Department of Endocrinology, Renmin Hospital of Wuhan University, Wuhan 430060, China.
Department of Endocrinology, Renmin Hospital of Wuhan University, Wuhan 430060, China.
Mol Metab. 2024 May;83:101927. doi: 10.1016/j.molmet.2024.101927. Epub 2024 Mar 27.
Hepatic insulin resistance, which leads to increased hepatic gluconeogenesis, is a major contributor to fasting hyperglycemia in type 2 diabetes mellitus (T2DM). However, the mechanism of impaired insulin-dependent suppression of hepatic gluconeogenesis remains elusive. Delta/Notch-like epidermal growth factor (EGF)-related receptor (DNER), firstly described as a neuron-specific Notch ligand, has been recently identified as a susceptibility gene for T2DM through genome-wide association studies. We herein investigated whether DNER regulates hepatic gluconeogenesis and whether this is mediated by enhanced insulin signaling.
The association between DNER, tribbles homolog 3 (TRB3) and Akt signaling was evaluated in C57BL/6J, ob/ob and db/db mice by western blot analysis. DNER loss-of-function and gain-of-function in hepatic gluconeogenesis were analyzed by western blot analysis, quantitative real-time PCR, glucose uptake and output assay in AML-12 cells and partially validated in primary mouse hepatocytes. Hepatic DNER knockdown mice were generated by tail vein injection of adenovirus to confirm the effects of DNER in vivo. The interaction between DNER and TRB3 was investigated by rescue experiments, cycloheximide chase analysis, co-immunoprecipitation and immunofluorescence. The potential insulin-stimulated phosphorylation sites of DNER were determined by co-immunoprecipitation, LC-MS/MS analysis and site-specific mutagenesis.
Here we show that DNER enhanced hepatic insulin signaling in gluconeogenesis by inhibiting TRB3, an endogenous Akt inhibitor, through the ubiquitin-proteasome degradation pathway. In AML-12 hepatocytes, insulin-stimulated activation of Akt and suppression of gluconeogenesis are attenuated by DNER knockdown, but potentiated by DNER over-expression. In C57BL/6J mice, hepatic DNER knockdown is accompanied by impaired glucose and pyruvate tolerance. Furthermore, the in vitro effects of DNER knockdown or over-expression on both Akt activity and hepatic gluconeogenesis can be rescued by TRB3 knockdown or over-expression, respectively. In response to insulin stimulation, DNER interacted directly with insulin receptor and was phosphorylated at Tyr. This site-specific phosphorylation is essential for DNER to upregulate Akt activity and then downregulate G6Pase and PEPCK expression, by interacting with TRB3 directly and inducing TRB3 proteasome-dependent degradation.
Taken together, the crosstalk between insulin-Akt and DNER-TRB3 pathways represents a previously unrecognized mechanism by which insulin regulates hepatic gluconeogenesis.
导致肝糖异生增加的肝胰岛素抵抗是 2 型糖尿病（T2DM）空腹高血糖的主要原因。然而，胰岛素依赖性抑制肝糖异生受损的机制仍不清楚。Delta/Notch-like 表皮生长因子（EGF）相关受体（DNER）最初被描述为一种神经元特异性 Notch 配体，最近通过全基因组关联研究被确定为 T2DM 的易感基因。本文研究了 DNER 是否调节肝糖异生，以及这种调节是否通过增强胰岛素信号转导来介导。
通过 Western blot 分析评估 C57BL/6J、ob/ob 和 db/db 小鼠中 DNER、tribbles 同源物 3（TRB3）和 Akt 信号之间的关联。通过 Western blot 分析、定量实时 PCR、AML-12 细胞中的葡萄糖摄取和输出测定以及在原代小鼠肝细胞中的部分验证，分析 DNER 在肝糖异生中的功能丧失和获得性功能。通过尾静脉注射腺病毒生成肝 DNER 敲低小鼠，以确认 DNER 在体内的作用。通过挽救实验、环己酰亚胺追踪分析、共免疫沉淀和免疫荧光研究 DNER 和 TRB3 之间的相互作用。通过共免疫沉淀、LC-MS/MS 分析和定点突变确定 DNER 的潜在胰岛素刺激磷酸化位点。
本文显示，DNER 通过泛素-蛋白酶体降解途径抑制内源性 Akt 抑制剂 TRB3，增强肝糖异生中的胰岛素信号。在 AML-12 肝细胞中，DNER 敲低可减弱胰岛素刺激的 Akt 激活和肝糖异生抑制，但 DNER 过表达可增强该作用。在 C57BL/6J 小鼠中，肝 DNER 敲低伴有葡萄糖和丙酮酸耐量受损。此外，DNER 敲低或过表达对 Akt 活性和肝糖异生的体外影响可分别通过 TRB3 敲低或过表达挽救。在胰岛素刺激下，DNER 直接与胰岛素受体相互作用并在 Tyr 上发生磷酸化。该位点特异性磷酸化对于 DNER 上调 Akt 活性，然后下调 G6Pase 和 PEPCK 表达至关重要，其通过直接与 TRB3 相互作用并诱导 TRB3 蛋白酶体依赖性降解。
综上所述，胰岛素-Akt 和 DNER-TRB3 通路之间的串扰代表了胰岛素调节肝糖异生的一个以前未被认识的机制。
