CEBPß regulation of endogenous IGF-1 in adult sensory neurons can be mobilized to overcome diabetes-induced deficits in bioenergetics and axonal outgrowth.

Aberrant insulin-like growth factor 1 (IGF-1) signaling has been proposed as a contributing factor to the development of neurodegenerative disorders including diabetic neuropathy, and delivery of exogenous IGF-1 has been explored as a treatment for Alzheimer's disease and amyotrophic lateral sclerosis. However, the role of autocrine/paracrine IGF-1 in neuroprotection has not been well established. We therefore used in vitro cell culture systems and animal models of diabetic neuropathy to characterize endogenous IGF-1 in sensory neurons and determine the factors regulating IGF-1 expression and/or affecting neuronal health. Single-cell RNA sequencing (scRNA-Seq) and in situ hybridization analyses revealed high expression of endogenous IGF-1 in non-peptidergic neurons and satellite glial cells (SGCs) of dorsal root ganglia (DRG). Brain cortex and DRG had higher IGF-1 gene expression than sciatic nerve. Bidirectional transport of IGF-1 along sensory nerves was observed. Despite no difference in IGF-1 receptor levels, IGF-1 gene expression was significantly (P < 0.05) reduced in liver and DRG from streptozotocin (STZ)-induced type 1 diabetic rats, Zucker diabetic fatty (ZDF) rats, mice on a high-fat/ high-sugar diet and db/db type 2 diabetic mice. Hyperglycemia suppressed IGF-1 gene expression in cultured DRG neurons and this was reversed by exogenous IGF-1 or the aldose reductase inhibitor sorbinil. Transcription factors, such as NFAT1 and CEBPß, were also less enriched at the IGF-1 promoter in DRG from diabetic rats vs control rats. CEBPß overexpression promoted neurite outgrowth and mitochondrial respiration, both of which were blunted by knocking down or blocking IGF-1. Suppression of endogenous IGF-1 in diabetes may contribute to neuropathy and its upregulation at the transcriptional level by CEBPß can be a promising therapeutic approach.

Salicylic acid in ginseng root alleviates skin hyperpigmentation disorders by inhibiting melanogenesis and melanosome transport.

Abnormal melanogenesis and melanosome transport can cause skin pigmentation disorders that are often treated using ginseng-based formulation. We previously found that phenolic acid compounds in ginseng root could inhibit melanin production and as a skin-whitening agents. However, mechanisms of action underlying effects of ginseng phenolic acid monomers on melanogenesis remain unclear. This study was conducted to investigate effects of salicylic acid, a main ginseng root phenolic acid component, on melanogenesis and melanosome functions in melanocytes of zebrafish and other species. Salicylic acid exhibited no cytotoxicity and reduced melanin levels and tyrosinase activity in B16F10 murine melanoma cells and normal human epidermal melanocytes regardless of prior cell stimulation with α-melanocyte stimulating hormone. Additionally, salicylic acid treatment reduced expression of melanogenic enzymes tyrosinase, tyrosinase-related protein 1 and tyrosinase-related protein 2, while reducing expression of their master transcriptional regulator, microphthalmia-associated transcription factor. Moreover, reduced phosphorylation of cAMP response-element binding protein was observed due to reduced cAMP levels resulting from salicylic acid inhibition of upstream signal regulators (adenylyl cyclase and protein kinase A). Furthermore, salicylic acid treatment suppressed expression of transport complex-associated proteins melanophilin and myosin Va in two UVB-treated melanocytic cell lines, suppressed phagocytosis of fluorescent microspheres by UVB-stimulated human keratinocytes (HaCaT), inhibited protease-activated receptor 2 activation by reducing both Ca2+ release and activation of phosphoinositide 3 kinase/AKT and mitogen-activated protein kinases and induced anti-melanogenic effects in zebrafish. Collectively, these results indicate that salicylic acid within ginseng root can inhibit melanocyte melanogenesis and melanin transport, while also suppressing keratinocyte phagocytic function.

Identification of Insulin-Mimetic Plant Extracts: From an In Vitro High-Content Screen to Blood Glucose Reduction in Live Animals.

Type 2 diabetes mellitus (T2DM) is linked to insulin resistance and a loss of insulin sensitivity, leading to millions of deaths worldwide each year. T2DM is caused by reduced uptake of glucose facilitated by glucose transporter 4 (GLUT4) in muscle and adipose tissue due to decreased intracellular translocation of GLUT4-containing vesicles to the plasma membrane. To treat T2DM, novel medications are required. Through a fluorescence microscopy-based high-content screen, we tested more than 600 plant extracts for their potential to induce GLUT4 translocation in the absence of insulin. The primary screen in CHO-K1 cells resulted in 30 positive hits, which were further investigated in HeLa and 3T3-L1 cells. In addition, full plasma membrane insertion was examined by immunostaining of the first extracellular loop of GLUT4. The application of appropriate inhibitors identified PI3 kinase as the most important signal transduction target relevant for GLUT4 translocation. Finally, from the most effective hits in vitro, four extracts effectively reduced blood glucose levels in chicken embryos (in ovo), indicating their applicability as antidiabetic pharmaceuticals or nutraceuticals.

Malayoside, a cardenolide glycoside extracted from Antiaris toxicaria Lesch, induces apoptosis in human non-small lung cancer cells via MAPK-Nur77 signaling pathway.

Lung cancer is the leading cause of cancer deaths in the world. Non-small cell lung cancer (NSCLC), with poor prognosis and resistance to chemoradiotherapy, is the most common histological type of lung cancer. Therefore, it is necessary to develop new and more effective treatment strategy for NSCLC. Nur77, an orphan member of the nuclear receptor superfamily, induces apoptosis in cancer cells including NSCLC cells, by high expression and translocation to mitochondria. Small molecules trigger expression and mitochondrial localization of Nur77 may be an ideal anti-cancer drug candidate. Here, we report malayoside, a cardiac glycoside in the extract of Antiaris toxicaria Lesch., had different sensitivities to NSCLC cells. Malayoside induced apoptosis in NCI-H460 cells. Meanwhile, malayoside induced Nur77 expression and mitochondrial localization, and its induction of apoptosis was Nur77-dependent. To investigate the molecular mechanism of malayoside inducing Nur77 and apoptosis, we found that malayoside activated MAPK signaling pathway, including both ERK and p38 phosphorylation. The suppression of MAPK signaling activation inhibited the expression of Nur77 and apoptosis induced by malayoside. Our studies in nude mice showed that malayside potently inhibited the growth of tumor cells in vivo. Furthermore, the anti-cancer effect of malayosidwas in vivo was also related to the elevated expression of Nur77, p-ERK, and p-p38 proteins. Our results suggest that malayoside possesses an anti-NSCLC activity in vitro and in vivo mainly via activation of MAPK-Nur77 signaling pathway, indicating that malayoside is a promising chemotherapeutic candidate for NSCLC.

Anti-inflammatory Effects of S. cumini Seed Extract on Gelatinase-B (MMP-9) Regulation against Hyperglycemic Cardiomyocyte Stress.

Black berry (Syzygium cumini) fruit is useful in curing diabetic complications; however, its role in diabetes-induced cardiomyopathy is not yet known. In this study, we investigated the regulation of gelatinase-B (MMP-9) by S. cumini methanol seed extract (MSE) in diabetic cardiomyopathy using real-time PCR, RT-PCR, immunocytochemistry, gel diffusion assay, and substrate zymography. The regulatory effects of MSE on NF-κB, TNF-α, and IL-6 were also examined. Identification and estimation of polyphenol constituents present in S. cumini extract were carried out using reverse-phase HPLC. Further, in silico docking studies of identified polyphenols with gelatinase-B were performed to elucidate molecular level interaction in the active site of gelatinase-B. Docking studies showed strong interaction of S. cumini polyphenols with gelatinase-B. Our findings indicate that MSE significantly suppresses gelatinase-B expression and activity in high-glucose- (HG-) stimulated cardiomyopathy. Further, HG-induced activation of NF-κB, TNF-α, and IL-6 was also remarkably reduced by MSE. Our results suggest that S. cumini MSE may be useful as an effective functional food and dietary supplement to regulate HG-induced cardiac stress through gelatinase.

Punicalagin induces ROS-mediated apoptotic cell death through inhibiting STAT3 translocation in lung cancer A549 cells.

Lung cancer is a noxious disease with substandard overall survival. Despite this, there are several treatment strategies for lung cancer include chemotherapy, radiotherapy, surgery; however, the overall survival remains poor. Punicalagin has been documented as a potential phytomedicine to selectively inhibit the progression and expansion of numerous cancers. In the present study, we evaluated the antiproliferative ability of punicalagin against lung cancer A549 cells by inducing apoptosis by inhibiting STAT-3 activation. Punicalagin induces toxic effects of A549 cells in a dose-associated manner after 24 h treatment. And we also observed that punicalagin (10, 20, and 30 µM) induced reactive oxygen species generation, alters the mitochondrion membrane potential and apoptotic morphological changes in A549 cells. The STAT-3 overexpression regulates apoptosis, proliferation, and angiogenesis. Here, the punicalagin inhibited STAT-3 translocation and thereby induces apoptosis by inhibiting expression Bcl-2 and enhanced expression of Bax, cytochrome-c, caspase-9, and caspase-3 in A549 cells. Hence, we stated that the punicalagin is a possible therapy for non-small cell lung, malignancies. Altogether, the punicalagin is a promising phytomedicine in malignancy treatment and further endeavors are needed to unveil the complete potential.

Anthocyanins from Hibiscus syriacus L. Inhibit NLRP3 Inflammasome in BV2 Microglia Cells by Alleviating NF-κB- and ER Stress-Induced Ca2+ Accumulation and Mitochondrial ROS Production.

Anthocyanins from the petals of Hibiscus syriacus L. (PS) possess anti-inflammatory, antioxidant, and antimelanogenic activities. However, it remains unclear whether PS inhibit the NLR family pyrin domain-containing 3 (NLRP3) inflammasome activation and assembly. This study is aimed at investigating whether PS downregulate NLRP3-mediated inflammasome by inhibiting nuclear factor-κB (NF-κB) and endoplasmic reticulum (ER) stress. BV2 microglia cells were treated with PS in the presence of lipopolysaccharide and adenosine triphosphate (LPS/ATP), and the NLRP3-related signaling pathway was investigated. In this study, we found that LPS/ATP treatment activated the NLRP3 inflammasome, which resulted in the release of interleukin-1ß (IL-1ß) and IL-18. Meanwhile, PS reduced LPS/ATP-mediated NLRP3 inflammasome at 12 h by inhibiting ER stress-mediated Ca2+ accumulation and subsequent mitochondrial reactive oxygen species (mtROS) production, which, in turn, decreased IL-1ß and IL-18 release. Furthermore, PS inhibited the NLRP3 inflammasome 1 h after LPS/ATP treatment by suppressing the NF-κB pathway, which downregulated Ca2+ accumulation and mtROS production. These data showed that PS negatively regulated activation of the NLRP3 inflammasome in a time-different manner by inhibiting the NF-κB signaling pathway in the early stage and the ER stress response in the late stage. The pathways shared Ca2+ accumulation-mediated mtROS production, which was significantly inhibited in the presence of PS. In conclusion, our results suggested that PS has potential as a supplement against NLRP3 inflammasome-related inflammatory disorders; nevertheless, further studies are needed to determine the effect of PS in the noncanonical NLRP3 inflammasome pathways and pathological conditions in vivo.

Rosa canina L. Can Restore Endoplasmic Reticulum Alterations, Protein Trafficking and Membrane Integrity in a Dextran Sulfate Sodium-Induced Inflammatory Bowel Disease Phenotype.

Rosa canina L. is a natural polyphenol-rich medicinal plant that exhibits antioxidant and anti-inflammatory activities. Recent in vivo studies have demonstrated that a methanol extract of Rosa canina L. (RCME) has reversed an inflammatory bowel disease (IBD)-like phenotype that has been triggered by dextran sulfate sodium (DSS) in mice. In the current study, we investigated the effects of RCME on perturbations of cellular mechanisms induced by DSS-treatment of intestinal Caco-2 cells, including stress response in the endoplasmic reticulum (ER), protein trafficking and sorting as well as lipid rafts integrity and functional capacities of an intestinal enzyme. 6 days post-confluent cells were treated for 24 h with DSS (3%) or simultaneously with DSS (3%) and RCME (100 µg/mL) or exclusively with RCME (100 µg/mL) or not treated. The results obtained demonstrate the ability of RCME to counteract the substantial increase in the expression levels of several ER stress markers in DSS-treated cells. Concomitantly, the delayed trafficking of intestinal membrane glycoproteins sucrase-isomaltase (SI) and dipeptidyl peptidase 4 (DPP4) induced by DSS between the ER and the Golgi has been compromised by RCME. Furthermore, RCME restored the partially impaired polarized sorting of SI and DPP4 to the brush border membrane. An efficient sorting mechanism of SI and DPP4 is tightly associated with intact lipid rafts structures in the trans-Golgi network (TGN), which have been distorted by DSS and normalized by RCME. Finally, the enzymatic activities of SI are enhanced in the presence of RCME. Altogether, DSS treatment has triggered ER stress, impaired trafficking and function of membrane glycoproteins and distorted lipid rafts, all of which can be compromised by RCME. These findings indicate that the antioxidants in RCME act at two major sites in Caco-2 cells, the ER and the TGN and are thus capable of maintaining the membrane integrity by correcting the sorting of membrane-associated proteins.

Olive Leaf Extract (OLE) impaired vasopressin-induced aquaporin-2 trafficking through the activation of the calcium-sensing receptor.

Vasopressin (AVP) increases water permeability in the renal collecting duct through the regulation of aquaporin-2 (AQP2) trafficking. Several disorders, including hypertension and inappropriate antidiuretic hormone secretion (SIADH), are associated with abnormalities in water homeostasis. It has been shown that certain phytocompounds are beneficial to human health. Here, the effects of the Olive Leaf Extract (OLE) have been evaluated using in vitro and in vivo models. Confocal studies showed that OLE prevents the vasopressin induced AQP2 translocation to the plasma membrane in MCD4 cells and rat kidneys. Incubation with OLE decreases the AVP-dependent increase of the osmotic water permeability coefficient (Pf). To elucidate the possible effectors of OLE, intracellular calcium was evaluated. OLE increases the intracellular calcium through the activation of the Calcium Sensing Receptor (CaSR). NPS2143, a selective CaSR inhibitor, abolished the inhibitory effect of OLE on AVP-dependent water permeability. In vivo experiments revealed that treatment with OLE increases the expression of the CaSR mRNA and decreases AQP2 mRNA paralleled by an increase of the AQP2-targeting miRNA-137. Together, these findings suggest that OLE antagonizes vasopressin action through stimulation of the CaSR indicating that this extract may be beneficial to attenuate disorders characterized by abnormal CaSR signaling and affecting renal water reabsorption.

Ghrelin protects against rotenone-induced cytotoxicity: Involvement of mitophagy and the AMPK/SIRT1/PGC1α pathway.

Parkinson's disease (PD) is the second most common neurodegenerative disorder, characterized by the loss of dopaminergic neurons in the substantia nigra and the deposition of Lewy bodies. Mitochondrial dysfunction, oxidative stress, and autophagy dysfunction are involved in the pathogenesis of PD. Ghrelin is a brain-gut peptide that has been reported that protected against 1-methyl-4-phenyl-1,2,3,6- tetrahydropyran (MPTP)/MPP+-induced toxic effects. In the present work, human neuroblastoma SH-SY5Y cells were exposed to rotenone as a PD model to explore the underlying mechanism of ghrelin. We found that ghrelin inhibited rotenone-induced cytotoxicity, mitochondrial dysfunction, and apoptosis by improving cell viability, increasing the ratio of red/green of JC-1, inhibiting the production of reactive oxidative species (ROS), and regulating Bcl-2, Bax, Cytochrome c, caspase-9, and caspase-3 expression. Besides, ghrelin promoted mitophagy accompanied by up-regulating microtubule-associated protein 1 Light Chain 3B-II/I(LC3B-II/I) and Beclin1 but decreasing the expression of p62. Moreover, ghrelin promoted PINK1/Parkin mitochondrial translocation. Additionally, we investigated that ghrelin activated the AMPK/SIRT1/PGC1α pathway and pharmacological inhibition of AMPK and SIRT1 abolished the cytoprotection of ghrelin, decreased the level of mitophagy, and PINK1/Parkin mitochondrial translocation. Taken together, our findings suggested that mitophagy and AMPK/SIRT1/PGC1α pathways were related to the cytoprotection of ghrelin. These findings provided novel insights into the underlying mechanisms of ghrelin, further mechanistic studies on preclinical and clinical levels are required to be conducted with ghrelin to avail and foresee it as a potential agent in the treatment and management of PD.

Cottonseed Oil Alleviates Ischemic Stroke-Induced Oxidative Stress Injury Via Activating the Nrf2 Signaling Pathway.

Oxidative stress is believed to be one of the primary causes in ischemic stroke injury, and the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway is the most important endogenous antioxidative stress damage pathway. Cottonseed oil (CSO), which is used mostly as a solvent for lipid-soluble drugs, has been shown to exert antioxidative effects against peripheral tissue injury. However, the effects and mechanisms of CSO on ischemic stroke-induced oxidative stress injury and the Nrf2 signaling pathway remain largely unknown. In this study, we investigated the potential of CSO in regulating oxidative stress injury induced by middle cerebral artery occlusion and reperfusion (MCAO-R), or oxygen and glucose deprivation and reperfusion (OGD-R). We found that 1.3 mL/kg CSO treatment of male rats with a subcutaneous injection once every other day for 3 weeks significantly improved neurological deficit; reduced infarction volume; alleviated neuronal injuries; reduced the content of ROS and MDA; increased the activity of SOD, GSH, and GSH-PX; and markedly increased the expression of Nrf2. Furthermore, treatment with 10-9 µL/mL CSO to a neuron cell line (HT-22) for 24 h significantly increased cell viability and decreased cell apoptosis after OGD-R injury; significantly reduced the levels of ROS and MDA; increased the activity of SOD, GSH, and GSH-PX; and induced an increase in Nrf2 nuclear translocation. Based on our findings, we conclude that CSO treatment alleviates ischemic stroke injury-induced oxidative stress via activating the Nrf2 signaling pathway, highlighting the potential that CSO has as a therapeutic for ischemic strokes.

A novel quantitative assay for analysis of GLUT4 translocation using high content screening.

Insulin resistance is associated with obesity and can lead to several metabolic disorders including type II diabetes, nonalcoholic fatty liver disease and cardiovascular problems. Search for the small molecules which can either induce or mimic the insulin action are of great interest and can be utilized to manage insulin resistance. There are several dietary phytochemicals which can potentially have insulinomimetic action. Nevertheless, high throughput screening methods to test efficiency of small molecules to act as an insulinomimetic are not fully established. In this paper we have performed chemical screen analysis based on GLUT4 translocation using a cell line CHO-HIRC-myc-GLUT4 eGFP that expresses GLUT4-GFP in association with human Insulin receptor. We have established a high content screening-based method which can track and quantify the GLUT4 translocation from perinuclear area to the cell membrane. The assay involves measuring fluorescence intensity in a defined perinuclear area and a defined area along the cell membrane; and the results are expressed as the ratio of fluorescence intensity in the perinuclear to membrane area. The assay could collect real time data of GLUT4 translocation from thousand of cells/ sample and from many such samples in one experiment. We validated the assay using Insulin, insulin mimics/sensitizers and insulin inhibitors. The agonist or antagonists were analyzed for their ability to enhance or block the GLUT4 translocation independent of insulin. The outcome of the assay was correlated by performing glucose uptake assay using differentiated 3T3L1 cells. Using this platform we further identified several plant extracts which had the insulin mimetic action. We confirmed that these plant extracts were non-toxic to the beta cells using RIN mf5cells and 3T3L1 cells. We have identified plant extracts with the potential insulinomimetic action using novel high-content screening approach; these can be further tested for their efficiency in-vivo in pre-clinical trials.

Unconventional endocytosis and trafficking of transferrin receptor induced by iron.

The posttranslational regulation of transferrin receptor (TfR1) is largely unknown. We investigated whether iron availability affects TfR1 endocytic cycle and protein stability in HepG2 hepatoma cells exposed to ferric ammonium citrate (FAC). NH4Cl and bafilomycin A1, but not the proteasomal inhibitor MG132, prevented the FAC-mediated decrease in TfR1 protein levels, thus indicating lysosomal involvement. Knockdown experiments showed that TfR1 lysosomal degradation is independent of 1) endocytosis mediated by the clathrin adaptor AP2; 2) Tf, which was suggested to facilitate TfR1 internalization; 3) H-ferritin; and 4) MARCH8, previously implicated in TfR1 degradation. Notably, FAC decreased the number of TfR1 molecules at the cell surface and increased the Tf endocytic rate. Colocalization experiments confirmed that, upon FAC treatment, TfR1 was endocytosed in an AP2- and Tf-independent pathway and trafficked to the lysosome for degradation. This unconventional endocytic regulatory mechanism aimed at reducing surface TfR1 may represent an additional posttranslational control to prevent iron overload. Our results show that iron is a key regulator of the trafficking of TfR1, which has been widely used to study endocytosis, often not considering its function in iron homeostasis.

Clinacanthus nutans Mitigates Neuronal Death and Reduces Ischemic Brain Injury: Role of NF-κB-driven IL-1ß Transcription.

Neuroinflammation has been shown to exacerbate ischemic brain injury, and is considered as a prime target for the development of stroke therapies. Clinacanthus nutans Lindau (C. nutans) is widely used in traditional medicine for treating insect bites, viral infection and cancer, due largely to its anti-oxidative and anti-inflammatory properties. Recently, we reported that an ethanol extract from the leaf of C. nutans could protect the brain against ischemia-triggered neuronal death and infarction. In order to further understand the molecular mechanism(s) for its beneficial effects, two experimental paradigms, namely, in vitro primary cortical neurons subjected to oxygen-glucose deprivation (OGD) and in vivo rat middle cerebral artery (MCA) occlusion, were used to dissect the anti-inflammatory effects of C. nutans extract. Using promoter assays, immunofluorescence staining, and loss-of-function (siRNA) approaches, we demonstrated that transient OGD led to marked induction of IL-1ß, IL-6 and TNFα, while pretreatment with C. nutans suppressed production of inflammatory cytokines in primary neurons. C. nutans inhibited IL-1ß transcription via preventing NF-κB/p65 nuclear translocation, and siRNA knockdown of either p65 or IL-1ß mitigated OGD-mediated neuronal death. Correspondingly, post-ischemic treatment of C. nutans attenuated IκBα degradation and decreased IL-1ß, IL-6 and TNFα production in the ischemic brain. Furthermore, IL-1ß siRNA post-ischemic treatment reduced cerebral infarct, thus mimicking the beneficial effects of C. nutans. In summary, our findings demonstrated the ability for C. nutans to suppress NF-κB nuclear translocation and inhibit IL-1ß transcription in ischemic models. Results further suggest the possibility for using C. nutans to prevent and treat stroke patients.

A Multi-Omics Approach Identifies Key Regulatory Pathways Induced by Long-Term Zinc Supplementation in Human Primary Retinal Pigment Epithelium.

In age-related macular degeneration (AMD), both systemic and local zinc levels decline. Elevation of zinc in clinical studies delayed the progression to end-stage AMD. However, the molecular pathways underpinning this beneficial effect are not yet identified. In this study, we used differentiated primary human fetal retinal pigment epithelium (RPE) cultures and long-term zinc supplementation to carry out a combined transcriptome, proteome and secretome analysis from three genetically different human donors. After combining significant differences, we identified the complex molecular networks using Database for Annotation, Visualization and Integrated Discovery (DAVID) and Ingenuity Pathway Analysis (IPA). The cell cultures from the three donors showed extensive pigmentation, development of microvilli and basal infoldings and responded to zinc supplementation with an increase in transepithelial electrical resistance (TEER) (apical supplementation: 443.2 ± 79.3%, basal supplementation: 424.9 ± 116.8%, compared to control: 317.5 ± 98.2%). Significant changes were observed in the expression of 1044 genes, 151 cellular proteins and 124 secreted proteins. Gene set enrichment analysis revealed changes in specific molecular pathways related to cell adhesion/polarity, extracellular matrix organization, protein processing/transport, and oxidative stress response by zinc and identified a key upstream regulator effect similar to that of TGFB1.

Structure-Function Analysis of SMAX1 Reveals Domains That Mediate Its Karrikin-Induced Proteolysis and Interaction with the Receptor KAI2.

Karrikins (KARs) are butenolides found in smoke that can influence germination and seedling development of many plants. The KAR signaling mechanism is hypothesized to be very similar to that of the plant hormone strigolactone (SL). Both pathways require the F-box protein MORE AXILLARY GROWTH2 (MAX2), and other core signaling components have shared ancestry. Putatively, KAR activates the receptor KARRIKIN INSENSITIVE2 (KAI2), triggering its association with the E3 ubiquitin ligase complex SCFMAX2 and downstream targets SUPPRESSOR OF MAX2 1 (SMAX1) and SMAX1-LIKE2 (SMXL2). Polyubiquitination and proteolysis of SMAX1 and SMXL2 then enable growth responses to KAR. However, many of the assumptions of this model have not been demonstrated. Therefore, we investigated the posttranslational regulation of SMAX1 from the model plant Arabidopsis (Arabidopsis thaliana). We find evidence that SMAX1 is degraded by KAI2-SCFMAX2 but is also subject to MAX2-independent turnover. We identify SMAX1 domains that are responsible for its nuclear localization, KAR-induced degradation, association with KAI2, and ability to interact with other SMXL proteins. KAI2 undergoes MAX2-independent degradation after KAR treatment, which we propose results from its association with SMAX1 and SMXL2. Finally, we discover an SMXL domain that mediates receptor-target interaction preferences in KAR and SL signaling, laying the foundation for understanding how these highly similar pathways evolved to fulfill different roles.

Green tea polyphenols ameliorate metabolic abnormalities and insulin resistance by enhancing insulin signalling in skeletal muscle of Zucker fatty rats.

In the present study, we evaluated the metabolic effects of green tea polyphenols (GTPs) in high-fat diet (HFD) fed Zucker fatty (ZF) rats, in particular the effects of GTP on skeletal muscle insulin sensitivity. Body weight, visceral fat, glucose tolerance, lipid profiles and whole-body insulin sensitivity were measured in HFD-fed ZF rats after 8-week-treatment with GTP (200 mg/kg of body weight) or saline (5 ml/kg of body weight). Zucker lean rats were studied as controls. Ex vivo insulin-mediated muscle glucose uptake was assessed. Immunoblotting was used to evaluate the expression of key insulin signalling proteins in skeletal muscle. GTP treatment attenuated weight gain (P<0.05) and visceral fat accumulation (27.6%, P<0.05), and significantly reduced fasting serum glucose (P<0.05) and insulin (P<0.01) levels. Homoeostasis model assessment of insulin resistance (HOMA-IR), a measure of insulin resistance, was lower (P<0.01) in GTP-treated animals compared with ZF controls. Moreover, insulin-stimulated glucose uptake by isolated soleus muscle was increased (P<0.05) in GTP-ZF rats compared with ZF-controls. GTP treatment attenuated the accumulation of ectopic lipids (triacyl- and diacyl-glycerols), enhanced the expression and translocation of glucose transporter-4, and decreased pSer612IRS-1 and increased pSer473Akt2 expression in skeletal muscle. These molecular changes were also associated with significantly decreased activation of the inhibitory (muscle-specific) protein kinase (PKC) isoform, PKC-θ. Taken together, the present study has shown that regular ingestion of GTP exerts a number of favourable metabolic and molecular effects in an established animal model of obesity and insulin resistance. The benefits of GTP are mediated in part by inhibiting PKC-θ and improving muscle insulin sensitivity.

Cyclovirobuxine D protects against diabetic cardiomyopathy by activating Nrf2-mediated antioxidant responses.

Diabetic cardiomyopathy (DCM) is the principal cause of death in people with diabetes. However, there is currently no effective strategy to prevent the development of DCM. Although cyclovirobuxine D (CVB-D) has been widely used to treat multiple cardiovascular diseases, the possible beneficial effects of CVB-D on DCM remained unknown. The present aim was to explore the potential effects and underlying mechanisms of CVB-D on DCM. We explored the effects of CVB-D in DCM by using high fat high sucrose diet and streptozotocin-induced rat DCM model. Cardiac function and survival in rats with DCM were improved via the amelioration of oxidative damage after CVB-D treatment. Our data also demonstrated that pre-treatment with CVB-D exerted a remarkable cytoprotective effect against high glucose -or H2O2 -induced neonatal rat cardiomyocyte damage via the suppression of reactive oxygen species accumulation and restoration of mitochondrial membrane potential; this effect was associated with promotion of Nrf2 nuclear translocation and its downstream antioxidative stress signals (NQO-1, Prdx1). Overall, the present data has provided the first evidence that CVB-D has potential therapeutic in DCM, mainly by activation of the Nrf2 signalling pathway to suppress oxidative stress. Our findings also have positive implications on the novel promising clinical applications of CVB-D.

Acacetin enhances glucose uptake through insulin-independent GLUT4 translocation in L6 myotubes.

BACKGROUND: Lowering blood glucose levels by increasing glucose uptake in insulin target tissues, such as skeletal muscle and adipose tissue, is one strategy to discover and develop antidiabetic drugs from natural products used as traditional medicines. PURPOSE: Our goal was to reveal the mechanism and activity of acacetin (5,7-dihydroxy-4'-methoxyflavone), one of the major compounds in Agastache rugose, in stimulating glucose uptake in muscle cells. METHODS: To determine whether acacetin promotes GLUT4-dependent glucose uptake in cultured L6 skeletal muscle cells, we performed a [14C] 2-deoxy-D-glucose (2-DG) uptake assay after treating differentiated L6-GLUT4myc cells with acacetin. RESULTS: Acacetin dose-dependently increased 2-DG uptake by enhancing GLUT4 translocation to the plasma membrane. Our results revealed that acacetin activated the CaMKII-AMPK pathway by increasing intracellular calcium concentrations. We also found that aPKCλ/ζ phosphorylation and intracellular reactive oxygen species (ROS) production were involved in acacetin-induced GLUT4 translocation. Moreover, acacetin-activated AMPK inhibited intracellular lipid accumulation and increased 2-DG uptake in HepG2 cells. CONCLUSION: Taken together, these results suggest that acacetin might be useful as an antidiabetic functional ingredient. Subsequent experiments using disease model animals are needed to verify our results.

(-)-Catechin-7-O-ß-d-Apiofuranoside Inhibits Hepatic Stellate Cell Activation by Suppressing the STAT3 Signaling Pathway.

Hepatic fibrosis is characterized by the abnormal deposition of extracellular matrix (ECM) proteins. During hepatic fibrogenesis, hepatic stellate cell (HSC) activation followed by chronic injuries is considered a key event in fibrogenesis, and activated HSCs are known to comprise approximately 90% of ECM-producing myofibroblasts. Here, we demonstrated that (-)-catechin-7-O-ß-d-apiofuranoside (C7A) significantly inhibited HSC activation via blocking the signal transducer and activator of transcription 3 (STAT3) signaling pathway. This is the first study to show the hepatic protective effects of C7A with possible mechanisms in vitro and in vivo. In our bioactivity screening, we figured out that the EtOH extract of Ulmusdavidiana var. japonica root barks, which have been used as a Korean traditional medicine, inhibited collagen synthesis in HSCs. Four catechins isolated from the EtOAc fraction of the EtOH extract were compared with each other in terms of reduction in collagen, which is considered as a marker of hepatic protective effects, and C7A showed the strongest inhibitory effects on HSC activation in protein and qPCR analyses. As a possible mechanism, we investigated the effects of C7A on the STAT3 signaling pathway, which is known to activate HSCs. We found that C7A inhibited phosphorylation of STAT3 and translocation of STAT3 to nucleus. C7A also inhibited expressions of MMP-2 and MMP-9, which are downstream genes of STAT3 signaling. Anti-fibrotic effects of C7A were evaluated in a thioacetamide (TAA)-induced liver fibrosis model, which indicated that C7A significantly inhibited ECM deposition through inhibiting STAT3 signaling. C7A decreased serum levels of aspartate amino transferase and alanine transaminase, which were markedly increased by TAA injection. Moreover, ECM-associated proteins and mRNA expression were strongly suppressed by C7A. Our study provides the experimental evidence that C7A has inhibitory effects on HSC activation after live injury and has preventive and therapeutic potentials for the management of hepatic fibrosis.