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Ectopic lipids in peripheral tissues have been implicated in attenuating insulin action in vivo. The botanical extract of Artemisia dracunculus L. (PMI 5011) improves insulin action, yet the precise mechanism is not known. We sought to determine whether the mechanism by which PMI 5011 improves insulin signaling is through regulation of lipid metabolism. After differentiation, cells were separately preincubated with free fatty acids (FFAs) and ceramide C2, and the effects on glycogen content, insulin signaling, and ceramide profiles were determined. The effect of PMI 5011 on ceramide accumulation and ceramide-induced inhibition of insulin signaling was evaluated. FFAs resulted in increased levels of total ceramides and ceramide species in L6 myotubes. Saturated FFAs and ceramide C2 inhibited insulin-stimulated phosphorylation of protein kinase B/Akt and reduced glycogen content. PMI 5011 had no effect on ceramide formation or accumulation but increased insulin sensitivity via restoration of Akt phosphorylation. PMI 5011 also attenuated the FFA-induced upregulation of a negative inhibitor of insulin signaling, i.e., protein tyrosine phosphatase 1B (PTP1B), and increased phosphorylation of PTP1B. PMI 5011 attenuates the reduction in insulin signaling induced by ceramide accumulation, but the mechanism of improved insulin signaling is independent of ceramide formation. [ABSTRACT FROM AUTHOR]

An ethanolic extract of Artemisia dracunculus L. (PMI 5011) has been observed to decrease glucose and insulin levels in animal models, but the cellular mechanisms by which insulin action is enhanced in vivo are not precisely known. In this study, we evaluated the effects of PMI 5011 to modulate gene expression and cellular signaling through the insulin receptor in skeletal muscle of KK-A(y) mice. Eighteen male KK-A(y) mice were randomized to a diet (w/w) mixed with PMI 5011 (1%) or diet alone for 8 weeks. Food intake, adiposity, glucose and insulin were assessed over the study, and at study completion, vastus lateralis muscle was obtained to assess insulin signaling parameters and gene expression. Animals randomized to PMI 5011 were shown to have enhanced insulin sensitivity and increased insulin receptor signaling, i.e., IRS-associated PI-3 kinase activity, Akt-1 activity and Akt phosphorylation, in skeletal muscle when compared to control animals (P<.01, P<.01 and P<.001, respectively). Gene expression for insulin signaling proteins, i.e., IRS-1, PI-3 kinase and Glut-4, was not increased, although a relative increase in protein abundance was noted with PMI 5011 treatment. Gene expression for specific ubiquitin proteins and specific 20S proteasome activity, in addition to skeletal muscle phosphatase activity, i.e., PTP1B activity, was significantly decreased in mice randomized to PMI 5011 relative to control. Thus, the data demonstrate that PMI 5011 increases insulin sensitivity and enhances insulin receptor signaling in an animal model of insulin resistance. PMI 5011 may modulate skeletal muscle protein degradation and phosphatase activity as a possible mode of action. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The fatty acid and natural product content of hemp seed oil was analyzed by GC-MS and LC-MS. The presence of linoleic acid (LA) and a-linolenic acid (LNA) were confirmed in their previously reported ratio of 3:1 LA:LNA. The presence of (3-caryophyllene (740 mg/L), myrcene (160 mg/L), (3-sitosterol (100-148 g/L) and trace amounts of methyl salicylate was observed in the oil which had not been previously reported. Trace amounts of cannabidiol (CBD) were also detected. Bioassays were performed with the oil to determine its effectiveness as an antimicrobial agent. Some bioactivity was observed during the primary screening. [ABSTRACT FROM AUTHOR]

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., (Copyright © 2021 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Two new diprenylated coumaric acid isomers ( 1a and 1b ) and two known congeners, capillartemisin A ( 2 ) and B ( 3 ), were isolated from Artemisia scoparia as bioactive markers using bioactivity-guided HPLC fractionation. Their structures were determined by spectroscopic means, including 1D and 2D NMR methods and LC-MS, with their purity assessed by 1D 1 H pure shift qNMR spectroscopic analysis. The bioactivity of compounds was evaluated by enhanced accumulation of lipids, as measured using Oil Red O staining, and by increased expression of several adipocyte marker genes, including adiponectin in 3T3-L1 adipocytes relative to untreated negative controls. Compared to the plant's 80% EtOH extract, these purified compounds showed significant but still weaker inhibition of TNFα-induced lipolysis in 3T3-L1 adipocytes. This suggests that additional bioactive substances are responsible for the multiple metabolically favorable effects on adipocytes observed with Artemisia scoparia extract.

This study aims to document the dual mode of pharmacological action of moringa isothiocyanate-1 (MIC-1) derived from seeds of Moringa oleifera Lam. Oral administration of chemically stable MIC-1 (80 mg/kg) significantly reduced the expression of inflammatory markers (Tnf-α, Ifn-α, IL-1β, IL-6) in the liver, kidney, spleen, and colon and decreased spleen weight in the lipopolysaccharide (LPS)-induced sepsis / acute inflammation model in mice. Transcriptomic analysis of the effect of MIC-1 on the liver and in the LPS-induced RAW264.7 murine macrophage showed that MIC-1 decreases inflammation via inflammation, immunity, and oxidative stress pathways. These results are supported by the immunocytochemical observations that MIC-1 increased the nuclear accumulation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) transcription factor and decreased the nuclear accumulation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) in the LPS-induced macrophages. Transcriptional activation of antioxidant genes by MIC-1 translated into a reduction of reactive oxygen species (ROS) in the cytoplasm, decrease of mitochondrial superoxide content, and restoration of the mitochondrial membrane potential in the LPS-induced macrophages. Our data indicate that MIC-1 affects inflammation and oxidative stress, two key processes involved in the etiology of many chronic diseases. These effects involve upstream regulation of two key transcriptional factors regulating responses to these processes at a gene expression level., Competing Interests: The authors have declared that no competing interests exist.

Complementing classical drug discovery, phytochemicals act on multiple pharmacological targets, especially in botanical extracts, where they form complex bioactive mixtures. The reductionist approach used in bioactivity-guided fractionation to identify single bioactive phytochemicals is inadequate for capturing the full therapeutic potential of the (bio)chemical interactions present in such complex mixtures. This study used a DESIGNER (Deplete and Enrich Select Ingredients to Generate Normalized Extract Resources) approach to selectively remove the known bioactives, 4'- O -methyldavidigenin ( 1 ; 4,2'-dihydroxy-4'-methoxydihydrochalcone, syn. DMC-1) and its isomer 4- O -methyldavidigenin ( 2 ; syn. DMC-2), from the mixture of phytochemicals in an ethanol extract from Artemisia dracunculus to determine to what degree the more abundant 2 accounts for the established antidiabetic effect of the A. dracunculus extract. Using an otherwise chemically intact "knock-out extract" depleted in 2 and its regioisomer, 1 , in vitro and in vivo outcomes confirmed that 2 (and likely 1 ) acts as major bioactive(s) that enhance(s) insulin signaling in skeletal muscle, but also revealed that 2 does not account for the breadth of detectable biological activity of the extract. This is the first report of generating, at a sufficiently large preparative scale, a "knock-out extract" used as a pharmacological tool for in vitro and in vivo studies to dissect the biological impact of a designated bioactive in a complex phytochemical mixture.

Stress-induced peripheral inflammation contributes to depression-like behaviors in both human and experimental models. PMI 5011, a botanical extract of Artemisia dracunculus L., was previously shown to have multiple bioactivities, including anti-inflammatory activity. In this work, using a repeated social defeat stress (RSDS) model of depression, we demonstrate that oral administration of the botanical extract PMI 5011 promotes resilience to RSDS-mediated depression-like phenotypes. We also show that the behavioral improvements are associated with attenuation of stress-mediated induction of inflammatory cytokines in the periphery and alteration of synaptic plasticity in the nucleus accumbens (NAc). Our studies provide experimental evidence that botanical extracts such as PMI 5011, which target pathological mechanisms (i.e., peripheral inflammation) not addressed by currently available antidepressants, could be further developed as novel therapeutics for the treatment of stress disorders and anxiety in humans.

To assess the metabolically beneficial effects of fenugreek (Trigonella foenum-graecum), C57BL/6J mice were fed a low- or high-fat diet for 16 weeks with or without 2% (w/w) fenugreek supplementation. Body weight, body composition, energy expenditure, food intake, and insulin/glucose tolerance were measured regularly, and tissues were collected for histological and biochemical analysis after 16 weeks of diet exposure. Fenugreek did not alter body weight, fat mass, or food intake in either group, but did transiently improve glucose tolerance in high fat-fed mice. Fenugreek also significantly improved high-density lipoprotein to low-density lipoprotein ratios in high fat-fed mice without affecting circulating total cholesterol, triglycerides, or glycerol levels. Fenugreek decreased hepatic expression of fatty acid-binding protein 4 and increased subcutaneous inguinal adipose tissue expression of adiponectin, but did not prevent hepatic steatosis. Notably, fenugreek was not as effective at improving glucose tolerance as was four days of voluntary wheel running. Overall, our results demonstrate that fenugreek promotes metabolic resiliency via significant and selected effects on glucose regulation, hyperlipidemia, and adipose pathology; but may not be as effective as behavioral modifications at preventing the adverse metabolic consequences of a high fat diet.

Scope: The ability of high phenolic Rutgers Scarlet Lettuce (RSL) to attenuate metabolic syndrome and gut dysbiosis was studied in very high fat diet (VHFD)-fed mice. Phenolic absorption was assessed in vivo and in a gastrointestinal tract model., Methods and Results: Mice were fed VHFD, VHFD supplemented with RSL (RSL-VHFD) or store-purchased green lettuce (GL-VHFD), or low-fat diet (LFD) for 13 weeks. Compared to VHFD or GL-VHFD-fed groups, RSL-VHFD group showed significantly improved oral glucose tolerance (p<0.05). Comparison of VHFD, RSL-VHFD, and GL-VHFD groups revealed no significant differences with respect to insulin tolerance, hepatic lipids, body weight gain, fat mass, plasma glucose, triglycerides, free fatty acid, and lipopolysaccharide levels, as well as relative abundances of major bacterial phyla from 16S rDNA amplicon data sequences (from fecal and cecal samples). However, RSL and GL-supplementation increased abundance of several taxa involved in plant polysaccharide degradation/fermentation. RSL phenolics chlorogenic acid, quercetin-3-glucoside, and quercetin-malonyl-glucoside were bioaccessible in the TIM-1 digestion model, but had relatively low recovery., Conclusions: RSL phenolics contributed to attenuation of post-prandial hyperglycemia. Changes in gut microbiota were likely due to microbiota accessible carbohydrates in RSL and GL rather than RSL phenolics, which may be metabolized, absorbed, or degraded before reaching the colon., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Objective: Excess dietary lipids result in the accumulation of lipid metabolites including ceramides that can attenuate insulin signaling. There is evidence that a botanical extract of Urtica dioica L. (stinging nettle) improves insulin action, yet the precise mechanism(s) are not known. Hence, we examined the effects of Urtica dioica L. (UT) on adipocytes., Research Design: We investigated the effects of an ethanolic extract of UT on free fatty acid (palmitic acid) induced inhibition of insulin-stimulated Akt serine phosphorylation and modulation of ceramidase expression in 3T3-L1 adipocytes. Adipocytes were exposed to excess FFAs in the presence or absence of UT. Effects on adiponectin expression, ceramidase expression, ceramidase activity, ceramide accumulation and insulin signaling were determined., Results: As expected, FFAs reduced adiponectin expression and increased the expression of ceramidase enzymes but not their activity. FFA also induced the accumulation of ceramides and reduced insulin-stimulated phosphorylation of Akt in adipocytes. The effects of FFA were partially reversed by UT. UT enhanced adiponectin expression and ceramidase activity in the presence of excess FFAs. UT abated ceramide accumulation and increased insulin sensitivity via enhanced Akt phosphorylation. A siRNA knockdown of adiponectin expression prevented UT from exerting positive effects on ceramidase activity but not Akt phosphorylation., Conclusions: In adipocytes, the ability of UT to antagonize the negative effects of FFA by modulating ceramidase activity and ceramide accumulation is dependent on the presence of adiponectin. However, the ability of UT to enhance Akt phosphorylation is independent of adiponectin expression. These studies demonstrate direct effects of UT on adipocytes and suggest this botanical extract is metabolically beneficial.

The leaf extract of Urtica dioica L. (UT) has been reported to improve glucose homeostasis in vivo, but definitive studies on efficacy and mechanism of action are lacking. We investigated the effects of UT on obesity- induced insulin resistance in skeletal muscle. Male C57BL/6J mice were divided into three groups: low-fat diet (LFD), high-fat diet (HFD) and HFD supplemented with UT. Body weight, body composition, plasma glucose and plasma insulin were monitored. Skeletal muscle (gastrocnemius) was analyzed for insulin sensitivity, ceramide accumulation and the post translational modification and activity of protein phosphatase 2A (PP2A). PP2A is activated by ceramides and dephosphorylates Akt. C2C12 myotubes exposed to excess free fatty acids with or without UT were also evaluated for insulin signaling and modulation of PP2A. The HFD induced insulin resistance, increased fasting plasma glucose, enhanced ceramide accumulation and PP2A activity in skeletal muscle. Supplementation with UT improved plasma glucose homeostasis and enhanced skeletal muscle insulin sensitivity without affecting body weight and body composition. In myotubes, UT attenuated the ability of FFAs to induce insulin resistance and PP2A hyperactivity without affecting ceramide accumulation and PP2A expression. UT decreased PP2A activity through posttranslational modification that was accompanied by a reduction in Akt dephosphorylation.

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.

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., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Objective: Plant-based therapies have been used in medicine throughout recorded history. Information about the therapeutic properties of plants often can be found in local cultures as folk medicine is communicated from one generation to the next. The aim of this study was to identify native Louisiana plants from Creole folk medicine as a potential source of therapeutic compounds for the treatment of insulin resistance, type 2 diabetes, and related disorders., Methods: We used an interdisciplinary approach combining expertise in disciplines ranging from cultural anthropology and botany to biochemistry and endocrinology to screen native southwest Louisiana plants. Translation of accounts of Creole folk medicine yielded a list of plants with documented use in treating a variety of conditions, including inflammation. These plants were collected, vouchered, and catalogued before extraction of soluble components. Extracts were analyzed for bioactivity in regulating inflammatory responses in macrophages or fatty acid-induced insulin resistance in C2C12 skeletal muscle cells., Results: Several extracts altered gene expression of inflammatory markers in macrophages. Multiplex analysis of kinase activation in insulin-signaling pathways in skeletal muscle also identified a subset of extracts that alter insulin-stimulated protein kinase B phosphorylation in the presence of fatty-acid-induced insulin resistance., Conclusion: An interdisciplinary approach to screening botanical sources of therapeutic agents can be successfully applied to identify native plants used in folk medicine as potential sources of therapeutic agents in treating insulin resistance in skeletal muscle or inflammatory processes associated with obesity-related insulin resistance., (Copyright © 2014 Elsevier Inc. All rights reserved.)

We sought to evaluate the effects of Momordica charantia (bitter melon, BM) extract on insulin sensitivity, NAFLD, hepatic FGF21 and AMPK signaling in mice fed a high-fat diet. Male C57/B6 mice were randomly divided into HFD and HFD supplementation with BM for 12 week. Body weight, plasma glucose, FGF21 and insulin levels, hepatic FGF21 and AMPK signaling proteins were measured. The results showed that plasma FGF21 and insulin concentrations were significantly decreased and hepatic FGF21 content was significantly down-regulated, while FGF receptors 1, 3 and 4 (FGFR1, FGFR3 and FGFR4) were greatly up-regulated in BM group compared to the HFD group (P < 0.05 and P < 0.01). BM also significantly increased hepatic AMPK p, AMPK α1 AMPK α2 and Sirt1 content compared to the HFD mice. We, for the first time, demonstrated that BM extract attenuated hepatic steatosis in mice by enhancing hepatic FGF21 and AMPK/Sirt1 signaling.

Objective: Nonalcoholic fatty liver disease (NAFLD) is a common liver disease which has no standard treatment. In this regard, we sought to evaluate the effects of extracts of Artemisia santolinaefolia (SANT) and Artemisia scoparia (SCO) on hepatic lipid deposition and cellular signaling in a diet-induced obesity (DIO) animal model., Materials/methods: DIO C57/B6J mice were randomly divided into three groups, i.e. HFD, SANT and SCO. Both extracts were incorporated into HFD at a concentration of 0.5% (w/w). Fasting plasma glucose, insulin, adiponectin, and FGF21 concentrations were measured., Results: At the end of the 4-week intervention, liver tissues were collected for analysis of insulin, AMPK, and FGF21 signaling. SANT and SCO supplementation significantly increased plasma adiponectin levels when compared with the HFD mice (P<0.001). Fasting insulin levels were significantly lower in the SCO than HFD mice, but not in SANT group. Hepatic H&E staining showed fewer lipid droplets in the SCO group than in the other two groups. Cellular signaling data demonstrated that SCO significantly increased liver IRS-2 content, phosphorylation of IRS-1, IR β, Akt1 and Akt2, AMPK α1 and AMPK activity and significantly reduced PTP 1B abundance when compared with the HFD group. SCO also significantly decreased fatty acid synthase (FAS), HMG-CoA Reductase (HMGR), and Sterol regulatory element-binding protein 1c (SREBP1c), but not Carnitine palmitoyltransferase I (CPT-1) when compared with HFD group. Neither SANT nor SCO significantly altered plasma FGF21 concentrations and liver FGF21 signaling., Conclusion: This study suggests that SCO may attenuate liver lipid accumulation in DIO mice. Contributing mechanisms were postulated to include promotion of adiponectin expression, inhibition of hepatic lipogenesis, and/or enhanced insulin and AMPK signaling independent of FGF21 pathway., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Impaired insulin signaling is a key feature of type 2 diabetes and is associated with increased ubiquitin-proteasome-dependent protein degradation in skeletal muscle. An extract of Artemisia dracunculus L. (termed PMI5011) improves insulin action by increasing insulin signaling in skeletal muscle. We sought to determine if the effect of PMI5011 on insulin signaling extends to regulation of the ubiquitin-proteasome system. C2C12 myotubes and the KK-A(y) murine model of type 2 diabetes were used to evaluate the effect of PMI5011 on steady-state levels of ubiquitylation, proteasome activity and expression of Atrogin-1 and MuRF-1, muscle-specific ubiquitin ligases that are upregulated with impaired insulin signaling. Our results show that PMI5011 inhibits proteasome activity and steady-state ubiquitylation levels in vitro and in vivo. The effect of PMI5011 is mediated by PI3K/Akt signaling and correlates with decreased expression of Atrogin-1 and MuRF-1. Under in vitro conditions of hormonal or fatty acid-induced insulin resistance, PMI5011 improves insulin signaling and reduces Atrogin-1 and MuRF-1 protein levels. In the KK-A(y) murine model of type 2 diabetes, skeletal muscle ubiquitylation and proteasome activity is inhibited and Atrogin-1 and MuRF-1 expression is decreased by PMI5011. PMI5011-mediated changes in the ubiquitin-proteasome system in vivo correlate with increased phosphorylation of Akt and FoxO3a and increased myofiber size. The changes in Atrogin-1 and MuRF-1 expression, ubiquitin-proteasome activity and myofiber size modulated by PMI5011 in the presence of insulin resistance indicate the botanical extract PMI5011 may have therapeutic potential in the preservation of muscle mass in type 2 diabetes.

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., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Plant extracts continue to represent an untapped source of renewable therapeutic compounds for the treatment and prevention of illnesses including chronic metabolic disorders. With the increase in worldwide obesity and its related morbidities, the need for identifying safe and effective treatments is also rising. As such, use of primary human adipose-derived stem cells represents a physiologically relevant cell system to screen for bioactive agents in the prevention and treatment of obesity and its related complications. By using these cells in a primary screen, the risk and cost of identifying artifacts due to interspecies variation and immortalized cell lines is eliminated. We demonstrate that these cells can be formatted into 384-well high throughput screens to rapidly identify botanical extracts that affect lipogenesis and lipolysis. Additionally, counterscreening with human primary stem cells from distinct adipose depots can be routinely performed to identify tissue specific responses. In our study, over 500 botanical extracts were screened and 16 (2.7%) were found to affect lipogenesis and 4 (0.7%) affected lipolysis., (Copyright © 2012 Elsevier Inc. All rights reserved.)

We used a murine model of type II diabetes, which reproduces the major features of the human disease, and a number of cellular models to study the antidiabetic effect of ANC, a standardised anthocyanin-rich formulation from maqui berry ( Aristotelia chilensis ). We also isolated delphinidin 3-sambubioside-5-glucoside (D3S5G), a characteristic anthocyanin from maqui berry, and studied its antidiabetic properties. We observed that oral administration of ANC improved fasting blood glucose levels and glucose tolerance in hyperglycaemic obese C57BL/6J mice fed a high fat diet. In H4IIE rat liver cells, ANC decreased glucose production and enhanced the insulin-stimulated down regulation of the gluconeogenic enzyme, glucose-6-phosphatase. In L6 myotubes ANC treatment increased both insulin and non-insulin mediated glucose uptake. As with the ACN, oral administration of pure D3S5G dose-dependently decreased fasting blood glucose levels in obese C57BL/6J mice, and decreased glucose production in rat liver cells. D3S5G also increased glucose uptake in L6 myotubes and is at least partially responsible for ANC's anti-diabetic properties.

Bioactive components from bitter melon (BM) have been reported to improve glucose metabolism in vivo, but definitive studies on efficacy and mechanism of action are lacking. We sought to investigate the effects of BM bioactives on body weight, muscle lipid content and insulin signaling in mice fed a high-fat diet and on insulin signaling in L6 myotubes. Male C57BL/6J mice were randomly divided into low-fat diet control (LFD), high-fat diet (HFD) and HFD plus BM (BM) groups. Body weight, body composition, plasma glucose, leptin, insulin and muscle lipid profile were determined over 12 weeks. Insulin signaling was determined in the mouse muscle taken at end of study and in L6 myotubes exposed to the extract. Body weight, plasma glucose, insulin, leptin levels and HOMA-IR values were significantly lower in the BM-fed HFD group when compared to the HFD group. BM supplementation significantly increased IRS-2, IR β, PI 3K and GLUT4 protein abundance in skeletal muscle, as well as phosphorylation of IRS-1, Akt1 and Akt2 when compared with HFD (P<.05 and P<.01). BM also significantly reduced muscle lipid content in the HFD mice. BM extract greatly increased glucose uptake and enhanced insulin signaling in L6 myotubes. This study shows that BM bioactives reduced body weight, improved glucose metabolism and enhanced skeletal muscle insulin signaling. A contributing mechanism to the enhanced insulin signaling may be associated with the reduction in skeletal muscle lipid content. Nutritional supplementation with this extract, if validated for human studies, may offer an adjunctive therapy for diabetes., (Copyright © 2011 Elsevier Inc. All rights reserved.)

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., (Copyright 2010 John Wiley & Sons, Ltd.)

Background: Phenethylisothiocyanate (PEITC) is produced by Brassica food plants. PEO is a PEITC Essential Oil containing >95% natural PEITC. PEITC is known to produce various health benefits but its effect in alleviation of ulcerative colitis signs is unknown., Results: In two efficacy studies (acute and chronic) oral administration of PEO was effective at remitting acute and chronic signs of ulcerative colitis (UC) in mice. Disease activity, histology and biochemical characteristics were measured in the treated animals and were compared with appropriate controls. PEO treatment significantly improved body weights and stool consistency as well as decreased intestinal bleeding. PEO treatment also reduced mucosal inflammation, depletion of goblet cells and infiltration of inflammatory cells. Attenuation of proinflammatory interleukin1beta production was observed in the colons of PEO-treated animals. Expression analyses were also carried out for immune function related genes, transcription factors and cytokines in lipopolysaccharide-activated mouse macrophage cells. PEO likely affects an intricate network of immune signaling genes including a novel concentration dependent reduction of total cellular Signal Transducer and Activator of Transcription 1 (STAT1) as well as nuclear phosphorylated-STAT1 (activated form of STAT1). A PEO-concentration dependent decrease of mRNA of C-X-C motif ligand 10 (a STAT1 responsive chemokine) and Interleukin 6 were also observed., Conclusions: PEO might be a promising candidate to develop as a treatment for ulcerative colitis patients. The disease attenuation by PEO is likely associated with suppression of activation of STAT1 transcription and inhibition of pro-inflammatory cytokines.

Wild berries are integral dietary components for Alaska Native people and a rich source of polyphenolic metabolites that can ameliorate metabolic disorders such as obesity and diabetes. In this study, five species of wild Alaskan berries (Vaccinium ovalifolium , Vaccinium uliginosum , Rubus chamaemorus , Rubus spectabilis , and Empetrum nigrum) were screened for bioactivity through a community-participatory research method involving three geographically distinct tribal communities. Compositional analysis by HPLC and LC-MS(2) revealed substantial site-specific variation in anthocyanins (0.01-4.39 mg/g of FW) and proanthocyanidins (0.74-6.25 mg/g of FW) and identified A-type proanthocyanidin polymers. R. spectabilis increased expression levels of preadipocyte factor 1 (182%), and proanthocyanidin-enriched fractions from other species reduced lipid accumulation in 3T3-L1 adipocytes. Selected extracts reduced serum glucose levels in C57BL/6J mice by up to 45%. Local observations provided robust insights into effects of climatic fluctuations on berry abundance and quality, and preliminary site-specific compositional and bioactivity differences were noted, suggesting the need to monitor this Alaska Native resource as climate shifts affect the region.

Quercetin, a polyphenolic compound and a major bioflavonoid in the human diet, has anti-inflammatory properties and has been postulated to enhance energy expenditure (EE). We sought to determine whether quercetin alters body weight, body composition, EE, and circulating markers of inflammation. At 6 weeks (W) of age, 2 cohorts of C57BL/6J mice (N = 80) were placed on one of 2 diets for 3W or 8W: (1) high fat (HF) (45% kcal fat) or (2) high fat + quercetin (HF + Q) (45% kcal fat + 0.8% quercetin). Quercetin concentrations in the diet and plasma were evaluated using mass spectrometry. Body weight, composition (nuclear magnetic resonance), and food consumption were measured weekly. Energy expenditure was measured by indirect calorimetry at 3 and 8W, and inflammatory markers were measured in plasma obtained at 8W. The presence of quercetin in the HF diet did not alter food consumption over time in the HF + Q group and did not differ from the HF group at any time point. However, circulating plasma quercetin concentrations declined between 3 and 8W. At 3W, EE was higher during both day and night phases (P < .0001) in the HF + Q group compared with the HF group; but this difference was not detected at 8W and did not translate into significant differences between the HF + Q and HF groups with respect to body weight or body composition. During the night phase, concentrations of the inflammatory markers (interferon-gamma, interleukin-1alpha, and interleukin-4) were significantly lower when compared with HF treatment group (P < .05). Dietary supplementation with quercetin produces transient (3W) increases in EE that are not detected after 8W on the diet. A corresponding decrease in circulating quercetin between 3 and 8W suggests that metabolic adaptation may have diminished the impact of quercetin's early effect on EE and diminished its overall effect on nutrient partitioning and adiposity. However, quercetin at the levels provided was effective in reducing circulating markers of inflammation observed in animals on an HF diet at 8W.

An alcoholic extract of Artemisia dracunculus L (PMI 5011) has been shown to decrease glucose and improve insulin levels in animal models, suggesting an ability to enhance insulin sensitivity. We sought to assess the cellular mechanism by which this botanical affects carbohydrate metabolism in primary human skeletal muscle culture. We measured basal and insulin-stimulated glucose uptake, glycogen accumulation, phosphoinositide 3 (PI-3) kinase activity, and Akt phosphorylation in primary skeletal muscle culture from subjects with type 2 diabetes mellitus incubated with or without various concentrations of PMI 5011. We also analyzed the abundance of insulin receptor signaling proteins, for example, IRS-1, IRS-2, and PI-3 kinase. Glucose uptake was significantly increased in the presence of increasing concentrations of PMI 5011. In addition, glycogen accumulation, observed to be decreased with increasing free fatty acid levels, was partially restored with PMI 5011. PMI 5011 treatment did not appear to significantly affect protein abundance for IRS-1, IRS-2, PI-3 kinase, Akt, insulin receptor, or Glut-4. However, PMI 5011 significantly decreased levels of a specific protein tyrosine phosphatase, that is, PTP1B. Time course studies confirmed that protein abundance of PTP1B decreases in the presence of PMI 5011. The cellular mechanism of action to explain the effects by which an alcoholic extract of A dracunculus L improves carbohydrate metabolism on a clinical level may be secondary to enhancing insulin receptor signaling and modulating levels of a specific protein tyrosine phosphatase, that is, PTP1B.

Epigenetics refers to heritable changes in gene expression that are not attributable to changes in DNA sequence and impacts many areas of applied and basic biology including developmental biology, gene therapy, somatic cell nuclear transfer, somatic cell reprogramming, and stem cell biology. Epigenetic changes are known to contribute to aging in addition to multiple disease states. Epigenetic changes can be influenced by environmental factors that in turn can be inherited by daughter cells during cell division and can also be inherited through the germ line. Thus, it is intriguing to consider that epigenetics, in general, may play a role in human conditions that are strongly influenced by changes in the environment and lifestyle. In particular, metabolic syndrome, a condition increasing in prevalence around the world, is one such condition for which epigenetics is postulated to contribute. Epigenetic defects (epimutations) are thought to be more easily reversible (when compared with genetic defects) and, as such, have inspired efforts to identify novel compounds that correct epimutations or prevent progression to the disease state. These efforts have resulted in the development of a rapidly growing new field being referred to as epigenetic therapy. To date, 2 classes of drugs have received the most attention, that is, DNA methyltransferase inhibitors and histone deacetylase inhibitors; but recent data suggest that botanical sources may be a rich source of agents that can potentially modulate the epigenome and related pathways and potentially be useful in attenuating the progression of many factors related to development of metabolic syndrome. This review will provide an overview of the field of epigenetics, epigenetic therapy, and the molecules currently receiving the most interest with respect to treatment, and review data on botanical compounds that show promise in this regard.

An ethanolic extract of Russian tarragon, Artemisia dracunculus L., with antihyperglycemic activity in animal models was reported to decrease phosphoenolpyruvate carboxykinase (PEPCK) mRNA expression in STZ-induced diabetic rats. A quantitative polymerase chain reaction (qPCR) assay was developed for the bioactivity-guided purification of the compounds within the extract that decrease PEPCK expression. The assay was based on the inhibition of dexamethasone-stimulated PEPCK upregulation in an H4IIE hepatoma cell line. Two polyphenolic compounds that inhibited PEPCK mRNA levels were isolated and identified as 6-demethoxycapillarisin and 2',4'-dihydroxy-4-methoxydihydrochalcone with IC(50) values of 43 and 61 muM, respectively. The phosphoinositide-3 kinase (PI3K) inhibitor LY-294002 showed that 6-demethoxycapillarisin exerts its effect through the activation of the PI3K pathway, similarly to insulin. The effect of 2',4'-dihydroxy-4-methoxydihydrochalcone is not regulated by PI3K and dependent on activation of AMPK pathway. These results indicate that the isolated compounds may be responsible for much of the glucose-lowering activity of the Artemisia dracunculus extract.

Winter cress (Barbarea verna) seed preparations rich in phenethylisothiocyanate (PEITC) had strong in vivo and in vitro anti-inflammatory activity, significantly reducing the size of carrageenan-induced rat paw edema. This in vivo effect was comparable with that of the nonsteroidal anti-inflammatory drug aspirin. The seed preparation, in a concentration-dependent manner, reduced the mRNA levels of inflammation-related genes such as the inducible forms of cyclooxygenase and nitric-oxide synthase and the proinflammatory cytokine interleukin in lipopolysaccharide-stimulated mouse macrophage cell line RAW 264.7. Activity of the seed preparation was similar to that of the synthetic PEITC. PEITC was the most active of five different forms of isothiocyanate tested for their effects on in vitro proinflammatory gene expression. In vitro activity of the seed preparation was also compared with that of two known anti-inflammatory drugs. We conclude that Barbarea verna seed preparation may function as a potent anti-inflammatory agent, interfering with the transcription of proinflammatory genes.

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.

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.