The di-peptide Trp-His (WH) has vasorelaxant and anti-atherosclerotic functions. We hypothesized that WH has multiple biological functions and may aid AMP-activated protein kinase (AMPK) activation and affect the glucose transport system in skeletal muscle. First, we examined whether WH or His-Trp (HW) can activate AMPKα. Treatment of L6 myotubes with WH or HW significantly increased phosphorylation of AMPKα. WH activatedAMPK independently of insulin and significantly increased glucose uptake into L6 myotubes following translocation of glucose transporter 4 (Glut4) to the plasma membrane. This activation was induced by the LKB1 pathway but was independent of changes in intracellular Ca(2+) levels and the Ca(2+)/calmodulin-dependent kinase pathway. L6 myotubes express only one type of oligopeptide transporter, peptide/histidine transporter 1 (PHT1, also known as SLC15a4), and WH is incorporated into cells and activates AMPKα following PHT1-mediated cell uptake. These findings indicate that (1) WH activates AMPK and insulin independently enhances glucose uptake following translocation of Glut4 to the plasma membrane, (2) activation of AMPKα by WH is mediated by the LKB1 pathway, without altering the Ca(2+)-dependent pathway, and (3) L6 myotubes express only one type of peptide transporter (PHT1; SLC15a4), which incorporates WH into cells to activate AMPKα.
Soga M, Ohashi A, Taniguchi M et al., FEBS Open Bio. 2014 Oct 22;4:898-904. doi: 10.1016/j.fob.2014.10.008. eCollection 2014.
OBJECTIVE: Liraglutide is an anti-diabetic drug and human glucagon-like peptide-1 (GLP-1) analog that primarily functions in the pancreas. However, its extra-pancreatic functions are not clear. Skeletal muscle tissue is an important determinant of blood glucose and cells take in approximately 80% of dietary glucose via glucose transporter 4 (GLUT4) on the plasma membrane. Insulin and muscle contraction are two physiological stimuli of GLUT4 translocation to the cell membrane from intracellular storage compartments, but the signaling mechanisms that mediate these processes are different. AMP-activated protein kinase (AMPK) and Akt are the key signal molecules mediating the effects of muscle contraction and insulin, respectively, on GLUT4 translocation. Here, we investigate the effect of liraglutide on GLUT4 translocation and the roles of AMPK and Akt in this mechanism in skeletal muscle cells by stably expressing GLUT4myc with an exofacial myc-epitope C(2)C(12)-GLUT4myc.
MATERIALS/METHODS: The cell surface GLUT4myc levels were determined by an antibody-coupled colorimetric assay. The phosphorylation levels of AMPK, Akt, AS160, TBC1D1, and GLUT4 were determined by western blotting. The cAMP levels were measured by an ELISA kit. siRNA was transfected with Lipofectamine 2000. Analysis of variance (ANOVA) was used for data analysis.
RESULTS: Liraglutide stimulated GLUT4 translocation in C(2)C(12)-GLUT4myc myotubes. Liraglutide increased the intracellular cAMP levels and the phosphorylation of AMPK, AS160, and TBC1D1. Akt phosphorylation and GLUT4 expression were not affected. Inhibition of AMPK by siRNA or Compound C reduced liraglutide-induced GLUT4 translocation.
CONCLUSION: Our results suggest that liraglutide may induce GLUT4 translocation by activation of AMPK in muscle cells.
Li Z, Ni CL, Yao Z et al., Metabolism. 2014 Aug;63(8):1022-30. doi: 10.1016/j.metabol.2014.05.008. Epub 2014 May 17.
BACKGROUND/AIMS: Glucagon-like peptide-1 (GLP-1), which counteracts insulin resistance in humans with type 2 diabetes, has been shown to ameliorate diabetic nephropathy in experimental models. However, the mechanisms through which GLP-1 modulates renal function remained illdefined. The present study investigated the putative mechanisms underlying effects of exendin-4, a GLP-1 analog, on mesangial cell proliferation and fibronectin.
METHODS: Rat mesangial cells (MCs) were treated with exendin-4 under high glucose conditions. AMP-activated protein kinase (AMPK) inhibitors (compound C) and agonists (AICAR) were used to analyze the role of this kinase. Cell proliferation was measured using a MTT assay. Fibronectin expression and AMPK-signaling pathway activity were assessed using ELISA and Western blotting, respectively. The production of matrix metalloproteinase (MMP)-2 and tissue inhibitors of metalloproteinases (TIMP)-2 was evaluated using quantitative real-time RT-PCR.
RESULTS: Exendin-4 inhibited cell proliferation and fibronectin secretion in high glucose-induced MCs. It also caused phosphorylation of AMPK and subsequently increased the ratio of MMP-2 to TIMP-2, which resulted in the degradation of fibronectin. Exendin-4 reversed extracellular signal-regulated kinase (ERK) phosphorylation and enhanced expression of mammalian target of rapamycin (mTOR) in MCs. Moreover, the activation of the AMPK pathway by exendin-4 was induced by AICAR, which was inhibited by compound C.
CONCLUSION: Exendin-4 exerts an inhibitory effect on cell proliferation and fibronectin secretion in rat MCs, partly through AMPK activation. These results may explain some of the beneficial effects of exendin-4 on the kidney.
Xu WW, Guan MP, Zheng ZJ et al., Cell Physiol Biochem. 2014;33(2):423-32. doi: 10.1159/000358623. Epub 2014 Feb 11.
Physical exercise has potent therapeutic and preventive effects against metabolic disorders. A number of studies have suggested that 5'-AMP-activated protein kinase (AMPK) plays a pivotal role in regulating carbohydrate and lipid metabolism in contracting skeletal muscles, while several genetically manipulated animal models revealed the significance of AMPK-independent pathways. To elucidate significance of AMPK and AMPK-independent signals in contracting skeletal muscles, we conducted a metabolomic analysis that compared the metabolic effects of 5-aminoimidazole-4-carboxamide-1-β-D-ribonucleoside (AICAR) stimulation with the electrical contraction ex vivo in isolated rat epitrochlearis muscles, in which both α1- and α2-isoforms of AMPK and glucose uptake were equally activated. The metabolomic analysis using capillary electrophoresis time-of-flight mass spectrometry detected 184 peaks and successfully annotated 132 small molecules. AICAR stimulation exhibited high similarity to the electrical contraction in overall metabolites. Principal component analysis (PCA) demonstrated that the major principal component characterized common effects whereas the minor principal component distinguished the difference. PCA and a factor analysis suggested a substantial change in redox status as a result of AMPK activation. We also found a decrease in reduced glutathione levels in both AICAR-stimulated and contracting muscles. The muscle contraction-evoked influences related to the metabolism of amino acids, in particular, aspartate, alanine, or lysine, are supposed to be independent of AMPK activation. Our results substantiate the significance of AMPK activation in contracting skeletal muscles and provide novel evidence that AICAR stimulation closely mimics the metabolomic changes in the contracting skeletal muscles.
Miyamoto L, Egawa T, Oshima R et al., Am J Physiol Cell Physiol. 2013 Dec 15;305(12):C1214-22. doi: 10.1152/ajpcell.00162.2013. Epub 2013 Oct 2.
AMP-activated protein kinase (AMPK) regulates metabolism in skeletal muscle, and myostatin (MSTN) negatively regulates skeletal muscle development and growth. In the present study, AMPK activation and the relationship between AMPK and MSTN during myogenic differentiation were investigated in cultures derived from bovine skeletal muscle. Myoblasts capable of forming myotubes were obtained from bovine skeletal muscle and treated with AICAR to activate AMPK, resulting in suppressed myotube formation. AICAR treatment significantly reduced the expression of MSTN mRNA during myogenic differentiation. Combined treatment with AICAR and MSTN suppressed myotube formation to a greater extent than AICAR alone. SB431542, an inhibitor of MSTN signaling, promoted myotube formation during myogenic differentiation. However, simultaneous treatment with AICAR blocked this effect of SB431542. Therefore, AMPK activation inhibits myogenic differentiation but may suppress MSTN expression to balance muscle development.
Miyake M, Takahashi H, Kitagawa E et al., Cell Tissue Res. 2012 Aug;349(2):615-23. doi: 10.1007/s00441-012-1422-8. Epub 2012 May 24.