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Bioactives of Artemisia dracunculus L. enhance insulin sensitivity by modulation of ceramide metabolism in rat skeletal muscle cells.

ABSTRACT: An increase in ectopic lipids in peripheral tissues has been implicated in attenuating insulin action. The botanical extract of Artemisia dracunculus L. (PMI 5011) improves insulin action, yet the precise mechanism is unknown. The aim of this study was to determine whether the mechanism by which the bioactive compounds in PMI 5011 improve insulin signaling is through regulation of ceramide metabolism.L6 Myotubes were separately preincubated with 250 ?M palmitic acid with or without PMI 5011 or four bioactive compounds isolated from PMI 5011 and postulated to be responsible for the effect. The effects on insulin signaling, ceramide, and glucosylceramide profiles were determined.Treatment of L6 myotubes with palmitic acid resulted in increased levels of total ceramides and glucosylceramides, and cell surface expression of gangliosides. Palmitic acid also inhibited insulin-stimulated phosphorylation of protein kinase B/Akt and reduced glycogen accumulation. Bioactives from PMI 5011 had no effect on ceramide formation but one active compound (DMC-2) and its synthetic analog significantly reduced glucosylceramide accumulation and increased insulin sensitivity via restoration of Akt phosphorylation.The observations suggest that insulin sensitization by PMI 5011 is partly mediated through moderation of glycosphingolipid accumulation.

SUBMITTER: Obanda DN

PROVIDER: S-EPMC4082801 | biostudies-literature | 2014 Jul-Aug

REPOSITORIES: biostudies-literature

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Bioactives of Artemisia dracunculus L. enhance insulin sensitivity by modulation of ceramide metabolism in rat skeletal muscle cells.

Obanda Diana N DN Ribnicky David M DM Raskin Ilya I Cefalu William T WT

Nutrition (Burbank, Los Angeles County, Calif.) 20140319 7-8 Suppl

<h4>Objective</h4>An increase in ectopic lipids in peripheral tissues has been implicated in attenuating insulin action. The botanical extract of Artemisia dracunculus L. (PMI 5011) improves insulin action, yet the precise mechanism is unknown. The aim of this study was to determine whether the mechanism by which the bioactive compounds in PMI 5011 improve insulin signaling is through regulation of ceramide metabolism.<h4>Methods</h4>L6 Myotubes were separately preincubated with 250 μM palmitic ...[more]

PMID: 24985108

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Project description:ObjectivesA 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.MethodsInsulin 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.ResultsInsulin 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.ConclusionThis 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:Adiponectin is an adipokine whose plasma levels are inversely related to degrees of insulin resistance (IR) or obesity. It enhances glucose disposal and mitochondrial substrate oxidation in skeletal muscle and its actions are mediated through binding to receptors, especially adiponectin receptor 1 (AdipoR1). However, the in vivo significance of adiponectin sensitivity and the molecular mechanisms of muscle insulin sensitization by adiponectin have not been fully established. We used in vivo electrotransfer to overexpress AdipoR1 in single muscles of rats, some of which were fed for 6 wk with chow or high-fat diet (HFD) and then subjected to hyperinsulinemic-euglycemic clamp. After 1 wk, the effects on glucose disposal, signaling, and sphingolipid metabolism were investigated in test vs. contralateral control muscles. AdipoR1 overexpression (OE) increased glucose uptake and glycogen accumulation in the basal and insulin-treated rat muscle and also in the HFD-fed rats, locally ameliorating muscle IR. These effects were associated with increased phosphorylation of insulin receptor substrate-1, Akt, and glycogen synthase kinase-3?. AdipoR1 OE also caused increased phosphorylation of p70S6 kinase, AMP-activated protein kinase, and acetyl-coA carboxylase as well as increased protein levels of adaptor protein containing pleckstrin homology domain, phosphotyrosine binding domain, and leucine zipper motif-1 and adiponectin, peroxisome proliferator activated receptor-? coactivator-1?, and uncoupling protein-3, indicative of increased mitochondrial biogenesis. Although neither HFD feeding nor AdipoR1 OE caused generalized changes in sphingolipids, AdipoR1 OE did reduce levels of sphingosine 1-phosphate, ceramide 18:1, ceramide 20:2, and dihydroceramide 20:0, plus mRNA levels of the ceramide synthetic enzymes serine palmitoyl transferase and sphingolipid ?-4 desaturase, changes that are associated with increased insulin sensitivity. These data demonstrate that enhancement of local adiponectin sensitivity is sufficient to improve skeletal muscle IR.

Dataset Information

Bioactives of Artemisia dracunculus L. enhance insulin sensitivity by modulation of ceramide metabolism in rat skeletal muscle cells.

Publications

Bioactives of Artemisia dracunculus L. enhance insulin sensitivity by modulation of ceramide metabolism in rat skeletal muscle cells.

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