دانلود رایگان مقاله هدف قرار دادن پروتئین برای گلیکوژن به عنوان تنظیم کننده سنتز گلیکوژن در آستروسیت

Abstract

The storage and use of glycogen, the main energy reserve in the brain, is a metabolic feature of astrocytes. Glycogen synthesis is regulated by Protein Targeting to Glycogen (PTG), a member of specific glycogen-binding subunits of protein phosphatase-1 (PPP1). It positively regulates glycogen synthesis through de-phosphorylation of both glycogen synthase (activation) and glycogen phosphorylase (inactivation). In cultured astrocytes, PTG mRNA levels were previously shown to be enhanced by the neurotransmitter noradrenaline. To achieve further insight into the role of PTG in the regulation of astrocytic glycogen, its levels of expression were manipulated in primary cultures of mouse cortical astrocytes using adenovirus-mediated overexpression of tagged-PTG or siRNA to downregulate its expression. Infection of astrocytes with adenovirus led to a strong increase in PTG expression and was associated with massive glycogen accumulation (>100 fold), demonstrating that increased PTG expression is sufficient to induce glycogen synthesis and accumulation. In contrast, siRNA-mediated downregulation of PTG resulted in a 2-fold decrease in glycogen levels. Interestingly, PTG downregulation strongly impaired long-term astrocytic glycogen synthesis induced by insulin or noradrenaline. Finally, these effects of PTG downregulation on glycogen metabolism could also be observed in cultured astrocytes isolated from PTG-KO mice. Collectively, these observations point to a major role of PTG in the regulation of glycogen synthesis in astrocytes and indicate that conditions leading to changes in PTG expression will directly impact glycogen levels in this cell type.

4. Discussion

PPP1-targeting subunits are key regulators of glycogen synthesis in mammalian cells. Here, we report that one of its members, PTG, plays a major role in glycogen metabolism regulation in astrocytes under both basal and glycogen synthesis stimulating conditions. By manipulating PTG expression levels in primary astrocyte cultures either by adenovirus-mediated overexpression of PTG or siRNA-mediated downregulation of its expression as well as using PTG KO cultured astrocytes, a direct correlation between PTG proteins levels in astrocyte and glycogen synthesis could be established. PPP1-glycogen targeting regulatory subunits (PPP1R3s) are scaffolding protein which co-localize the catalytic subunit of PPP1 (PPP1c) with specific glycogen-metabolic enzymes, enabling dephosphorylation of GS and GP hence fine-tuning glycogen synthesis flux. As PTG is highly expressed in astrocytes (Lovatt et al., 2007; Zhang et al., 2014) it represented a possible candidate as a regulator of glycogen synthesis in this cell type. Indeed, our results show that manipulating PTG levels (up or down) in astrocytes had a consistent and direct effect on glycogen content: i) overexpression of PTG led to a 100 fold increase in glycogen ii) the effects of PTG expression on protein levels and glycogen accumulation were counteracted by siRNA treatment to the same extent (75 and 63% decrease respectively) iii) downregulation of endogenous PTG expression by siRNA led to a 2 fold decrease in glycogen iv) ablation of PTG expression (PTG KO astrocytes) led to a 80% decrease in glycogen. No alteration or compensatory changes of expression of other genes involved in astrocytic glycogen metabolism was observed at the transcriptional levels (i.e. Pygb, Gys1, Gyg, PPP1R6), reinforcing the notion that the observed changes in glycogen metabolism were directly produced by changes in PTG expression.