Université Claude Bernard Lyon 1, Université de Lyon, INSERM UMR-S1213, F-69374, Lyon, France.
Université Claude Bernard Lyon 1, Université de Lyon, INSERM UMR-S1213, F-69374, Lyon, France.
Mol Metab. 2023 Apr;70:101700. doi: 10.1016/j.molmet.2023.101700. Epub 2023 Mar 2.
Deregulation of hepatic glucose production is a key driver in the pathogenesis of diabetes, but its short-term regulation is incompletely deciphered. According to textbooks, glucose is produced in the endoplasmic reticulum by glucose-6-phosphatase (G6Pase) and then exported in the blood by the glucose transporter GLUT2. However, in the absence of GLUT2, glucose can be produced by a cholesterol-dependent vesicular pathway, which remains to be deciphered. Interestingly, a similar mechanism relying on vesicle trafficking controls short-term G6Pase activity. We thus investigated whether Caveolin-1 (Cav1), a master regulator of cholesterol trafficking, might be the mechanistic link between glucose production by G6Pase in the ER and glucose export through a vesicular pathway.
Glucose production from fasted mice lacking Cav1, GLUT2 or both proteins was measured in vitro in primary culture of hepatocytes and in vivo by pyruvate tolerance tests. The cellular localization of Cav1 and the catalytic unit of glucose-6-phosphatase (G6PC1) were studied by western blotting from purified membranes, immunofluorescence on primary hepatocytes and fixed liver sections and by in vivo imaging of chimeric constructs overexpressed in cell lines. G6PC1 trafficking to the plasma membrane was inhibited by a broad inhibitor of vesicular pathways or by an anchoring system retaining G6PC1 specifically to the ER membrane.
Hepatocyte glucose production is reduced at the step catalyzed by G6Pase in the absence of Cav1. In the absence of both GLUT2 and Cav1, gluconeogenesis is nearly abolished, indicating that these pathways can be considered as the two major pathways of de novo glucose production. Mechanistically, Cav1 colocalizes but does not interact with G6PC1 and controls its localization in the Golgi complex and at the plasma membrane. The localization of G6PC1 at the plasma membrane is correlated to glucose production. Accordingly, retaining G6PC1 in the ER reduces glucose production by hepatic cells.
Our data evidence a pathway of glucose production that relies on Cav1-dependent trafficking of G6PC1 to the plasma membrane. This reveals a new cellular regulation of G6Pase activity that contributes to hepatic glucose production and glucose homeostasis.
肝葡萄糖生成的失调是糖尿病发病机制的关键驱动因素，但它的短期调节尚未完全阐明。根据教科书，葡萄糖是在内质网中由葡萄糖-6-磷酸酶（G6Pase）产生的，然后由葡萄糖转运蛋白 GLUT2 在血液中输出。然而，在没有 GLUT2 的情况下，葡萄糖可以通过胆固醇依赖的囊泡途径产生，这仍然需要阐明。有趣的是，一种依赖囊泡运输的类似机制控制着短期 G6Pase 活性。因此，我们研究了胆固醇转运的主要调节因子 Caveolin-1（Cav1）是否可能是 ER 中 G6Pase 产生的葡萄糖与通过囊泡途径输出的葡萄糖之间的机制联系。
在原代培养的肝细胞中以及通过丙酮酸耐量试验在体内测量缺乏 Cav1、GLUT2 或这两种蛋白的禁食小鼠的葡萄糖生成。通过从纯化的膜、原代肝细胞的免疫荧光和固定的肝切片以及在细胞系中过表达的嵌合构建体的体内成像研究 Cav1 和葡萄糖-6-磷酸酶（G6PC1）的催化单位的细胞定位。通过广泛的囊泡途径抑制剂或通过将 G6PC1 特异性锚定到内质网膜的锚定系统抑制 G6PC1 向质膜的转运。
在缺乏 Cav1 的情况下，肝细胞葡萄糖生成在 G6Pase 催化的步骤减少。在同时缺乏 GLUT2 和 Cav1 的情况下，糖异生几乎被完全抑制，表明这些途径可以被认为是从头产生葡萄糖的两个主要途径。从机制上讲，Cav1 与 G6PC1 共定位但不相互作用，并且控制 G6PC1 在高尔基体复合体和质膜中的定位。G6PC1 在质膜上的定位与葡萄糖生成相关。因此，将 G6PC1 保留在 ER 中会降低肝细胞的葡萄糖生成。
我们的数据证明了一种依赖于 Cav1 依赖性 G6PC1 向质膜转运的葡萄糖产生途径。这揭示了 G6Pase 活性的新细胞调节，有助于肝葡萄糖生成和血糖稳态。
