Bioactive compounds from Artemisia dracunculus L. activate AMPK signaling in skeletal muscle.

An extract from Artemisia dracunculus L. (termed PMI-5011) improves glucose homeostasis by enhancing insulin action and reducing ectopic lipid accumulation, while increasing fat oxidation in skeletal muscle tissue in obese insulin resistant male mice. A chalcone, DMC-2, in PMI-5011 is the major bioactive that enhances insulin signaling and activation of AKT. However, the mechanism by which PMI-5011 improves lipid metabolism is unknown. AMPK is the cellular energy and metabolic sensor and a key regulator of lipid metabolism in muscle. This study examined PMI-5011 activation of AMPK signaling using murine C2C12 muscle cell culture and skeletal muscle tissue. Findings show that PMI-5011 increases Thr172-phosphorylation of AMPK in muscle cells and skeletal muscle tissue, while hepatic AMPK activation by PMI-5011 was not observed. Increased AMPK activity by PMI-5011 affects downstream signaling of AMPK, resulting in inhibition of ACC and increased SIRT1 protein levels. Selective deletion of DMC-2 from PMI-5011 demonstrates that compounds other than DMC-2 in a "DMC-2 knock out extract" (KOE) are responsible for AMPK activation and its downstream effects. Compared to 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) and metformin, the phytochemical mixture characterizing the KOE appears to more efficiently activate AMPK in muscle cells. KOE-mediated AMPK activation was LKB-1 independent, suggesting KOE does not activate AMPK via LKB-1 stimulation. Through AMPK activation, compounds in PMI-5011 may regulate lipid metabolism in skeletal muscle. Thus, the AMPK-activating potential of the KOE adds therapeutic value to PMI-5011 and its constituents in treating insulin resistance or type 2 diabetes.

The Oral Administration of Sanguisorba officinalis Extract Improves Physical Performance through LDHA Modulation.

Muscle fatigue is induced by an acute or chronic physical performance inability after excessive physical activity often associated with lactate accumulation, the end-product of glycolysis. In this study, the water-extracted roots of Sanguisorba officinalis L., a herbal medicine traditionally used for inflammation and diarrhea, reduced the activities of lactate dehydrogenase A (LDHA) in in vitro enzyme assay myoblast C2C12 cells and murine muscle tissue. Physical performance measured by a treadmill test was improved in the S. officinalis-administrated group. The analysis of mouse serum and tissues showed significant changes in lactate levels. Among the proteins related to energy metabolism-related physical performance, phosphorylated-AMP-activated protein kinase alpha (AMPKα) and peroxisome proliferator-activated receptor-coactivator-1 alpha (PGC-1α) levels were enhanced, whereas the amount of LDHA was suppressed. Therefore, S. officinalis might be a candidate for improving physical performance via inhibiting LDHA and glycolysis.

Daidzein down-regulates ubiquitin-specific protease 19 expression through estrogen receptor ß and increases skeletal muscle mass in young female mice.

Ubiquitin-specific protease 19 (USP19) is a key player in the negative regulation of muscle mass during muscle atrophy. Loss-of-function approaches demonstrate that 17ß-estradiol (E2) increases USP19 expression through estrogen receptor (ER) α and consequently decreases soleus muscle mass in young female mice under physiological conditions. Daidzein is one of the main isoflavones in soy, and activates ERß-dependent transcription. Here, we investigated the effects of daidzein on E2-increased USP19 expression and E2-decreased soleus muscle mass in young female mice. Daidzein stimulated the transcriptional activity of ERß in murine C2C12 cells and down-regulated USP19 expression. Consistently, daidzein inhibited E2-induced USP19 expression in a reporter activity using a functional half-estrogen response element (hERE) from Usp19. Daidzein inhibited E2-induced recruitment of ERα and promoted recruitment of ERß to the Usp19 hERE. Dietary daidzein down-regulated the expression of USP19 at the mRNA and protein levels and increased soleus muscle mass in female mice, but not in males. In soleus muscle from ovariectomized (OVX) female mice, dietary daidzein inhibited E2-increased USP19 mRNA expression and E2-decreased muscle mass. Furthermore, E2 induced the recruitment of ERα and ERß to the hERE, whereas daidzein inhibited E2-induced recruitment of ERα, and enhanced E2-increased recruitment of ERß, to the Usp19 hERE. These results demonstrate that dietary daidzein decreases USP19 mRNA expression through ERß and increases soleus muscle mass in young female mice, but not in male mice, under physiological conditions.

Effect of rutin from tartary buckwheat sprout on serum glucose-lowering in animal model of type 2 diabetes.

This study investigates the anti-diabetic effects of rutin from tartary buckwheat sprout in type 2 diabetes mouse model. The rutin content in tartary buckwheat sprout (TBS) is five times higher than that found in common buckwheat sprout (CBS) as evident from high-performance liquid chromatography analysis. Administration of either rutin or TBS ethanolic extract to diabetes mice decreased the serum glucose level significantly. Rutin down-regulated the expression levels of protein-tyrosine phosphatase 1B; it is negative regulator of insulin pathway, both transcriptionally and translationally in myocyte C2C12 in a dose-dependent manner. In conclusion, rutin can play a critical role in down-regulation of serum glucose level in type 2 diabetes.

Regulation of TIMP-2, MT1-MMP, and MMP-2 expression during C2C12 differentiation.

Matrix metalloproteinases (MMPs) are zinc-dependent proteases capable of degrading extracellular matrix components. The activity of these proteases is tightly regulated through the actions of the tissue inhibitors of metalloproteinases (TIMPs). Although the regulation of MMPs and TIMPs during physiological and pathological remodeling has been investigated in a number of systems, almost nothing is known about their role in skeletal muscle differentiation. To investigate the role of MMP-mediated proteolysis during myogenesis, the regulation of TIMP-2, MT1-MMP, and MMP-2 expression was investigated during differentiation of the mouse myoblastic C2C12 cell line. We show that this trio is upregulated coincident with myogenesis. The more diffuse spatial distribution of TIMP-2 relative to MT1-MMP and MMP-2 suggests that TIMP-2 may exert MMP-independent functions during myogenesis. Elucidating the regulation of these molecules during muscle differentiation in vitro may lead to a better understanding of their role in pathological processes in muscle tissue in vivo.

Diverging regulation of pyruvate dehydrogenase kinase isoform gene expression in cultured human muscle cells.

The pyruvate dehydrogenase complex occupies a central and strategic position in muscle intermediary metabolism and is primarily regulated by phosphorylation/dephosphorylation. The identification of multiple isoforms of pyruvate dehydrogenase kinase (PDK1-4) and pyruvate dehydrogenase phosphatase (PDP1-2) has raised intriguing new possibilities for chronic pyruvate dehydrogenase complex control. Experiments to date suggest that PDK4 is the major isoenzyme responsible for changes in pyruvate dehydrogenase complex activity in response to various different metabolic conditions. Using a cultured human skeletal muscle cell model system, we found that expression of both PDK2 and PDK4 mRNA is upregulated in response to glucose deprivation and fatty acid supplementation, the effects of which are reversed by insulin treatment. In addition, insulin directly downregulates PDK2 and PDK4 mRNA transcript abundance via a phosphatidylinositol 3-kinase-dependent pathway, which may involve glycogen synthase kinase-3 but does not utilize the mammalian target of rapamycin or mitogen-activated protein kinase signalling pathways. In order to further elucidate the regulation of PDK, the role of the peroxisome proliferators-activated receptors (PPAR) was investigated using highly potent subtype selective agonists. PPARalpha and PPARdelta agonists were found to specifically upregulate PDK4 mRNA expression, whereas PPARgamma activation selectively decreased PDK2 mRNA transcript abundance. PDP1 mRNA expression was unaffected by all conditions analysed. These results suggest that in human muscle, hormonal and nutritional conditions may control PDK2 and PDK4 mRNA expression via a common signalling mechanism. In addition, PPARs appear to independently regulate specific PDK isoform transcipt levels, which are likely to impart important metabolic mediation of fuel utilization by the muscle.

Treatment of cells with the angiogenic inhibitor fumagillin results in increased stability of eukaryotic initiation factor 2-associated glycoprotein, p67, and reduced phosphorylation of extracellular signal-regulated kinases.

Fumagillin, an angiogenic inhibitor, binds to methionine aminopeptidase 2, which is the same as eukaryotic initiation factor 2-associated glycoprotein, p67. p67 protects eIF2alpha from phosphorylation by its kinases. To understand the importance of fumagillin binding to p67, we measured the level of p67 in mouse C2C12 myoblasts treated with fumagillin. We show that fumagillin increases the stability of p67 by decreasing its turnover rate. The increased levels of p67 result in inhibition of phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERKs 1 and 2). p67 binds to these ERKs, and the 108-480 amino acid segment is sufficient for this binding. p67's affinity to ERKs 1 and 2 also increases in fumagillin-treated myoblasts while its affinity for eIF2alpha remains unchanged. A mutant at the conserved amino acid residue D251A increases the phosphorylation of ERKs 1 and 2 without affecting the binding to p67, thus indicating the importance of this residue in the regulation of the phosphorylation of these ERKs. These results suggest that fumagillin increases the stability of p67 and its affinity to ERKs 1 and 2 and causes the inhibition of the phosphorylation of ERKs 1 and 2.

Ca2+ transients activate calcineurin/NFATc1 and initiate fast-to-slow transformation in a primary skeletal muscle culture.

The calcineurin-mediated signal transduction via nuclear factor of activated T cells (NFATc1) is involved in upregulating slow myosin heavy chain (MHC) gene expression during fast-to-slow transformation of skeletal muscle cells. This study aims to investigate the Ca2+ signal necessary to activate the calcineurin-NFATc1 cascade in skeletal muscle. Electrostimulation of primary myocytes from rabbit for 24 h induced a distinct fast-to-slow transformation at the MHC mRNA level and a full activation of the calcineurin-NFATc1 pathway, although resting Ca2+ concentration ([Ca2+]i) remained unaltered at 70 nM. During activation, the calcium transients of these myocytes reach a peak concentration of approximately 500 nM. Although 70 nM [Ca2+]i does not activate calcineurin-NFAT, we show by the use of Ca2+ ionophore that the system is fully activated when [Ca2+]i is >or=150 nM in a sustained manner. We conclude that the calcineurin signal transduction pathway and the slow MHC gene in cultured skeletal muscle cells are activated by repetition of the rapid high-amplitude calcium transients that are associated with excitation-contraction coupling rather than by a sustained elevation of resting Ca2+ concentration.