Artemisia dracunculus L. extract ameliorates insulin sensitivity by attenuating inflammatory signalling in human skeletal muscle culture.

AIMS: Bioactives 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. METHODS: Muscle 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. RESULTS: We 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. CONCLUSIONS: PMI 5011 improved insulin sensitivity in diabetic-obese myotubes to the level of normal-lean myotubes despite the presence of pro-inflammatory cytokines.

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.

Failure of dietary quercetin to alter the temporal progression of insulin resistance among tissues of C57BL/6J mice during the development of diet-induced obesity.

AIMS/HYPOTHESES: High-fat diets produce obesity and glucose intolerance by promoting the development of insulin resistance in peripheral tissues and liver. The present studies sought to identify the initial site(s) where insulin resistance develops using a moderately high-fat diet and to assess whether the bioflavonoid, quercetin, ameliorates progression of this sequence. METHODS: Four cohorts of male C57BL/6J mice were placed on diets formulated to be low-fat (10% of energy from fat), high-fat (45% of energy from fat) or high-fat plus 1.2% quercetin (wt/wt). After 3 and 8 weeks, cohorts were evaluated using euglycaemic-hyperinsulinaemic clamps, metabolomic analysis of fatty acylcarnitines and acute in vitro assessments of insulin signalling among tissues. RESULTS: After 3 and 8 weeks, the high-fat diet produced whole-body insulin resistance without altering insulin-dependent glucose uptake in peripheral tissues. The primary defect was impaired suppression of hepatic glucose production by insulin at both times. Quercetin initially exacerbated the effect of high-fat diet by further increasing hepatic insulin resistance, but by 8 weeks insulin resistance and hepatic responsiveness to insulin were similarly compromised in both high-fat groups. The high-fat diet, irrespective of quercetin, increased short-chain fatty acylcarnitines in liver but not in muscle, while reciprocally reducing hepatic long-chain fatty acylcarnitines and increasing them in muscle. CONCLUSIONS/INTERPRETATION: Failure of insulin to suppress hepatic glucose output is the initial defect that accounts for the insulin resistance that develops after short-term consumption of a high-fat (45% of energy) diet. Hepatic insulin resistance is associated with accumulation of short- and medium-, but not long-chain fatty acylcarnitines. Dietary quercetin does not ameliorate the progression of this sequence.

The Effects of Exogenous Auxins on Endogenous Indole-3-Acetic Acid Metabolism (The Implications for Carrot Somatic Embryogenesis).

The effect of auxin application on auxin metabolism was investigated in excised hypocotyl cultures of carrot (Daucus carota). Concentrations of both free and conjugated indole-3-acetic acid (IAA), [2H4]IAA, 2,4-dichlorophenoxyacetic acid, and naphthaleneacetic acid (NAA) were measured by mass spectroscopy using stable-isotope-labeled internal standards. [13C1]NAA was synthesized for this purpose, thus extending the range of auxins that can be assayed by stable-isotope techniques. 2,4-Dichlorophenoxyacetic acid promoted callus proliferation of the excised hypocotyls, accumulated as the free form in large quantities, and had minor effects on endogenous IAA concentrations. NAA promoted callus proliferation and the resulting callus became organogenic, producing both roots and shoots. NAA was found mostly in the conjugated form and had minor effects on endogenous IAA concentrations. [2H4]IAA had no visible effect on the growth pattern of cultured hypocotyls, possibly because it was rapidly metabolized to form inactive conjugates or possibly because it mediated a decrease in endogenous IAA concentrations by an apparent feedback mechanism. The presence of exogenous auxins did not affect tryptophan labeling of either the endogenous tryptophan or IAA pools. This suggested that exogenous auxins did not alter the IAA biosynthetic pathway, but that synthetic auxins did appear to be necessary to induce callus proliferation, which was essential for excised hypocotyls to gain the competence to form somatic embryos.

In vivo effects of dietary quercetin and quercetin-rich red onion extract on skeletal muscle mitochondria, metabolism, and insulin sensitivity.

Red onions and low doses of the flavonoid, quercetin, increase insulin sensitivity and improve glucose tolerance. We hypothesized that dietary supplementation with red onion extract (RO) would attenuate high fat diet (HFD)-induced obesity and insulin resistance similar to quercetin supplementation by increasing energy expenditure through a mechanism involving skeletal muscle mitochondrial adaptations. To test this hypothesis, C57BL/6J mice were randomized into four groups and fed either a low fat diet (LF), HFD (HF), HFD + quercetin (HF + Q), or HFD + RO (HF + RO) for 9 weeks. Food consumption and body weight and composition were measured weekly. Insulin sensitivity was assessed by insulin and glucose tolerance tests. Energy expenditure and physical activity were measured by indirect calorimetry. Skeletal muscle incomplete beta oxidation, mitochondrial number, and mtDNA-encoded gene expression were measured. Quercetin and RO supplementation decreased HFD-induced fat mass accumulation and insulin resistance (measured by insulin tolerance test) and increased energy expenditure; however, only HF + Q showed an increase in physical activity levels. Although quercetin and RO similarly increased skeletal muscle mitochondrial number and decreased incomplete beta oxidation, establishing mitochondrial function similar to that seen in LF, only HF + Q exhibited consistently lower mRNA levels of mtDNA-encoded genes necessary for complexes IV and V compared to LF. Quercetin- and RO-induced improvements in adiposity, insulin resistance, and energy expenditure occur through differential mechanisms, with quercetin-but not RO-induced energy expenditure being related to increases in physical activity. While both treatments improved skeletal muscle mitochondrial number and function, mtDNA-encoded transcript levels suggest that the antiobesogenic, insulin-sensitizing effects of purified quercetin aglycone, and RO may occur through differential mechanisms.

Influence of emulsifier structure on lipid bioaccessibility in oil-water nanoemulsions.

The influence of several nonionic surfactants (Tween-20, Tween-40, Tween-60, Span-20, Span-60, or Span-80) and anionic surfactants (sodium lauryl sulfate, sodium stearoyl lactylate, and sodium stearyl fumarate) showed drastic differences in the rank order of lipase activity/lipid bioaccessibility. The biophysical composition of the oil and water interface has a clear impact on the bioaccessibility of fatty acids (FA) by altering the interactions of lipase at the oil-water interface. It was found that the bioaccessibility was positively correlated with the hydrophilic/lipophilic balance (HLB) of the surfactant and inversely correlated to the surfactant aliphatic chain length. Furthermore, the induction time in the jejunum increased as the HLB value increased and decreased with increasing aliphatic chain length. The rate of lipolysis slowed in the jejunum with increasing HLB and with increasing aliphatic chain length.

Antihyperglycemic activity of Tarralin, an ethanolic extract of Artemisia dracunculus L.

The studies reported here were undertaken to examine the antihyperglycemic activity of an ethanolic extract of Artemisia dracunculus L., called Tarralin in diabetic and non-diabetic animals. In genetically diabetic KK-A(gamma) mice, Tarralin treatment by gavage (500 mg/kg body wt./day for 7 days) lowered elevated blood glucose levels by 24% from 479+/-25 to 352+/-16 mg/dl relative to control animals. In comparison, treatment with the known antidiabetic drugs, troglitazone (30 mg/kg body wt./day) and metformin (300 mg/kg body wt./day), decreased blood glucose concentrations by 28% and 41%, respectively. Blood insulin concentrations were reduced in the KK-A(gamma) mice by 33% with Tarralin, 48% with troglitazone and 52% with metformin. In (STZ)-induced diabetic mice, Tarralin treatment, (500 mg/kg body wt./day for 7 days), also significantly lowered blood glucose concentrations, by 20%, from 429+/-41 to 376+/-58 mg/dl relative to control. As a possible mechanism, Tarralin was shown to significantly decrease phosphoenolpyruvate carboxykinase (PEPCK) mRNA expression by 28% in STZ-induced diabetic rats. In non-diabetic animals, treatment with Tarralin did not significantly alter PEPCK expression, blood glucose or insulin concentrations. The extract was also shown to increase the binding of glucagon-like peptide (GLP-1) to its receptor in vitro. These results indicate that Tarralin has antihyperglycemic activity and a potential role in the management of diabetic states.

A microtechnique for the analysis of free and conjugated indole-3-acetic acid in milligram amounts of plant tissue using a benchtop gas chromatograph-mass spectrometer.

A microtechnique was developed for the quantification of indole-3-acetic acid (IAA) in plant samples of one milligram fresh weight or less. The method permitted quantification of both free and conjugated IAA using a benchtop gas chromatograph-mass spectrometer. New methods for sample purification with high recovery at microscale levels, together with simple changes that result in enhanced sensitivity of the instrumentation, allowed for a significant reduction in the amount of plant material required for analysis. Single oat (Avena sativa L.) coleoptile tips could be studied with this method and were found to contain free and total IAA levels of 137 and 399 pg.mg-1 fresh weight, respectively. A single 5-d-old Arabidopsis thaliana (L.) Heynh. seedling was shown to contain 61 pg.mg-1 fresh weight free IAA and 7850 pg.mg-1 fresh weight of total IAA following basic hydrolysis. This microtechnique provides a way to accurately measure IAA levels in very small structures and individual seedlings, thus making it a valuable research tool for elucidating the role and distribution of auxin in relation to growth and development.

Regulation of indole-3-acetic Acid biosynthetic pathways in carrot cell cultures.

2,4-Dichlorophenoxyacetic acid (2,4-D) promotes the accumulation of tryptophan-derived indole-3-acetic acid (IAA) in carrot cell cultures during callus proliferation by a biosynthetic pathway that is apparently not active during somatic embryo formation. The effects of 2,4-D were examined by measuring the isotopic enrichment of IAA due to the incorporation of stable isotope-labeled precursors (deuterium oxide, [(15)N]indole, and (2)H(5)-l-tryptophan). Enrichment of IAA from deuterium oxide is similar in both cultured hypocotyls and cell suspension cultures in the presence and absence of 2,4-D, despite the large differences in absolute IAA concentrations. The enrichment of IAA due to the incorporation of [(15)N]indole is also similar in callus proliferating in the presence of 2,4-D and in embryos developing in the absence of 2,4-D. The incorporation of (2)H(5)-l-tryptophan into IAA, however, is at least 7-fold higher in carrot callus cultures proliferating in the presence of 2,4-D than in embryos developing in the absence of 2,4-D. Other experiments demonstrated that this differential incorporation of (2)H(5)-l-tryptophan into IAA does not result from differential tryptophan uptake or its subsequent compartmentation. Thus, it appears that differential pathways for IAA synthesis operate in callus cultures and in developing embryos, which may suggest that a relationship exists between the route of IAA biosynthesis and development.