Beta-Caryophyllene Modifies Intracellular Lipid Composition in a Cell Model of Hepatic Steatosis by Acting through CB2 and PPAR Receptors.

Non-alcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease; however, no specific pharmacological therapy has yet been approved for this condition. Plant-derived extracts can be an important source for the development of new drugs. The aim of this study was to investigate the effects of (E)-ß-caryophyllene (BCP), a phytocannabinoid recently found to be beneficial against metabolic diseases, on HepG2 steatotic hepatocytes. Using a fluorescence-based lipid quantification assay and GC-MS analysis, we show that BCP is able to decrease lipid accumulation in steatotic conditions and to change the typical steatotic lipid profile by primarily reducing saturated fatty acids. By employing specific antagonists, we demonstrate that BCP action is mediated by multiple receptors: CB2 cannabinoid receptor, peroxisome proliferator-activated receptor α (PPARα) and γ (PPARγ). Interestingly, BCP was able to counteract the increase in CB2 and the reduction in PPARα receptor expression observed in steatotic conditions. Moreover, through immunofluorescence and confocal microscopy, we demonstrate that CB2 receptors are mainly intracellularly localized and that BCP is internalized in HepG2 cells with a maximum peak at 2 h, suggesting a direct interaction with intracellular receptors. The results obtained with BCP in normal and steatotic hepatocytes encourage future applications in the treatment of NAFLD.

Reproducibility of adipogenic responses to metabolism disrupting chemicals in the 3T3-L1 pre-adipocyte model system: An interlaboratory study.

The 3T3-L1 murine pre-adipocyte line is an established cell culture model for screening Metabolism Disrupting Chemicals (MDCs). Despite a need to accurately identify MDCs for further evaluation, relatively little research has been performed to comprehensively evaluate reproducibility across laboratories, assess factors that might contribute to varying degrees of differentiation between laboratories (media additives, plastics, cell source, etc.), or to standardize protocols. As such, the goals of this study were to assess interlaboratory variability of efficacy and potency outcomes for triglyceride accumulation and pre-adipocyte proliferation using the mouse 3T3-L1 pre-adipocyte cell assay to test chemicals. Ten laboratories from five different countries participated. Each laboratory evaluated one reference chemical (rosiglitazone) and three blinded test chemicals (tributyltin chloride, pyraclostrobin, and bisphenol A) using: 1) their Laboratory-specific 3T3-L1 Cells (LC) and their Laboratory-specific differentiation Protocol (LP), 2) Shared 3T3-L1 Cells (SC) with LP, 3) LC with a Shared differentiation Protocol (SP), and 4) SC with SP. Blinded test chemical responses were analyzed by the coordinating laboratory. The magnitude and range of bioactivities reported varied considerably across laboratories and test conditions, though the presence or absence of activity for each tested chemical was more consistent. Triglyceride accumulation activity determinations for rosiglitazone ranged from 90 to 100% across test conditions, but 30-70 % for pre-adipocyte proliferation; this was 40-80 % for triglyceride accumulation induced by pyraclostrobin, 80-100 % for tributyltin, and 80-100 % for bisphenol A. Consistency was much lower for pre-adipocyte proliferation, with 30-70 % active determinations for pyraclostrobin, 30-50 % for tributyltin, and 20-40 % for bisphenol A. Greater consistency was observed for the SC/SP assessment. As such, working to develop a standardized adipogenic differentiation protocol represents the best strategy for improving consistency of adipogenic responses using the 3T3-L1 model to reproducibly identify MDCs and increase confidence in reported outcomes.

Cannabinoid Receptor Modulation of Neurogenesis: ST14A Striatal Neural Progenitor Cells as a Simplified In Vitro Model.

The endocannabinoid system (ECS) is involved in the modulation of several basic biological processes, having widespread roles in neurodevelopment, neuromodulation, immune response, energy homeostasis and reproduction. In the adult central nervous system (CNS) the ECS mainly modulates neurotransmitter release, however, a substantial body of evidence has revealed a central role in regulating neurogenesis in developing and adult CNS, also under pathological conditions. Due to the complexity of investigating ECS functions in neural progenitors in vivo, we tested the suitability of the ST14A striatal neural progenitor cell line as a simplified in vitro model to dissect the role and the mechanisms of ECS-regulated neurogenesis, as well as to perform ECS-targeted pharmacological approaches. We report that ST14A cells express various ECS components, supporting the presence of an active ECS. While CB1 and CB2 receptor blockade did not affect ST14A cell number, exogenous administration of the endocannabinoid 2-AG and the synthetic CB2 agonist JWH133 increased ST14A cell proliferation. Phospholipase C (PLC), but not PI3K pharmacological blockade negatively modulated CB2-induced ST14A cell proliferation, suggesting that a PLC pathway is involved in the steps downstream to CB2 activation. On the basis of our results, we propose ST14A neural progenitor cells as a useful in vitro model for studying ECS modulation of neurogenesis, also in prospective in vivo pharmacological studies.

Microplastics and their associated organic pollutants from the coastal waters of the central Adriatic Sea (Italy): Investigation of adipogenic effects in vitro.

Even though microplastic (MP) pollution in aquatic environment is nowadays widely studied, a huge gap of knowledge exists on their actual biological effects. In this study we first reported environmental baseline data on the occurrence and characterization of floating MPs in Italian coastal waters of the Central Adriatic Sea by using a standardized monitoring protocol. Further, we analyzed the concentrations of MP-associated chemicals and evaluated their potential adipogenic effects using 3T3-L1 preadipocytes. MPs were found in each sampling stations showing the highest abundance (1.88 ± 1.78 items/m3) in the sites more distant from the coast with fragments as the most common shape category. All targeted organic pollutants (i.e. polychlorinated biphenyls - PCBs, polycyclic aromatic hydrocarbons -PAHs, organophosphorus - OP, and organochlorine - OC pesticides) have been detected on the surface of the collected MPs. The highest concentrations of PAHs were found on MPs from inshore (i.e. <1.5 NM) surface waters with low-ring PAHs as dominant components. Similarly, MPs from inshore waters had higher ΣPCB concentrations (64.72 ng/g plastic) than those found in offshore (i.e. >6 NM) waters (10.37 ng/g plastic). Among pesticides, all measured OPs were detected in each sample analyzed with pirimiphos-methyl as the most representative compound. For OCs, the sum of all concentrations of congeners was higher in coastal with respect to offshore waters. Moreover, in vitro 3T3-L1 screening of MP extracts indicated potential metabolic effects resulting in both adipogenesis and lipid uptake/storage.

Protective Effects of (E)-ß-Caryophyllene (BCP) in Chronic Inflammation.

(E)-ß-caryophyllene (BCP) is a bicyclic sesquiterpene widely distributed in the plant kingdom, where it contributes a unique aroma to essential oils and has a pivotal role in the survival and evolution of higher plants. Recent studies provided evidence for protective roles of BCP in animal cells, highlighting its possible use as a novel therapeutic tool. Experimental results show the ability of BCP to reduce pro-inflammatory mediators such as tumor necrosis factor-alfa (TNF-α), interleukin-1ß (IL-1ß), interleukin-6 (IL-6), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), thus ameliorating chronic pathologies characterized by inflammation and oxidative stress, in particular metabolic and neurological diseases. Through the binding to CB2 cannabinoid receptors and the interaction with members of the family of peroxisome proliferator-activated receptors (PPARs), BCP shows beneficial effects on obesity, non-alcoholic fatty liver disease/nonalcoholic steatohepatitis (NAFLD/NASH) liver diseases, diabetes, cardiovascular diseases, pain and other nervous system disorders. This review describes the current knowledge on the biosynthesis and natural sources of BCP, and reviews its role and mechanisms of action in different inflammation-related metabolic and neurologic disorders.

PipeNig®-FL, a Fluid Extract of Black Pepper (Piper Nigrum L.) with a High Standardized Content of Trans-ß-Caryophyllene, Reduces Lipid Accumulation in 3T3-L1 Preadipocytes and Improves Glucose Uptake in C2C12 Myotubes.

Trans-ß-caryophyllene (BCP) is a natural sesquiterpene hydrocarbon with several important pharmacological activities, including antioxidant, anti-inflammatory, anticancer, and cardioprotective functions. These properties are mainly due to its selective interaction with the peripherally expressed cannabinoid receptor 2. In addition, BCP activates peroxisome proliferated activator receptors α and γ and inhibits the Toll-like receptor signaling pathway. Given the growing scientific interest in BCP, the aim of our study was to investigate the metabolic effects of a black pepper extract (PipeNig®-FL), containing a high standardized content of BCP. In particular our interest was focused on its potential activity on lipid accumulation and glucose uptake. The extract PipeNig®-FL was chemically characterized by gas chromatography-mass spectrometry (GC-MS) and gas chromatography with flame-ionization detection (GC-FID), confirming a high content (814 mg/g) of BCP. Experiments were performed on 3T3-L1 preadipocytes and on C2C12 myotubes. Lipid content following 3T3-L1 adipogenic differentiation was quantified with AdipoRed fluorescence staining. Glucose uptake and GLUT4 membrane translocation were studied in C2C12 myotubes with the fluorescent glucose analog 2-NBDG and by immunofluorescence analysis. Here we show that PipeNig®-FL reduces 3T3-L1 adipocyte differentiation and lipid accumulation. Moreover, acute exposure of C2C12 myotubes to PipeNig®-FL improves glucose uptake activity and GLUT4 migration. Taken together, these results reveal interesting and novel properties of BCP, suggesting potential applications in the prevention of lipid accumulation and in the improvement of glucose uptake.