Bioactives from Artemisia dracunculus L. enhance insulin sensitivity via modulation of skeletal muscle protein phosphorylation.

OBJECTIVES: A botanical extract from Artemisia dracunculus L., termed PMI 5011, has been shown to improve insulin sensitivity by increasing cellular insulin signaling in in vitro and in vivo studies. These studies suggest that PMI 5011 effects changes in phosphorylation levels of proteins involved in insulin signaling. The aim of this study was to explore the effects of this promising botanical extract on the human skeletal muscle phosphoproteome, by evaluating changes in site-specific protein phosphorylation levels in primary skeletal muscle cultures from obese, insulin-resistant individuals stimulated with and without insulin. METHODS: Insulin resistance is a condition in which a normal or elevated insulin level results in an abnormal biologic response, e.g., glucose uptake. Using isobaric tagging for relative and absolute quantification (iTRAQ™) followed by phosphopeptide enrichment and liquid chromatography-tandem mass spectrometry, 125 unique phosphopeptides and 159 unique phosphorylation sites from 80 unique proteins were identified and quantified. RESULTS: Insulin stimulation of primary cultured muscle cells from insulin-resistant individuals resulted in minimal increase in phosphorylation, demonstrating impaired insulin action in this condition. Treatment with PMI 5011 resulted in significant up-regulation of 35 phosphopeptides that were mapped to proteins participating in the regulation of transcription, translation, actin cytoskeleton signaling, caveolae translocation, and translocation of glucose transporter 4. These data further showed that PMI 5011 increased phosphorylation levels of specific amino acids in proteins in the insulin-resistant state that are normally phosphorylated by insulin (thus, increasing cellular insulin signaling) and PMI 5011 also increased the abundance of phosphorylation sites of proteins regulating anti-apoptotic effects. CONCLUSION: This phosphoproteomics analysis demonstrated conclusively that PMI 5011 effects changes in phosphorylation levels of proteins and identified novel pathways by which PMI 5011 exerts its insulin-sensitizing effects in skeletal muscle.

Analysis of magnetic gradients to study gravitropism.

PREMISE OF THE STUDY: Gravitropism typically is generated by dense particles that respond to gravity. Experimental stimulation by high-gradient magnetic fields provides a new approach to selectively manipulate the gravisensing system. METHODS: The movement of corn, wheat, and potato starch grains in suspension was examined with videomicroscopy during parabolic flights that generated 20 to 25 s of weightlessness. During weightlessness, a magnetic gradient was generated by inserting a wedge into a uniform, external magnetic field that caused repulsion of starch grains. The resultant velocity of movement was compared with the velocity of sedimentation under 1 g conditions. RESULTS: The high-gradient magnetic fields repelled the starch grains and generated a force of at least 0.6 g. Different wedge shapes significantly affected starch velocity and directionality of movement. CONCLUSIONS: Magnetic gradients are able to move diamagnetic compounds under weightless or microgravity conditions and serve as directional stimulus during seed germination in low-gravity environments. Further work can determine whether gravity sensing is based on force or contact between amyloplasts and statocyte membrane system.

Proteomic analysis reveals cellular pathways regulating carbohydrate metabolism that are modulated in primary human skeletal muscle culture due to treatment with bioactives from Artemisia dracunculus L.

Insulin resistance is a major pathophysiologic abnormality that characterizes metabolic syndrome and type 2 diabetes. A well characterized ethanolic extract of Artemisia dracunculus L., termed PMI 5011, has been shown to improve insulin action in vitro and in vivo, but the cellular mechanisms remain elusive. Using differential proteomics, we have studied mechanisms by which PMI 5011 enhances insulin action in primary human skeletal muscle culture obtained by biopsy from obese, insulin-resistant individuals. Using iTRAQ™ labeling and LC-MS/MS, we have identified over 200 differentially regulated proteins due to treatment with PMI 5011 and insulin stimulation. Bioinformatics analyses determined that several metabolic pathways related to glycolysis, glucose transport and cell signaling were highly represented and differentially regulated in the presence of PMI 5011 indicating that this extract affects several pathways modulating carbohydrate metabolism, including translocation of GLUT4 to the plasma membrane. These findings provide a molecular mechanism by which a botanical extract improves insulin stimulated glucose uptake, transport and metabolism at the cellular level resulting in enhanced whole body insulin sensitivity.

Gel-based and gel-free proteomic technologies.

Proteomics refers to the analysis of expression, localization, functions, posttranslational modifications, and interactions of proteins expressed by a genome at a specific condition and at a specific time. Mass spectrometry (MS)-based proteomic methods have emerged as a key technology for unbiased systematic and high-throughput identification and quantification of complex protein mixtures. These methods have the potential to reveal unknown and novel changes in protein interactions and assemblies that regulate cellular and physiological processes. Both gel-based (one-dimensional [1D] gel electrophoresis, two-dimensional [2D] polyacrylamide gel electrophoresis, 2D difference in-gel electrophoresis [DIGE]) and gel-free (liquid chromatography [LC], capillary electrophoresis) approaches have been developed and utilized in a variety of combinations to separate proteins prior to mass spectrometric analysis. Detailed protocols for global proteomic analysis from adipose-derived stem cells (ASCs) using two central strategies, 2D-DIGE-MS and 2D-LC-MS, are presented here.

Solid phase gene extraction isolates mRNA at high spatial and temporal resolution.

Rapid, localized changes in gene expression require mRNA extraction at high temporal and spatial resolution. Current small-scale mRNA extractions depend on the removal of the cells/tissue from an organism or preserved specimens. What these methods have in common is that they are destructive and do not distinguish between genomic DNA and RNA. Therefore, extracted (m)RNA is typically contaminated by extracted cytoplasm, nuclear DNA, or other compounds, and the required purification leads to loss of especially low-abundant mRNA. The need to repeatedly remove mRNA from living material has led to the development of solid phase gene extraction (SPGE). SPGE sampling can be achieved using gene-specific or generic sequences and is not species-specific. Here we demonstrate the versatility and validity of this novel RNA extraction by simultaneously profiling nanos and bicoid mRNA in individual Drosophila eggs. The SPGE technique detects previously described distribution profiles of nanos and bicoid. Its low impact is underscored by the normal development of repeatedly sampled eggs. In our study, quantification of actin mRNA in germinating flax seeds linked gene expression to distinct developmental processes. These data demonstrate the universality of SPGE as a simple generic, analytical, and diagnostic procedure.

Auxin and cytoskeletal organization in algae.

Hormones affect growth and alter the cytoskeleton suggesting that hormones and the cytoskeleton interact with each other. The cytoskeleton of ancestral algae such as Chara showed similar sensitivity to auxin as higher plants, even in generative structures but the sensitivity differed between IAA and alpha-NAA and presumably other auxins. The ability of cells to elongate depends on microtubule organization during the transition from disorganized to perpendicular to longitudinal organization of the cytoskeleton. Because of the many functions of the cytoskeleton it is possible that its composition is influenced by selective gene expression and adaptation to growth regulators. Co-localization of microtubules and F-actin change at a high temporal and spatial scale. High resolution measurements of mRNA expression indicate rapid turnover that may affect the composition of the cytoskeleton.

Anisotropic viscosity of the Chara (Characeae) rhizoid cytoplasm.

To characterize cellular fluidity and mechanical processes, we determined the viscous properties of the cytoplasm of Chara contraria rhizoids in vivo by injecting and displacing superparamagnetic particles. After injection and a 24-h recovery period, the particles were moved to different positions within the rhizoid by an external magnet. The system was calibrated with solutions of known viscosities. The viscosity was determined based on the velocity at which individual beads moved toward the external magnet. The viscosity of the cytoplasm varied with direction of measurement (i.e., was highly anisotropic) and also varied between sites. The highest viscosity was observed near the endogenous statoliths (139 mP·s parallel and 78 mP·s perpendicular to the rhizoid axis). Depolymerization of actin filaments with latrunculin B reduced the viscosity significantly except around the nucleus but did not change the overall viscosity pattern. Microtubule depolymerization with oryzalin reduced viscosity especially between the nucleus and the statolith zone. The data indicate that F-actin but not microtubules affects statolith sedimentation and that cytoplasmic viscosity may be important for the gravisensing system.