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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.

ABSTRACT: 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.

SUBMITTER: Scherp P

PROVIDER: S-EPMC3367113 | biostudies-literature | 2012 Jun

REPOSITORIES: biostudies-literature

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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.

Scherp Peter P Putluri Nagireddy N LeBlanc Gary J GJ Wang Zhong Q ZQ Zhang Xian H XH Yu Yongmei Y Ribnicky David D Cefalu William T WT Kheterpal Indu I

Journal of proteomics 20120326 11

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 ...[more]

PMID: 22480907

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Project description: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.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.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.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.

Project description:AimsBioactives of Artemisia dracunculus L. (termed PMI 5011) have been shown to improve insulin action by increasing insulin signalling in skeletal muscle. However, it was not known if PMI 5011's effects are retained during an inflammatory condition. We examined the attenuation of insulin action and whether PMI 5011 enhances insulin signalling in the inflammatory environment with elevated cytokines.MethodsMuscle cell cultures derived from lean, overweight and diabetic-obese subjects were used. Expression of pro-inflammatory genes and inflammatory response of human myotubes were evaluated by real-time polymerase chain reaction (RT-PCR). Insulin signalling and activation of inflammatory pathways in human myotubes were evaluated by multiplex protein assays.ResultsWe found increased gene expression of monocyte chemoattractant protein 1 (MCP1) and TNFα (tumour necrosis factor alpha), and basal activity of the NFkB (nuclear factor kappa-light-chain-enhancer of activated B cells) pathway in myotubes derived from diabetic-obese subjects as compared with myotubes derived from normal-lean subjects. In line with this, basal Akt phosphorylation (Ser473) was significantly higher, while insulin-stimulated phosphorylation of Akt (Ser473) was lower in myotubes from normal-overweight and diabetic-obese subjects compared with normal-lean subjects. PMI 5011 treatment reduced basal phosphorylation of Akt and enhanced insulin-stimulated phosphorylation of Akt in the presence of cytokines in human myotubes. PMI 5011 treatment led to an inhibition of cytokine-induced activation of inflammatory signalling pathways such as Erk1/2 and IkBα (nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha)-NFkB and moreover, NFkB target gene expression, possibly by preventing further propagation of the inflammatory response within muscle tissue.ConclusionsPMI 5011 improved insulin sensitivity in diabetic-obese myotubes to the level of normal-lean myotubes despite the presence of pro-inflammatory cytokines.

Project description:Despite their reported therapeutic properties, not much is known about the immunomodulatory activity of essential oils present in Artemisia species. We isolated essential oils from the flowers and leaves of five Artemisia species: A. tridentata, A. ludoviciana, A. dracunculus, A. frigida, and A. cana. The chemical composition of the Artemisia essential oil samples had similarities and differences as compared to those previously reported in the literature. The main components of essential oils obtained from A. tridentata, A. ludoviciana, A. frigida, and A. cana were camphor (23.0-51.3%), 1,8-cineole (5.7-30.0%), camphene (1.6-7.7%), borneol (2.3-14.6%), artemisiole (1.2-7.5%), terpinen-4-ol (2.0-6.9%), α-pinene (0.8-3.9%), and santolinatriene (0.7-3.5%). Essential oils from A. dracunculus were enriched in methyl chavicol (38.8-42.9%), methyl eugenol (26.1-26.4%), terpinolene (5.5-8.8%), (E/Z)-β-ocimene (7.3-16.0%), β-phellandrene (1.3-2.2%), p-cymen-8-ol (0.9-2.3%), and xanthoxylin (1.2-2.2%). A comparison across species also demonstrated that some compounds were present in only one Artemisia species. Although Artemisia essential oils were weak activators of human neutrophils, they were relatively more potent in inhibiting subsequent neutrophil Ca2+ mobilization with N-formyl peptide receptor 1 (FPR1) agonist fMLF- and FPR2 agonist WKYMVM, with the most potent being essential oils from A. dracunculus. Further analysis of unique compounds found in A. dracunculus showed that farnesene, a compound with a similar hydrocarbon structure as lipoxin A4, inhibited Ca2+ influx induced in human neutrophils by fMLF (IC50 = 1.2 μM), WKYMVM (IC50 = 1.4 μM), or interleukin 8 (IC50 = 2.6 μM). Pretreatment with A. dracunculus essential oils and farnesene also inhibited human neutrophil chemotaxis induced by fMLF, suggesting these treatments down-regulated human neutrophil responses to inflammatory chemoattractants. Thus, our studies have identified farnesene as a potential anti-inflammatory modulator of human neutrophils.

Dataset Information

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.

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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.

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