Abstract：Vascular endothelial growth factor B (VEGFB) is an important regulator of glucose uptake in skeletal muscle. Knockout of VEGFB can promote glucose uptake in skeletal muscle tissue of diabetes mice (Mus musculus), and improve glucose intolerance and insulin resistance in diabetes mice. However, there is no research to clarify the specific regulation of VEGFB on glucose uptake of skeletal muscle cell. In order to explore the regulatory effect of VEGFB on glucose uptake by skeletal muscle cell, this study used mouse myoblast C2C12 as a model, and used qPCR to detect the gene expression of VEGF signaling related genes and glucose transporter 4 gene (GLUT4) during the differentiation process of C2C12. The effects of VEGFB, VEGF receptor (VEGFR) inhibitor Axitinib and PI3K inhibitor Wortmanin on glucose consumption and uptake during C2C12 differentiation were detected by glucose oxidase method and 2-(N-(7-nitrobenz-2-oxa-13-diazol-4-yl) amino)-2-deoxy-D-glucose (2-NBDG) method. Western blot was used to detect the expression of GLUT4 on the cell membrane and in the cytoplasm and the activation of PI3K/AKT. The results of qPCR showed that the gene expression of VEGFB (P<0.01), VEGFR2 (P<0.01) and GLUT4 (P<0.05) increased significantly after differentiation of C2C12 compared with that before differentiation. Exogenous addition of VEGFB significantly increased the glucose uptake of C2C12 during differentiation (P<0.05), and significantly promoted the expression of GLUT4 on the cell membrane (P<0.05), and activated the PI3K/AKT pathway. But the VEGF receptor inhibitor Axitinib could reverse the promotion of VEGFB on glucose uptake, GLUT4 membrane localization and PI3K/AKT activation. In addition, PI3K inhibitor Wotmanin treatment could also reverse the effect of VEGFB on glucose uptake and GLUT4 cell membrane expression. The results of this study indicate that VEGFB could promote GLUT4 translocation to C2C12 cell membranes through the VEGFR-PI3K/AKT signaling pathway, thereby promoting glucose uptake by C2C12 during differentiation. This study reveals the regulatory effect of VEGFB on glucose uptake during the differentiation of myoblast C2C12, providing a new scientific understanding of the regulation of skeletal muscle glucose metabolism, and a basic reference for in-depth research on the regulation mechanism of skeletal muscle glucose metabolism.
全露露, 凌明发, 李凡, 刘金好, 江青艳, 王松波. VEGFB对成肌细胞C2C12分化过程中葡萄糖摄取的调控作用[J]. 农业生物技术学报, 2024, 32(6): 1362-1370.
QUAN Lu-Lu, LING Ming-Fa, LI Fan, LIU Jin-Hao, JIANG Qing-Yan, WANG Song-Bo. Regulatory Effect of VEGFB on Glucose Uptake During the Differentiation of Myoblast C2C12. 农业生物技术学报, 2024, 32(6): 1362-1370.
[1] Alphonsus C S, Rodseth R N.2014. The endothelial glycocalyx: A review of the vascular barrier[J]. Anaesthesia, 69(7): 777-784. [2] Badu-Mensah A, Valinski P, Parsaud H, et al.2022. Hyperglycemia negatively affects IPSC-derived myoblast proliferation and skeletal muscle regeneration and function[J]. Cells, 11: 3674. [3] Bogan J S, Kandror K V.2010. Biogenesis and regulation of insulin-responsive vesicles containing glut4[J]. Current Opinion in Cell Biology, 22(4): 506-512. [4] Claesson-Welsh L.2016. VEGF receptor signal transduction - a brief update[J]. Vascular Pharmacology, 86: 14-17. [5] Dijkstra M H, Pirinen E, Huusko J, et al.2014. Lack of cardiac and high-fat diet induced metabolic phenotypes in two independent strains of VEGF-b knockout mice[J]. Scientific Reports, 4: 6238. [6] Espelage L, Al-Hasani H, Chadt A.2020. Rabgaps in skeletal muscle function and exercise[J]. Journal of Molecular Endocrinology, 64(1): R1-R19. [7] Fulco M, Cen Y, Zhao P, et al.2008. Glucose restriction inhibits skeletal myoblast differentiation by activating SIRT1 through AMPK-mediated regulation of NAMPT[J]. Developmental Cell, 14(5): 661-673. [8] Guo X, Sun W, Luo G, et al.2019. Panax notoginseng saponins alleviate skeletal muscle insulin resistance by regulating the IRS1-PI3K-AKT signaling pathway and Glut4 expression[J]. FEBS Open Bio, 9(5): 1008-1019. [9] Hagberg C E, Mehlem A, Falkevall A, et al.2012. Targeting VEGF-B as a novel treatment for insulin resistance and type 2 diabetes[J]. Nature, 490(7420): 426-430. [10] Hirata Y, Nomura K, Senga Y, et al.2019. Hyperglycemia induces skeletal muscle atrophy via a WWP1/KLF15 axis[J]. JCI Insight, 4(4): e124952 . [11] Hommelberg P P H, Plat J, Remels A H, et al.2010. Trans-10, cis-12 conjugated linoleic acid inhibits skeletal muscle differentiation and glut4 expression independently from NF-κB activation[J]. Molecular Nutrition & Food Research, 54(12): 1763-1772. [12] Kaliman P, Viñals F, Testar X, et al.1996. Phosphatidylinositol 3-kinase inhibitors block differentiation of skeletal muscle cells[J]. The Journal of Biological Chemistry, 271(32): 19146-19151. [13] Lal N, Puri K, Rodrigues B.2018. Vascular endothelial growth factor b and its signaling[J]. Frontiers in Cardiovascular Medicine, 5: 39. [14] Li X, Kumar A, Zhang F, et al.2012. Complicated life, complicated VEGF-B[J]. Trends in Molecular Medicine, 18(2): 119-127. [15] Ling M, Quan L, Lai X, et al.2021. VEGFB promotes myoblasts proliferation and differentiation through VEGFR1-PI3K/AKT signaling pathway[J]. International Journal of Molecular Sciences, 22(24): 13352. [16] Luo B, Wu Y, Liu S, et al.2020. Vagus nerve stimulation optimized cardiomyocyte phenotype, sarcomere organization and energy metabolism in infarcted heart through FoxO3A-VEGF signaling[J]. Cell Death & Disease, 11(11): 971. [17] Luo W, Ai L, Wang B, et al.2019. High glucose inhibits myogenesis and induces insulin resistance by down-regulating akt signaling[J]. Biomedicine & Pharmacotherapy, 120: 109498. [18] Luo X, Li R, Li Y, et al.2022. Reducing VEGFB expression regulates the balance of glucose and lipid metabolism[J]. Molecular Medicine Reports, 26(3): 285. [19] Marshall B A, Mueckler M M.1994. Differential effects of glut-1 or glut-4 overexpression on insulin responsiveness in transgenic mice[J]. The American Journal of Physiology, 267(5 Pt 1): E738-E744. [20] Merz K E, Thurmond D C.2020. Role of skeletal muscle in insulin resistance and glucose uptake[J]. Comprehensive Physiology, 10(3): 785-809. [21] Mitsumoto Y, Burdett E, Grant A, et al.1991. Differential expression of the glut1 and glut4 glucose transporters during differentiation of l6 muscle cells[J]. Biochemical and Biophysical Research Communications, 175(2): 652-659. [22] Moessinger C, Nilsson I, Muhl L, et al.2020. Vegf-B signaling impairs endothelial glucose transcytosis by decreasing membrane cholesterol content[J]. EMBO Reports, 21(7): e49343. [23] Moreno H, Serrano A L, Santalucía T, et al.2003. Differential regulation of the muscle-specific glut4 enhancer in regenerating and adult skeletal muscle[J]. The Journal of Biological Chemistry, 278(42): 40557-40564. [24] Richter E A, Hargreaves M.2013. Exercise, glut4, and skeletal muscle glucose uptake[J]. Physiological Reviews, 93(3): 993-1017. [25] Robciuc M R, Kivelä R, Williams I M, et al.2016. Vegfb/vegfr1-induced expansion of adipose vasculature counteracts obesity and related metabolic complications[J]. Cell Metabolism, 23(4): 712-724. [26] Robinson C J, Stringer S E.2001. The splice variants of vascular endothelial growth factor (VEGF) and their receptors[J]. Journal of Cell Science, 114(Pt 5): 853-865. [27] Simons M, Gordon E, Claesson-Welsh L.2016. Mechanisms and regulation of endothelial VEGF receptor signalling[J]. Nature Reviews. Molecular Cell Biology, 17(10): 611-625. [28] Yoo M, Kim B, Lee S, et al.2015. Syntaxin 4 regulates the surface localization of a promyogenic receptor CDO thereby promoting myogenic differentiation[J]. Skeletal Muscle, 5: 28. [29] Zhu H, Gao M, Gao X, et al.2018. Vascular endothelial growth factor-B: Impact on physiology and pathology[J]. Cell Adhesion & Migration, 12(3): 215-227.
