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/F2，6 BPase）是负责合成和降解果糖-2，6-二磷酸（F2，6 BP）的酶。F2，6 BP又是糖酵解限速酶6-磷酸果糖-1-激酶（PFK-1）的强大变构激活剂。至少有4个基因编码PFK-2/FBPase（PFKFB 1 -4），并且已经发现由PFKFB 3编码的诱导型PFK-2（iPFK-2）介导脂肪细胞中F2，6 BP的产生，表明iPFK-2在甘油三酯合成中的潜在作用。本研究以骨骼肌为研究对象，研究了iPFK-2/PFKFB 3基因在骨骼肌中的作用。我们分析了小鼠骨骼肌中存在的剪接变体，并观察了PFKFB 3-ACG、ACDG和AG三种变体的表达，其中PFKFB 3-ACDG是主要表达形式。为了研究LPS对肌肉糖酵解的刺激作用，采用腹腔注射LPS的方法，对8周龄BALB/c小鼠肌肉中PFKFB 3 mRNA的表达进行了研究。LPS处理后PFKFB 3 mRNA表达明显上调，且剪接变异体的表达模式与对照组不同。用编码PFKFB 3的不同剪接变体（PFKFB 3-ACCT、PFKFB 3-ACDG或PFKFB 3-AG）的质粒稳定转染的HEK 293细胞显示细胞内F2，6 BP含量显著升高。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