Role Of Class Ⅱ Histone Deacetylases In Mediating The Effect Of Exercise On Glucose Metabolism In Skeletal Muscle Cells

Objective:Glucose metabolic disorder of body is an important factor caused metabolism related disease. It is well known that regular aerobic exercise is an effective way to prevent and treat obesity, insulin resistance(IR) and type Ⅱ diabetes. Skeletal muscle is a major organ of human body movement, and is used to transport and metabolize 80% insulin-stimulated glucose. Therefore, enhanced the glucose metabolism of skeletal muscle increases insulin sensitivity of tissues and prevents the development of metabolism related disease.Class Ⅱ histone deacetylases(HDACs), as the transcription inhibitors, play an important role of regulating glucose homeostasis. Moreover, HDAC4 and HDAC5 are sensitive to exercise. Aerobic exercise has been wildly used as an effective method to treat IR and metabolic syndromes. And its underlying mechanism is associated with the regulation of Class Ⅱ HDACs in skeletal muscle.Our study focused on the regulation of Class Ⅱ HDACs to glucose and investigated the underlying mechanism of HDAC4 and HDAC5 in aerobic exercise improvement of glucose uptake and metabolism in skeletal muscle tissue. We investigated the effect of aerobic exercise on HDAC4 and HDAC5 regulation to the gene expression of GLUT4 in vivo and in vitro. And we explored the mechanism of mi R-206 mediated the down-regulation of HDAC4 gene expression in vitro. Moreover, AMPKα2-/-C57BL/6 mice were used to study the upstream kinase of HDAC4 and HDAC5, further providing the evidences above the mechanism of HDAC4 and HDAC5 in regulating glucose metabolism of skeletal muscle.Methods:1. Animals model5-week old C57BL/6 mice were divided into normal control group(NC), normal exercise group(NE). The genotypes of 5-week old knockout mice of AMPKα2 were diveded into wild type(WT), heterozygote(AMPKα2+/-) and homozygote(AMPKα2-/-), and further grouped NC and NE.2. Preparation of adenovirus vectorOver-expression plasmids were constructed by gateway clone technology. Recombinant adenovirus vectors were obtained by transfection of plasmids that were cut and then linearized by Pac I endonuclease. Dominant negative mutation adenovirus vectors(DN) were obtained by gateway clone, point mutation and then recombinant adenovirus.3.Cell culture and treatmentsC2C12 cells were differentiated in DMEM with 2% horse serum for 6 days into myotube cells. Over-expression adenovirus vectors of HDAC4, MEF2 A and HDAC4, and si RNA target sequences of HDAC4 and HDA5 were transfected into C2C12 myotubes, and then the effects on glucose metabolism were detected. After AMPK/DN-AMPK adenovirus transfection, the effect on HDAC4 was measured in C2C12 myotubes. In additional, glucose metabolism was detected when mi R-206 mimic and mi R-206 inhibitor were transfected into C2C12 myotubes.4.Biochemical indexes detectionWe used real-time PCR and western blot analysis to detect the genes and proteins expression, respectively. Glucose uptake of skeletal muscle was measured using a nonradioactive fluorescent glucose [2-(N-(7-nitrobenz-2-oxa-1, 3-diazol- 4-yl) amino)-2-deoxyglucose](2-NBDG) method. Immunoprecipitation was used to detect the interaction of proteins and immunofluorescence assay was used to measure the expression and location of proteins.Results:1. HDAC4/5 mediates the regulation of aerobic exercise on glucose metabolism6-week aerobic exercise significantly increased the gene and protein expression of GLUT4 and increased the phosphorylation level of HDAC Ser246, promoted the nuclear export of HDAC4 to plasm. Increased HDAC4 inhibited the transport of GLUT4, which was reversed by increasing MEF2 A in votro. In additional, increased HDAC4 also inhibited glucose uptake in skeletal muscle cells.After HDAC4 si RNA intervention, the transcription of GLUT4 was significantly increased in C2C12 myotubes, which trend were enhanced with HDAC5 si RNA together. Conversely, the inhibition rate on GLUT4 from combined over-expression adenovirus vectors of HDAC4 and HDAC5 transfection in C2C12 myotubes was significantly increased than the HDAC4 or HDAC5 alone groups. Therefore, the results indicated HDAC4 and HDAC5 may play a synergistic role in regulation of transcription of GLUT4 in skeletal muscle cells.6-week exercise training significantly decreased the protein expression of HDAC4 and HDAC5 in oxidative fiber but not glycolysis muscle fiber, which was identified again by immunofluorescent staining. And, the result of immunofluorescent staining suggested aerobic exercise promoted the nuclear export of HDAC4 and HDAC5 to plasm, and decreased the binding of HDAC4 and HDAC5 proteins. The findings suggested 6-week exercise training not only activated HDAC4 and HDAC5, and required specific skeletal muscle fibre type. HDAC4 and HDAC5 mediating the inhibition effect of exercise on downstream target gene may be associated with the synergistic interaction of the two proteins.2. Aerobic exercise activated HDAC4/5 in skeletal muscle via AMPKα2AMPKα2 deletion significantly increased HDAC4 Ser246/632 phosphorylation levels, and promoted the nuclear export of HDAC4 to plasm in vitro, indicated AMPKα2 was upstream kinase of HDAC4 in skeletal muscle cells.A single bout of exercise activated the AMPK-HDAC4 signaling pathway in skeletal muscle of wild type mice, but not p HDAC4 Ser246/632 in AMPKα2-/- mice. The result indicated exercise activated HDAC4 in skeletal muscle depending on AMPKα2.3. Aerobic exercise may regulate glucose metabolism in skeletal muscle via mi R-206/HDAC46-week exercise training increased the expression of mi R-206 in oxidative fiber, reduced the protein expression and activity of HDAC4, and significantly increased the gene and protein expression of GLUT4. Therefore, aerobic exercise may reduce the protein expression of HDAC4 via mi R-206, and then increase the transcription of GLUT4.mi R-206 mimic significantly decreased HDAC4 protein, but increased gene transcription of GLUT4 and glucose uptake in C2C12 myotubes. The finding suggested mi R-206 promoted the transcription of GLUT4 and glucose uptake through inhibition of HDAC4 in skeletal muscle cells.HDAC4 si RNA reversed the increase of GLUT4 and mi R-206 expression induced by mi R-206 mimic and when co-transfection in C2C12 myotubes. HDAC4 si RNA decreased the expression of mi R-206 in C2C12 myotubes, which similar as a mi R-206 Inhibitor. Those findings indicated HDAC4 si RNA inhibited the mi R-206 expression as a feedback.Moreover, the expression of GLUT4 increased depending on concentration gradient of mi R-206 mimic, and significantly increased compared with control, but significantly decreased compared with only mi R-206 mimic group following over-expression adenovirus of HDAC4 intervene.Conclusion:1. HDAC4 regulated the gene transcription of GLUT4 via MEF2 A in myotube cells and HDAC4/5 may play a synergistic role in this process. Aerobic exercise reduced the protein expression of HDAC4/5 in oxidative fiber and may regulate glucose metabolism in skeletal muscle tissue through synergistic action of HDAC4/5.2. AMPK increased the activity of HDAC4 in myotube cells. A single bout of aerobic exercise enhanced the activity of HDAC4 depending on AMPKα2 in skeletal muscle cells.3. mi R-206 inhibited the gene expression of HDAC4, increased the transcription of GLUT4, and then promoted the glucose uptake in myotube cells. In additional, the enhance effect of mi R-206 on glucose metabolism induced by aerobic exercise may require a specific of skeletal muscle fiber types.