Effect Of GLP-1 On Skeletal Muscle Myosin Heavy Chain Expression And Its Mechanisms Involved

Glucagon like peptide-1 (GLP-1) is a peptide hormone secreted from intestinal "L" cell in response to the ingestion of food. It is proved that GLP-1 can promote the secretion of insulin, improve glucose uptake and utilisation of periphery tissues. It is reported that temporal treatment with GLP-1 can increase glucose uptake of skeletal muscle, and GLP-1 treatment at early development stage can improve blood glucose and glucose tolerance of adult diabetic mice. In view of the close relationship betweeen skeletal muscle fiber type, body glucose utilization and development of diabetes, we hypothesized that GLP-1 treatment at early development stage may program skeletal muscle fiber type through regulation of myosin heavy chain expression. In this study, we tested the hypothesis with both in vivo and in vitro experiments. In vivo, a recombinant plasmid of secretory GLP-1 was transfected to the leg muscle of neonatal rats, and the programming effects of neonatal GLP-1 exposure on muscle myosin heavy chain types and other related gene expression were investigated in adult rats. In vitro, insulin resistant C2C12 myotubes were treated with GLP-1 analogues, Exendin 4 and Exendin 9, and myosin heavy chain and other related gene expression were detected and the possible mechanisms were studied.1 Programming effects of neonatal GLP-1 recombinant plasmid transfection on muscle myosin heavy chain expression and other related gene expression in adult ratsIn this study, GLP-1 cDNA was inserted into pcDNA3 plasmid together with the signal peptide to constitute a secretory GLP-1 expressiong plasmid named as sig-glp-1-pcDNA3. Newborn male Wistar rats were divided into three groups at random after weighed at 1 day of age. Two-day-old rats were transfected intramuscularly in vivo with vacant vector plasmid (VP), or recombinant plasmid expressing secretory GLP-1 at the doses of 60 p,g (low GLP-1, LG) and 120μg (high GLP-1, HG), respectively, and reared under the same condition until 8 weeks of age. Expression of GLP-1 in skeletal muscle was checked at 8 days of age, serum hormone levels and muscle gene expression were detected at 56 days of age. The results showed that:(1) Both LG and HG rats were detected over-expression of GLP-1 7 days after transfection, the expression being higher in LG rats. It is proved that recombinant plasmid was constructed correctly and transfected successfully. (2) Compared to VP rats, LG rats showed significant growth retardation but no significant differences were seen in muscle and pancreas weight. HG rats showed only a tendency of body weight reduction. (3) The growth retardation was accompanied with significantly reduced serum insulin concentration at 8 weeks of age, yet glucose tolerance maintained normal. Serum T3 level was significantly reduced in HG rats. (4) No significant alterations were observed for muscle weight, glycogen content and percentage of type I muscle fiber detected with ATPase staining in either soleus or gastrocnemius. The lactic acid content in gastrocnemius in both treatment groups were significantly reduced, but in soleus significant reduction was shown only in HG rats. (5) In LG rats, a fiber type switch from fast to slow was seen in gastrocnemius of LG rats, whereas up-regulations of PGC-1αand GLUT4 gene expression were found in soleus. In conclusion, neonatal exposure of healthy pups to ectopic expression of GLP-1 causes growth retardation with decreased serum insulin, as well as muscle type dependent modifications in MyHC type composition and metabolic gene expression in adult rats, leading to increased glucose uptake and oxidation in skeletal muscle which contributes to the maintenance of glucose homeostasis.2 Effects of GLP-1 analogues on glucose uptake, myosin heavy chain and other related gene expression in myotubes, and possible mechanisms involvedC2C12 myoblast cells were induced to differentiate in culture. On the 4th day of differentiation, GLP-1 analogues,10-9mol/L Exendin 4 (Ex4),10-9mol/L Exendin 9 (Ex9) and 10-9 mol/L Ex4+10-9 mol/L Ex9 were added directly in culture medium. Culture medium was refreshed every 24 h and treatment lasted for 72 h. At the end of treatment, culture medium was collected for measuring glucose concentration, and the cells were harvested for detecting glucose uptake and gene expression. The results showed that:(1) Ex4 or Ex9 treatment did not alter the basal glucose uptake. However, Ex9 treatment significantly increased insulin-stimulated glucose uptake. Combined treatment with Ex4 and Ex9 increaseed basal glucose uptake but had no effect on insulin-stimulated glucose uptake. (2) Four isoforms of myosin heavy chain were all expressed in myotubes. MyHC I, MyHC 2A mRNA expression, as well as the percentage of MyHC I protein, were significantly increased in Ex9 treated cells. The total content of MyHCⅡprotein was also increased in Ex9 treated group. On the contrary, Ex4, alone or in combination with Ex9, caused significant reduction in MyHC 2A mRNA expression and the percentage of MyHC I protein with no alteration in total MyHCⅡprotein content. (3) GLUT4 gene expression did not show significant variation, while PGC-la gene expression was promoted by Ex9 and inhibited by Ex4, alone or in combination with Ex9; PDK4 gene expression was reduced in Ex4+Ex9 group. Protein contents were in agreement with the mRNA abundances. No changes were seen for GLUT4 protein content, whereas PGC-la protein content was increased in Ex9 group, but maintained unaltered in Ex4 and Ex4+Ex9 groups. In conclusion, Ex9 improves the insulin sensitivity of C2C12 myotube, promotes MyHC I and PGC-1αexpression. Ex4 does not influence myotube glucose uptake but inhibits PGC-la, MyHC I and MyHC 2A expression. In general, Ex4 treated in combination with Ex9 showed similar effects to Ex4 treated alone, no antagonistic effects were observed between Ex9 and Ex4.Transcriptome microarray analysis was performed in order to identify the genes differentially expressed by Ex4 and Ex9 treatment in C2C12 myotubes. The results indicated that:(1) Most of the differentially expressed genes were up-regulated in Ex9 group, among which were Ca2+related K+voltage-gated channel and Calumenin. (2) In Ex4 and Ex4+Ex9 groups, most of the differentially expressed genes were down-regulated, which including some commonly down-regulated genes, such as skeleton protein Neblin and titin. In addition, significant up-reglulation of DNA fragmentation factor was seen in both Ex4 and Ex4+Ex9 groups, suggesting the possible involvement of cell apoptosis in Ex4 action. (3) Neither of the treatments caused significant alterations in casepase 3/7 activity. However, increased intracellular Ca2+cocentration was observed in all the treatment groups compared with control, but only Ex9 improved calcineurin A protein content in the cells. In conclusion, Ex9 may promote MyHC I expression through Ca2+-calcineurin-NFAT pathway. In addition, Ex9 may also promote MyHC I expression by activating PGC-1α.