Hillman Cancer Center, Cancer Virology Program, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America.
School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America.
PLoS One. 2023 Mar 10;18(3):e0282914. doi: 10.1371/journal.pone.0282914. eCollection 2023.
Cyclin-dependent kinase 1 (CDK1)/cyclin B1 phosphorylates many of the same substrates as mTORC1 (a key regulator of glucose metabolism), including the eukaryotic initiation factor 4E-binding protein 1 (4E-BP1). Only mitotic CDK1 phosphorylates 4E-BP1 at residue S82 in mice (S83 in humans), in addition to the common 4E-BP1 phospho-acceptor sites phosphorylated by both CDK1 and mTORC1. We examined glucose metabolism in mice having a single aspartate phosphomimetic amino acid knock in substitution at the 4E-BP1 serine 82 (4E-BP1S82D) mimicking constitutive CDK1 phosphorylation.
Knock-in homozygous 4E-BP1S82D and 4E-BP1S82A C57Bl/6N mice were assessed for glucose tolerance testing (GTT) and metabolic cage analysis on regular and on high-fat chow diets. Gastrocnemius tissues from 4E-BP1S82D and WT mice were subject to Reverse Phase Protein Array analysis. Since the bone marrow is one of the few tissues typically having cycling cells that transit mitosis, reciprocal bone-marrow transplants were performed between male 4E-BP1S82D and WT mice, followed by metabolic assessment, to determine the role of actively cycling cells on glucose homeostasis.
Homozygous knock-in 4E-BP1S82D mice showed glucose intolerance that was markedly accentuated with a diabetogenic high-fat diet (p = 0.004). In contrast, homozygous mice with the unphosphorylatable alanine substitution (4E-BP1S82A) had normal glucose tolerance. Protein profiling of lean muscle tissues, largely arrested in G0, did not show protein expression or signaling changes that could account for these results. Reciprocal bone-marrow transplantation between 4E-BP1S82D and wild-type littermates revealed a trend for wild-type mice with 4E-BP1S82D marrow engraftment on high-fat diets to become hyperglycemic after glucose challenge.
4E-BP1S82D is a single amino acid substitution that induces glucose intolerance in mice. These findings indicate that glucose metabolism may be regulated by CDK1 4E-BP1 phosphorylation independent from mTOR and point towards an unexpected role for cycling cells that transit mitosis in diabetic glucose control.
细胞周期蛋白依赖性激酶 1（CDK1）/细胞周期蛋白 B1 磷酸化了许多与 mTORC1（葡萄糖代谢的关键调节剂）相同的底物，包括真核起始因子 4E 结合蛋白 1（4E-BP1）。只有有丝分裂 CDK1 会在小鼠中磷酸化 4E-BP1 的丝氨酸 82 残基（人类为 S83），而不是 CDK1 和 mTORC1 共同磷酸化的常见 4E-BP1 磷酸化接受位点。我们在模拟 CDK1 磷酸化的 4E-BP1 丝氨酸 82 （4E-BP1S82D）上有单个天冬氨酸磷酸模拟氨基酸取代的 4E-BP1S82D 敲入纯合子小鼠中检查了葡萄糖代谢。
对 4E-BP1S82D 和 4E-BP1S82A C57Bl/6N 敲入纯合子小鼠进行葡萄糖耐量试验（GTT）和高脂肪饮食代谢笼分析。来自 4E-BP1S82D 和 WT 小鼠的腓肠肌组织进行反向蛋白质阵列分析。由于骨髓是通常具有有丝分裂细胞周期的少数组织之一，因此在雄性 4E-BP1S82D 和 WT 小鼠之间进行了骨髓相互移植，然后进行代谢评估，以确定活跃的有丝分裂细胞对葡萄糖稳态的作用。
纯合敲入 4E-BP1S82D 小鼠表现出葡萄糖不耐受，高脂肪饮食（p = 0.004）使这种不耐受明显加重。相比之下，具有不可磷酸化的丙氨酸取代（4E-BP1S82A）的纯合子小鼠具有正常的葡萄糖耐量。主要处于 G0 期的瘦肌肉组织的蛋白质谱分析并未显示出可以解释这些结果的蛋白质表达或信号变化。4E-BP1S82D 和野生型同窝仔之间的骨髓相互移植表明，在高脂肪饮食中，带有 4E-BP1S82D 骨髓移植物的野生型小鼠在葡萄糖挑战后有成为高血糖的趋势。
4E-BP1S82D 是一种单一氨基酸取代，可诱导小鼠葡萄糖不耐受。这些发现表明，葡萄糖代谢可能受 CDK1 4E-BP1 磷酸化调控，与 mTOR 无关，并指出有丝分裂细胞周期过渡在糖尿病葡萄糖控制中的作用出乎意料。
