The Anti-Obesity Effects of Xanthohumol Are Mediated Partly by the Adenosine Monophosphate-protein Kinase Signaling Pathway

Xanthohumol (XN), a flavonoid compound extracted from the hop plant Humulus lupus, has been studied for its anti-cancer and anti-adipogenic effects. In this study, we have investigated the effects of XN on the inhibition of adipogenesis and the induction of browning in 3T3-L1 adipocytes. Furthermore, we provide evidence for the first time on the role of adenosine monophosphate-activated protein kinase (AMPK) signaling pathway in XN-induced anti-obesity effects. Browning of white adipose tissue, WAT, is emerging as a novel approach to address obesity. AMPK is activated in response to stress-like exposure to cold and has been shown to induce browning of WAT. 3T3-L1 preadipocytes were differentiated using a differentiating media supplemented with 10% FBS for 8-10 days following a standard differentiation protocol. Mature adipocytes were treated with either 0.01% DMSO or varying doses of XN for 24-48hrs. Treatment of mature 3T3-L1 adipocytes with XN 6.25µM and 25µM decreased lipid content during adipogenesis and increased the expression of uncoupling protein 1 (UCP1), in a dose-dependent manner in mature adipocytes. XN further increased mitochondrial activity in mature adipocytes. To demonstrate the role of AMPK pathway in XN-induced anti-obesity effects, mature adipocytes were treated with either 0.01% DMSO or XN 25µM in the presence or absence of dorsomorphin, an established inhibitor of the AMPK pathway, and AICAR, an AMPK stimulator. XN increased the expression of phospho-AMPK and this XN-induced increase in AMPK activation was diminished in the presence of dorsomorphin. In contrast, XN+AICAR demonstrated an additive effect on the activation of AMPK. Likewise, dorsomorphin reversed XN-induced inhibition of adipogenesis while XN+AICAR demonstrated an additive effect on the inhibition of lipid content during adipogenesis. These results provide evidence for the potential role of AMPK pathway in XN-induced anti-obesity effects in 3T3-L1 adipocytes

Cytoprotective Effects of Natural Compounds against Oxidative Stress

Oxidative stress, an imbalance between reactive oxygen species and antioxidants, has been witnessed in pathophysiological states of many disorders. Compounds identified from natural sources have long been recognized to ameliorate oxidative stress due to their inherent antioxidant activities. Here, we summarize the cytoprotective effects and mechanisms of natural or naturally derived synthetic compounds against oxidative stress. These compounds include: caffeic acid phenethyl ester (CAPE) found in honey bee propolis, curcumin from turmeric roots, resveratrol abundant in grape, and 1-[2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl] imidazole (CDDO-Im), a synthetic triterpenoid based on naturally occurring oleanolic acid. Cytoprotective effects of these compounds in diseases conditions like cardiovascular diseases and obesity to decrease oxidative stress are discussed

Novel Anti-Obesity Effects of Beer Hops Compound Xanthohumol: Role of AMPK Signaling Pathway

Background: Obesity alters adipose tissue metabolic and endocrine functioning, leading to an increased adiposity and release of pro-inflammatory cytokines. Various phytochemicals have been reported to contribute to the beiging of white adipose tissue in order to ameliorate obesity by increasing thermogenesis. Here, we show that the prenylated chalcone, xanthohumol (XN), induces beiging of white adipocytes, stimulates lipolysis, and inhibits adipogenesis of murine 3T3-L1 adipocytes and primary human subcutaneous preadipocytes and these effects are partly mediated by the activation of the AMP-activated protein kinase (AMPK) signaling pathway. Methods: 3T3-L1 adipocytes and primary human subcutaneous preadipocytes were differentiated using a standard protocol and were treated with various concentrations of XN, dorsomorphin, an AMPK inhibitor, or AICAR, an AMPK activator, to investigate the effects on adipogenesis, beiging and lipolysis. Results: XN induced beiging of white adipocytes as witnessed by the increased expression of beige markers CIDE-A and TBX-1. XN increased mitochondrial biogenesis, as evidenced by increased mitochondrial content, enhanced expression of PGC-1α, and the thermogenic protein UCP1. Following 24 h of treatment, XN also increased oxygen consumption rate. XN stimulated lipolysis of mature 3T3-L1 and primary human subcutaneous adipocytes and inhibited adipogenesis of maturing adipocytes. XN activated AMPK and in turn, XN-induced upregulation of UCP1, p-ACC, HSL, and ATGL was downregulated in the presence of dorsomorphin. Likewise, an XN-induced decrease in adipogenesis was reversed in the presence of dorsomorphin. Conclusions: Taken together, XN demonstrates anti-obesity effects by not only inducing beiging but also decreasing adipogenesis and inducing lipolysis. The anti-obesity effects of XN are partly mediated by AMPK signaling pathway suggesting that XN may have potential therapeutic implications for obesity

Xanthohumol Stimulates the Secretion of Catecholamines and Induces M2 Polarization in Raw 264.7 Macrophages

Anti-inflammatory, anti-oxidant, and anti-cancer effects of xanthohumol (XN), a prenylated chalcone extracted from common hop plants, are gaining attention and research has been expanding on the beneficial effects of this compound. In this study, we have investigated the anti-inflammatory effects of XN using a mouse monocytic cell line, RAW 264.7. We hypothesized that the anti-inflammatory effects of XN are due to M2 polarization of macrophages which, in turn, is mediated partly through the adenosine monophosphate-activated protein kinase (AMPK) signaling pathway. RAW 264.7 cells were treated with either 0.1% DMSO or XN at varying concentrations for 24hrs. Cell culture supernatant was collected for ELISA and whole cell lysates were collected for Western blotting experiments. Our results suggest that XN upregulated the secretion of interleukin 10 (IL10), a signature cytokine for M2 polarization, in RAW 264.7 cells after 24 hours. We further demonstrated that XN increased arginase expression, a marker for M2 polarization, and failed to increase inducible nitric oxide synthase (iNOS) expression, a marker for M1 polarization. XN decreased interferon-γ (IFNγ) induced elevation of nitrite release, indicating the inhibitory effects of XN against M1 polarization. Additionally, XN at 25µM increased the secretion of catecholamines from macrophages comparable to interleukin 4 (IL4), an inducer of the M2 phenotype. Finally, XN upregulated the expression of phospho-AMPK in RAW 264.7 cells, indicating the role of AMPK signaling pathway in XN-induced effects. These results provide evidence for the anti-inflammatory properties of XN–mediated induction of M2 polarization. The M2 macrophage mediated anti-inflammatory effects, coupled with catecholamine secretion, and previously anti-adipogenic effects, makes XN an attractive molecule to study its beneficial effects on metabolic disease, like obesity and diabetes, that are associated with underlying chronic, low-grade inflammation

Phytochemicals as Novel Agents for the Induction of Browning in White Adipose Tissue

Obesity and its associated metabolic syndrome continue to be a health epidemic in westernized societies and is catching up in the developing world. Despite such increases, little headway has been made to reverse adverse weight gain in the global population. Few medical options exist for the treatment of obesity which points to the necessity for exploration of anti-obesity therapies including pharmaceutical and nutraceutical compounds. Defects in brown adipose tissue, a major energy dissipating organ, has been identified in the obese and is hypothesized to contribute to the overall metabolic deficit observed in obesity. Not surprisingly, considerable attention has been placed on the discovery of methods to activate brown adipose tissue. A variety of plant-derived, natural compounds have shown promise to regulate brown adipose tissue activity and enhance the lipolytic and catabolic potential of white adipose tissue. Through activation of the sympathetic nervous system, thyroid hormone signaling, and transcriptional regulation of metabolism, natural compounds such as capsaicin and resveratrol may provide a relatively safe and effective option to upregulate energy expenditure. Through utilizing the energy dissipating potential of such nutraceutical compounds, the possibility exists to provide a therapeutic solution to correct the energy imbalance that underlines obesity

Lipocalin-2 deficiency may predispose to the progression of spontaneous age-related adiposity in mice

Lipocalin-2 (Lcn2) is an innate immune protein elevated by several orders of magnitude in various inflammatory conditions including aging and obesity. Recent studies have shown that Lcn2 is secreted by adipocytes in response to inflammation and is categorized as a new adipokine cross-linking innate immunity and metabolic disorders including obesity. However, the involvement of Lcn2 and its function during the progression of obesity is largely unknown. Recently, browning of white adipose tissue (WAT) has gained attention as a therapeutic strategy to combat obesity. Herein, we have shown that treatment of mature 3T3-L1 adipocytes with recombinant Lcn2 (rec-Lcn2) resulted in the up-regulation of thermogenic and beige/brown markers (UCP1, PRDM16, ZIC-1 and TBX1) and increased mitochondrial activity. Additionally, global Lcn2 genetic knockout (Lcn2KO) mice exhibited accelerated weight gain and visceral fat deposition with age, when compared to wild type (WT) mice. Taken together, both in vitro and in vivo studies suggest that Lcn2 is a naturally occurring adipokine, and may serve as an anti-obesity agent by upregulating the thermogenic markers resulting in the browning of WAT. Therefore, Lcn2 and its downstream signaling pathways could be a potential therapeutic target for obesity

Direct and Indirect Effects of Guggulsterone on the Induction of Beiging in Mature 3T3-L1 Adipocytes

Phytochemicals have long demonstrated anti-obesity properties in adipocytes. Their ability, however, to induce browning in white adipose tissue is only beginning to emerge. We have recently established that the white adipocyte cell line, 3T3-L1, is capable of beiging under beta-adrenergic conditions. Using this information, we sought to investigate if the plant steroid guggulsterone (GS) can induce beiging in 3T3-L1s. 3T3-L1 preadipocytes were differentiated using established protocols supplemented with rosiglitazone and thyroid hormone. Direct effects of GS were measured by treating mature 3T3-L1s for 24 hours. Indirect effects were measured by treating mature 3T3-L1s with conditioned media from GS-treated RAW 264.7 macrophages. Direct treatment of 3T3-L1s with GS resulted in increased lipolysis, increased mitochondrial activity (11%), and increased peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) levels by 250% more than control. 3T3-L1s also demonstrated an increase in uncoupling protein 1 (UCP1) expression by 200% and beige marker, T-box protein 1 (TBX1) expression by 80% more than control. Furthermore, this was accompanied by increased levels of G protein-coupled bile acid receptor (TGR5) and its downstream target iodothyronine deiodinase 2 (DIO2). Treatment of RAW 264.7 macrophages with GS induced a 60% increase in catecholamine release into the media compared to control. Using this conditioned media from macrophages, 3T3-L1 adipocytes increased the expression of DIO2 and UCP1 following 24 hours of incubation. Results from this study demonstrate that GS can potentially induce beiging in white adipose tissue through two distinct mechanisms: (1) direct signaling through the TGR5-cAMP-DIO2 pathway and (2) indirectly through stimulating catecholamine release in macrophages. Thus, it is reasonable to conclude that GS may improve the metabolic capacity of adipose tissue thereby counteracting the effects of obesity

Magnetic Drug Delivery of Xanthohumol to Adipocytes Using Ultrasmall Superparamagnetic Iron Oxide Nanoparticles.

According to the CDC’s National Center for Health Statistics, more than one-third (36.5%) of U.S adults are obese. It is the main risk factor for type-2-diabetes, hypertension, dyslipidemia and atherosclerosis. Nutraceuticals such as Xanthohumol have shown potential to inhibit adipogeneis, however, their bioavailability has remained controversial. Hence there is a need to develop targeted therapy which will increase the concentration of Xanthohumol on adipose tissues. Currently, magnetic drug delivery has been used to develop targeted therapies where conventional therapies have proven to be less effective. Among various types of nanoparticles ultrasmall superparamagnetic iron oxide nanoparticles (USPIO) have found considerable attention in magnetic drug delivery as they are easy to synthesize, inert, and are biocompatible. Hence to deliver Xanthohumol to adipose tissues we synthesized USPIO tagged Xanthohumol. The synthesized USPIO nanoparticles were amine functionalized using 3-aminotripropyl ethoxysilane. The presence of amine functional groups on the surface of USPIO was confirmed qualitatively using FTIR and quantitatively using ninhydrin Assay. The ninhydrin assay revealed that 1 mg of amine functionalized UPSIO had 22mg of amine groups. Xanthohumol was then conjugated to the surface of amine-USPIO using via a polyethylene glycol linker. The presence of Xanthohumol on the surface of nanoparticles was confirmed via FTIR. The amount of Xanthohumol tagged onto the surface of doped nanoparticles was quantified using HPLC. The particle size of the synthesized nanoparticles will be evaluated using TEM and the toxicity and therapeutic potency of Xanthohumol tagged to USPIO will be evaluated in vitro

Guggulsterone Activates Adipocyte Beiging through Direct Effects on 3T3-L1 Adipocytes and Indirect Effects Mediated through RAW264.7 Macrophages.

Background: Plant-derived phytochemicals have been of emerging interest as anti-obesity compounds due to their apparent effects on promoting reduced lipid accumulation in adipocytes. Despite such promising evidence, little is known about the potential mechanisms behind their anti-obesity effects. The aim of this study is to establish potential anti-obesity effects of the phytochemical guggulsterone (GS). Methods: Mature 3T3-L1 adipocytes were treated with GS, derived from the guggul plant native in northern India, to investigate its effects on mitochondrial biogenesis and adipocyte beiging. Further, to explore the relationship between macrophages and adipocytes, 3T3-L1s were treated with conditioned media from GS-treated RAW264.7 macrophages. Markers of mitochondrial biogenesis and beiging were measured by western blot. Results: GS treatment in adipocytes resulted in increased mitochondrial density, biogenesis (PGC1α and PPARγ), and increased markers of a beige adipocyte phenotype (UCP1, TBX1, and β-3AR). This upregulation in mitochondrial expression was accompanied by increases oxygen consumption. In GS-treated macrophages, markers of M2 polarization were elevated (e.g., arginase and IL-10), along with increased catecholamine release into the media. Lastly, 3T3-L1 adipocytes treated with conditioned media from macrophages induced a 167.8% increase in UCP1 expression, suggestive of a role of macrophages in eliciting an anti-adipogenic response to GS. Conclusions: Results from this study provide the first mechanistic understanding of the anti-obesity effects of GS and suggests a role for both direct GS-signaling and indirect stimulation of M2 macrophage polarization in this model

Isoproterenol Increases Uncoupling, Glycolysis, and Markers of Beiging in Mature 3T3-L1 Adipocytes.

Beta-adrenergic activation stimulates uncoupling protein 1 (UCP1), enhancing metabolic rate. In vitro, most work has studied brown adipocytes, however, few have investigated more established adipocyte lines such as the murine 3T3-L1 line. To assess the effect of beta-adrenergic activation, mature 3T3-L1s were treated for 6 or 48 hours with or without isoproterenol (10 and 100 μM) following standard differentiation supplemented with thyroid hormone (T3; 1 nM). The highest dose of isoproterenol increased lipid content following 48 hours of treatment. This concentration enhanced UCP1 mRNA and protein expression. The increase in UCP1 following 48 hours of isoproterenol increased oxygen consumption rate. Further, coupling efficiency of the electron transport chain was disturbed and an enhancement of glycolytic rate was measured alongside this, indicating an attempt to meet the energy demands of the cell. Lastly, markers of beige adipocytes (protein content of CD137 and gene transcript of CITED1) were also found to be upregulated at 48 hours of isoproterenol treatment. This data indicates that mature 3T3-L1 adipocytes are responsive to isoproterenol and induce UCP1 expression and activity. Further, this finding provides a model for further pharmaceutical and nutraceutical investigation of UCP1 in 3T3-L1s