Expression of Inducible 6-phosphofructo-2-kinase Isoforms in Skeletal Muscle and Their Potential Role in Glycolytic Regulation

6-磷酸果糖-2-激酶/果糖- 2,6 -二磷酸酶（PFK-2/F2, 6BPase）是负责果糖- 2,6 -二磷酸（F2, 6BP）合成和降解的酶。F2, 6BP反过来是6-磷酸果糖-1激酶（PFK-1）的强效变构激活剂，PFK-1是糖酵解的限速酶。至少有4个基因编码PFK-2/FBPase (PFKFB1-4)，由PFKFB3编码的诱导型PFK-2 （iPFK-2）已被发现介导脂肪细胞中F2, 6BP的产生，这表明iPFK-2在甘油三酯合成中的潜在作用。在本研究中，我们研究了iPFK-2/PFKFB3在骨骼肌中的作用。PFKFB3基因通过7外显子（A-G）的选择性剪接产生多个同种异构体。我们分析了小鼠骨骼肌中存在的剪接变体，并观察了PFKFB3-ACG、ACDG和AG三种变体的表达，其中PFKFB3-ACDG是主要表达形式。为了研究肌肉糖酵解刺激物LPS对PFKFB3 mRNA表达的影响，8周龄BALB/c小鼠腹腔注射LPS， 3小时后去肌，采用实时RT-PCR分析PFKFB3 mRNA的表达。LPS处理显著诱导了PFKFB3 mRNA的表达，且LPS处理小鼠剪接变异的表达模式与对照小鼠不同。转染编码PFKFB3不同剪接变体的质粒（PFKFB3- acct、PFKFB3- acdg或PFKFB3- ag）后，HEK293细胞内F2、6BP含量显著升高。这些数据与过量产生F2, 6BP在诱导代谢改变中的作用一致。此外，体内数据表明，刺激骨骼肌中的iPFK-2可能是治疗糖尿病的潜在靶点

6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase (PFK-2/F2, 6BPase) is the enzyme responsible for the synthesis and degradation of fructose-2, 6-bisphosphate (F2, 6BP). F2, 6BP in turn is a powerful allosteric activator of 6-phosphofructo-1-kinase (PFK-1), which is the rate-limiting enzyme for glycolysis. At least 4 genes encode PFK-2/FBPase (PFKFB1-4), and an inducible PFK-2 (iPFK-2), encoded by PFKFB3, has been discovered to mediate F2, 6BP production in adipocytes suggesting the potential role of iPFK-2 in triglyceride synthesis. In this study, we investigated the role of iPFK-2/PFKFB3 in skeletal muscle.The PFKFB3 gene generates multiple isoforms by the alternative splicing of 7 exon (A-G). We analyzed the splicing variants present in mouse skeletal muscle, and observed the expression of the three variants, PFKFB3-ACG, ACDG, and AG, with PFKFB3-ACDG being the dominantly expressed form. To investigate the effect of LPS, which is a stimulant of glycolysis in muscle, on the expression of PFKFB3 mRNA, eight-wk old BALB/c mice were injected intraperitoneally with LPS and three hours later the muscle was removed and the expression of PFKFB3 mRNA analyzed by real-time RT-PCR. A significant induction of PFKFB3 mRNA was observed after LPS treatment, and the expression pattern of splicing variants in LPS treated mice was different from that of control mice. HEK293 cells stably transfected with plasmids encoding the different splice variants of PFKFB3 (PFKFB3-ACCT, PFKFB3-ACDG or PFKFB3-AG) showed a marked elevation in intracellular F2, 6BP content.These data are consistent with a role for the overproduction of F2, 6BP in the induction of metabolic alteration. Moreover, the in vivo data suggest that the stimulation of iPFK-2 in skeletal muscle may be a potential target for the treatment of diabetes