Background & Aims

Obesity-induced nonalcoholic fatty liver disease (NAFLD) develops, in part, via excess insulin-stimulated hepatic de novo lipogenesis, which increases, paradoxically, in patients with obesity-induced insulin resistance. Pleckstrin homology domain leucine-rich repeat protein phosphatase 2 (PHLPP2) terminates insulin signaling by dephosphorylating Akt; levels of PHLPP2 are reduced in livers from obese mice. We investigated whether loss of hepatic PHLPP2 is sufficient to induce fatty liver in mice, mechanisms of PHLPP2 degradation in fatty liver, and expression of genes that regulate PHLPP2 in livers of patients with NAFLD.

Methods

C57BL/6J mice (controls), obese db/db mice, and mice with liver-specific deletion of PHLPP2 (L-PHLPP2) fed either normal chow or high-fat diet (HFD) were analyzed for metabolic phenotypes, including glucose tolerance and hepatic steatosis. PHLPP2-deficient primary hepatocytes or CRISPR/Cas9-mediated PHLPP2-knockout hepatoma cells were analyzed for insulin signaling and gene expression. We performed mass spectrometry analyses of liver tissues from C57BL/6J mice transduced with Ad-HA-Flag-PHLPP2 to identify posttranslational modifications to PHLPP2 and proteins that interact with PHLPP2. We measured levels of mRNAs by quantitative reverse transcription polymerase chain reaction in liver biopsies from patients with varying degrees of hepatic steatosis.

Results

PHLPP2-knockout hepatoma cells and hepatocytes from L-PHLPP2 mice showed normal initiation of insulin signaling, but prolonged insulin action. Chow-fed L-PHLPP2 mice had normal glucose tolerance but hepatic steatosis. In HFD-fed C57BL/6J or db/db obese mice, endogenous PHLPP2 was degraded by glucagon and PKA-dependent phosphorylation of PHLPP2 (at Ser1119 and Ser1210), which led to PHLPP2 binding to potassium channel tetramerization domain containing 17 (KCTD17), a substrate-adaptor for Cul3-RING ubiquitin ligases. Levels of KCTD17 mRNA were increased in livers of HFD-fed C57BL/6J or db/db obese mice and in liver biopsies patients with NAFLD, compared with liver tissues from healthy control mice or patients without steatosis. Knockdown of KCTD17 with small hairpin RNA in primary hepatocytes increased PHLPP2 protein but not Phlpp2 mRNA, indicating that KCTD17 mediates PHLPP2 degradation. KCTD17 knockdown in obese mice prevented PHLPP2 degradation and decreased expression of lipogenic genes.

Conclusions

In mouse models of obesity, we found that PHLPP2 degradation induced lipogenesis without affecting gluconeogenesis. KCTD17, which is up-regulated in liver tissues of obese mice and patients with NAFLD, binds to phosphorylated PHLPP2 to target it for ubiquitin-mediated degradation; this increases expression of genes that regulate lipogenesis to promote hepatic steatosis. Inhibitors of this pathway might be developed for treatment of patients with NAFLD.

中文翻译：

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KCTD17通过胰高血糖素依赖性途径降解PHLPP2会促进肝脂肪变性

背景与目标

肥胖引起的非酒精性脂肪性肝病（NAFLD）部分是由于胰岛素刺激的新生肝脂肪形成过多而引起的，而肥胖引起的胰岛素抵抗患者则反而增加。Pleckstrin同源结构域富含亮氨酸的重复蛋白磷酸酶2（PHLPP2）通过使Akt磷酸化来终止胰岛素信号传导。肥胖小鼠肝脏中PHLPP2的水平降低。我们调查了肝脏PHLPP2的丧失是否足以诱导小鼠脂肪肝，脂肪肝中PHLPP2降解的机制以及NAFLD患者肝脏中调节PHLPP2的基因的表达。

方法

对C57BL / 6J小鼠（对照组），肥胖db / db小鼠以及肝脏正常缺失PHLPP2（L-PHLPP2）的小鼠（饲喂普通食物或高脂饮食（HFD））进行了代谢表型分析，包括葡萄糖耐量和肝脂肪变性。分析了缺乏PHLPP2的原代肝细胞或CRISPR / Cas9介导的PHLPP2敲除肝癌细胞的胰岛素信号传导和基因表达。我们对用Ad-HA-Flag-PHLPP2转导的C57BL / 6J小鼠的肝脏组织进行了质谱分析，以鉴定对PHLPP2和与PHLPP2相互作用的蛋白质的翻译后修饰。我们通过定量逆转录聚合酶链反应在不同程度的肝脂肪变性患者的肝活检中测量了mRNA的水平。

结果

PHLPP2敲除的肝癌细胞和来自L-PHLPP2小鼠的肝细胞显示正常的胰岛素信号传导，但是胰岛素作用延长。喂养L-PHLPP2的小鼠的葡萄糖耐量正常，但肝脂肪变性。在由HFD喂养的C57BL / 6J或db / db肥胖小鼠中，胰高血糖素和依赖PKA的PHLPP2磷酸化作用（在Ser1119和Ser1210处）降解了内源性PHLPP2，这导致PHLPP2与包含17的钾通道四聚化结构域结合， Cul3-RING泛素连接酶的底物适配器。喂食HFD的C57BL / 6J或db / db的肝脏中KCTD17 mRNA的水平升高肥胖小鼠和NAFLD肝活检患者，与健康对照小鼠或无脂肪变性患者的肝组织相比。用原发性肝细胞中的小发夹RNA敲除KCTD17会增加PHLPP2蛋白，但不会增加Phlpp2 mRNA，表明KCTD17介导了PHLPP2降解。肥胖小鼠中的KCTD17敲低阻止了PHLPP2降解并降低了生脂基因的表达。

结论

在肥胖的小鼠模型中，我们发现PHLPP2降解诱导脂肪生成而不影响糖原异生。肥胖小鼠和患有NAFLD的患者肝脏组织中上调的KCTD17与磷酸化的PHLPP2结合，使其靶向泛素介导的降解。这增加了调节脂肪生成以促进肝脂肪变性的基因的表达。该途径的抑制剂可能被开发用于NAFLD患者的治疗。