity, and with that, FA synthesis, in adipose tissue.

中文翻译：

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抑制AMPK响应脂肪细胞中的胰岛素：涉及AMPK pS485，PDE和细胞能量水平。

肥胖症和2型糖尿病（T2D）中的胰岛素抵抗已被证明与脂肪组织中从头脂肪酸（FA）合成减少有关。众所周知，胰岛素可以刺激脂肪细胞中FA的合成，但是尚不清楚这种作用的机制。FA合成中的限速步骤由乙酰辅酶A羧化酶（ACC）催化，该酶已知受AMP激活的蛋白激酶（AMPK）在S79处的抑制性磷酸化作用调节。我们实验室的先前结果显示，胰岛素可抑制AMPK活性，并伴随S485时AMPK的PKB依赖性磷酸化。但是，如果胰岛素诱导的AMPK抑制需要S485磷酸化，还是激酶活性降低的其他机制尚不清楚。为了对此进行调查，用编码AMPK-WT或不可磷酸化的AMPK S485A突变体的重组腺病毒转导原代大鼠脂肪细胞。通过蛋白质印迹和体外激酶测定法测量的AMPK活性表明，WT和S485A AMPK被胰岛素抑制的程度相似，表明胰岛素诱导的AMPK抑制不需要AMPK S485磷酸化。进一步的分析表明，降低的AMP / ATP比例与胰岛素诱导的AMPK活性抑制有关，而磷酸二酯酶的可能作用被排除在外。此外，我们表明胰岛素诱导的AMPK S485磷酸化也发生在人的脂肪细胞中，这表明它的重要性尚待揭示。总而言之，这项研究增进了我们对胰岛素如何调节AMPK活性以及FA合成，