Laboratory of Pediatrics, University of Groningen, University Medical Center Groningen, The Netherlands.
Department of Laboratory Medicine, University of Groningen, University Medical Center Groningen, The Netherlands.
Mol Metab. 2024 Jan;79:101838. doi: 10.1016/j.molmet.2023.101838. Epub 2023 Nov 22.
Carbohydrate Response Element Binding Protein (ChREBP) is a glucose 6-phosphate (G6P)-sensitive transcription factor that acts as a metabolic switch to maintain intracellular glucose and phosphate homeostasis. Hepatic ChREBP is well-known for its regulatory role in glycolysis, the pentose phosphate pathway, and de novo lipogenesis. The physiological role of ChREBP in hepatic glycogen metabolism and blood glucose regulation has not been assessed in detail, and ChREBP's contribution to carbohydrate flux adaptations in hepatic Glycogen Storage Disease type 1 (GSD I) requires further investigation.
The current study aimed to investigate the role of ChREBP as a regulator of glycogen metabolism in response to hepatic G6P accumulation, using a model for acute hepatic GSD type Ib. The immediate biochemical and regulatory responses to hepatic G6P accumulation were evaluated upon G6P transporter inhibition by the chlorogenic acid S4048 in mice that were either treated with a short hairpin RNA (shRNA) directed against ChREBP (shChREBP) or a scrambled shRNA (shSCR). Complementary stable isotope experiments were performed to quantify hepatic carbohydrate fluxes in vivo.
ShChREBP treatment normalized the S4048-mediated induction of hepatic ChREBP target genes to levels observed in vehicle- and shSCR-treated controls. In parallel, hepatic shChREBP treatment in S4048-infused mice resulted in a more pronounced accumulation of hepatic glycogen and further reduction of blood glucose levels compared to shSCR treatment. Hepatic ChREBP knockdown modestly increased glucokinase (GCK) flux in S4048-treated mice while it enhanced UDP-glucose turnover as well as glycogen synthase and phosphorylase fluxes. Hepatic GCK mRNA and protein levels were induced by shChREBP treatment in both vehicle- and S4048-treated mice, while glycogen synthase 2 (GYS2) and glycogen phosphorylase (PYGL) mRNA and protein levels were reduced. Finally, knockdown of hepatic ChREBP expression reduced starch domain binding protein 1 (STBD1) mRNA and protein levels while it inhibited acid alpha-glucosidase (GAA) activity, suggesting reduced capacity for lysosomal glycogen breakdown.
Our data show that ChREBP activation controls hepatic glycogen and blood glucose levels in acute hepatic GSD Ib through concomitant regulation of glucose phosphorylation, glycogenesis, and glycogenolysis. ChREBP-mediated control of GCK enzyme levels aligns with corresponding adaptations in GCK flux. In contrast, ChREBP activation in response to acute hepatic GSD Ib exerts opposite effects on GYS2/PYGL enzyme levels and their corresponding fluxes, indicating that GYS2/PYGL expression levels are not limiting to their respective fluxes under these conditions.
碳水化合物反应元件结合蛋白（ChREBP）是一种葡萄糖 6-磷酸（G6P）敏感的转录因子，作为一种代谢开关，维持细胞内葡萄糖和磷酸盐的稳态。肝 ChREBP 以其在糖酵解、戊糖磷酸途径和从头合成脂肪中的调节作用而闻名。ChREBP 在肝糖原代谢和血糖调节中的生理作用尚未详细评估，ChREBP 在肝糖原贮积症 1 型（GSD I）中碳水化合物通量适应中的贡献需要进一步研究。
本研究旨在通过急性肝 GSD Ib 模型，研究 ChREBP 作为糖原代谢调节剂在肝 G6P 积累时的作用。在氯原酸 S4048 抑制肝 G6P 转运后，评估 ChREBP 短发夹 RNA（shRNA）（shChREBP）或乱序 shRNA（shSCR）处理的小鼠的即时生化和调节反应。进行互补的稳定同位素实验以定量体内肝碳水化合物通量。
shChREBP 处理将 S4048 介导的肝 ChREBP 靶基因诱导归一化为与载体和 shSCR 处理对照中观察到的水平。平行地，在 S4048 输注的小鼠中，肝 shChREBP 处理导致肝糖原的积累更加明显，并且与 shSCR 处理相比，血糖水平进一步降低。肝 ChREBP 敲低在 S4048 处理的小鼠中适度增加了葡萄糖激酶（GCK）通量，同时增强了 UDP-葡萄糖周转率以及糖原合酶和磷酸化酶通量。在载体和 S4048 处理的小鼠中，shChREBP 处理诱导了肝 GCK mRNA 和蛋白水平，而糖原合酶 2（GYS2）和糖原磷酸化酶（PYGL）mRNA 和蛋白水平降低。最后，肝 ChREBP 表达的敲低降低了淀粉域结合蛋白 1（STBD1）mRNA 和蛋白水平，同时抑制了酸性α-葡萄糖苷酶（GAA）活性，表明溶酶体糖原分解的能力降低。
我们的数据表明，ChREBP 激活通过葡萄糖磷酸化、糖生成和糖原分解的协同调节来控制急性肝 GSD Ib 中的肝糖原和血糖水平。ChREBP 对 GCK 酶水平的调节与 GCK 通量的相应适应一致。相比之下，急性肝 GSD Ib 中 ChREBP 的激活对 GYS2/PYGL 酶水平及其相应通量产生相反的影响，表明在这些条件下，GYS2/PYGL 的表达水平不受其各自通量的限制。
