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.

Muscle-specific deletion of carnitine acetyltransferase compromises glucose tolerance and metabolic flexibility.

The concept of "metabolic inflexibility" was first introduced to describe the failure of insulin-resistant human subjects to appropriately adjust mitochondrial fuel selection in response to nutritional cues. This phenomenon has since gained increasing recognition as a core component of the metabolic syndrome, but the underlying mechanisms have remained elusive. Here, we identify an essential role for the mitochondrial matrix enzyme, carnitine acetyltransferase (CrAT), in regulating substrate switching and glucose tolerance. By converting acetyl-CoA to its membrane permeant acetylcarnitine ester, CrAT regulates mitochondrial and intracellular carbon trafficking. Studies in muscle-specific Crat knockout mice, primary human skeletal myocytes, and human subjects undergoing L-carnitine supplementation support a model wherein CrAT combats nutrient stress, promotes metabolic flexibility, and enhances insulin action by permitting mitochondrial efflux of excess acetyl moieties that otherwise inhibit key regulatory enzymes such as pyruvate dehydrogenase. These findings offer therapeutically relevant insights into the molecular basis of metabolic inflexibility.

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.

Regulation of insulin action by an extract of Artemisia dracunculus L. in primary human skeletal muscle culture: a proteomics approach.

An ethanolic extract of Artemisia dracunculus L. (PMI 5011) has been observed to decrease glucose and insulin levels in animal models and enhance cellular signaling in cultured cells. To determine the mechanism of action of PMI-5011, we have measured changes in protein expression in human primary skeletal muscle culture (HSMC) from subjects with Type 2 diabetes. After obtaining skeletal muscle biopsies, HSMCs were initiated, grown to confluence, and exposed to 10 microg/mL PMI 5011 overnight. Two-dimensional difference in-gel electrophoresis was used to separate proteins, and liquid chromatography mass spectrometry was used to identify differentially regulated proteins. Additionally, real-time polymerase chain reaction (PCR) was used to confirm candidate proteins identified. These data demonstrate that a well characterized botanical extract of Artemisia dracunculus L. significantly modulates proteins involved in regulating inflammatory pathways, particularly the NFkappaB complex system.

Mass spectrometric quantification of MIF-1 in mouse brain by multiple reaction monitoring.

MIF-1 (Pro-Leu-Gly-NH(2)) has potent therapeutic effects in depression and Parkinson's disease, but its CNS sites of production are not yet clear. In this study, the concentration of MIF-1 in different brain regions was measured by the multiple reaction monitoring technique on a 4000 QTRAP mass spectrometer. The limit of quantification was 300 fg of MIF-1, and limit of detection was 60 fg. The low molecular weight fractions of tissue homogenates from different regions of mouse brain were analyzed. The concentration of MIF-1 ranged from 22+/-3 fg/microg protein in cerebral cortex to 930+/-60 fg/microg protein in the hypothalamus. Moderate concentrations were also detected in all other regions tested, including the striatum, thalamus, and hippocampus. By incubation of stable isotope-labeled oxytocin with tissue preparations, it was also confirmed that oxytocin at least partially contributed to the production of MIF-1 in the hypothalamus by action of peptidases. Regional differences were also found. The results are the first to show the ultrasensitive quantification of MIF-1 in different brain regions, and support the neuromodulatory actions of MIF-1 in the striatum.