Lemongrass essential oil and its major constituent citral isomers modulate adipogenic gene expression in 3T3-L1 cells.

Obesity is a predisposing factor to diseases such as diabetes mellitus, hypertension, and coronary artery disease. Lemongrass essential oil (LEO), from Cymbopogon flexuosus, possesses numerous therapeutic properties including modulation of obesity in vivo. This experiment investigated the effect of LEO and its major components citral (3,7-dimethyl-2,6-octadienal), citral dimethyl acetal (1,1-dimethoxy-3,7-dimethylocta-2,6-diene), and citral diethyl acetal (1,1-diethoxy-3,7-dimethylocta-2,6-diene) in modulation of adipogenesis and genetic expression in adipocytes. Adipogenesis was induced from murine 3T3-L1 preadipocytes procured from ATCC and maintained in Dulbecco's modified Eagle's medium (DMEM) enriched with calf serum. Differentiation was conducted using DMEM enriched with 10% fetal bovine serum, Dexamethasone 0.25 µM, 3-isobutyl-methylxanthine 0.5 mM, and insulin 10 mg/ml for 2 days, followed by 5 days of insulin 10 mg/ml alone. Samples were subjected to experimental treatments at a concentration of 2.5 × 10-3 . Intracellular triglycerides were quantified and photomicrographs were obtained following Oil red O (ORO) staining procedure. Total ribonucleic acid was extracted and expression of genes effecting in lipid metabolism were quantitated using real-time polymerase chain reaction. ORO staining procedure and spectrophotometric analysis demonstrated decreased lipid accumulation following treatments. LEO and its major constituents significantly inhibited expression of sterol response binding protein 2, cluster of differentiation 36, fatty acid binding protein 4, and peripilin. These results indicate modulation of lipid accumulation through decreased lipid uptake, increased lipolysis, decreased differentiation, and downregulated lipid biosynthesis. This investigation suggests that LEO and its constituents exert effects on adipocyte metabolism and are important for understanding metabolic disease. Further investigation is required to elucidate the degree that each mechanism implicated contributes to the observed effect.

Induction of Adipogenic Genes by Novel Serum-Free Conditions From Pre-adipocyte 3T3-L1 and ST2 Cells.

Introduction Obesity, defined as a condition of excessive fat accumulation in adipose tissue, is a global epidemic implicated in a myriad of processes deleterious to human health. It has become one of the leading impediments to public health globally. The study of obesity necessitates adipocyte models, which commonly employ a medium enriched with adipogenic hormones and fetal bovine serum (FBS) to culture terminal adipocytes. In the current study, we developed a novel protocol for serum-free differentiation of 3T3-L1 and ST2 pre-adipocytes using media enriched with free fatty acids (FFA) and bovine serum albumin (BSA). Differentiation was characterized by measuring FFA uptake and changes in expression of adipogenic genes. The novel protocol was also compared against the existing serum-inclusive method. Methods The National Institutes of Health (NIH)-3T3-L1 and ST2 pre-adipocyte cells were maintained in Dulbecco's Modified Eagle Medium (DMEM) containing 10% calf serum and 1% penicillin-streptomycin and Roswell Park Memorial Institute Medium (RPMI) with 10% FBS and 1% penicillin-streptomycin mixture, respectively, at 37℃, 5% CO2 in a humidified atmosphere. Differentiation was induced using a mixture of 0.25 µM dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 10 µg/mL insulin, or 1% insulin-transferrin-selenium (ITS). Cells were cultured in serum-free media containing DMEM with BSA (2.5%) and lipid mixture 1 (LM1 1%) as well as serum-inclusive media enriched with 10% FBS. Total RNA was extracted, and quantitative reverse transcription-polymerase chain reaction (RT-PCR) was performed using delta-delta Ct method, also known as the 2-∆∆Ct  method. Ribosomal protein, large, P0 (RPLP0) was used as a house-keeping gene for quantitation of relative expressions. Results We observed an increase in fatty acid accumulation relative to controls using Oil Red O neutral lipid staining and spectrophotometry. This result was consistent with the effects of the serum-inclusive method. Differentiation was further confirmed by increased gene expression of adipogenic transcription factors - peroxisome proliferator-activated receptor gamma (PPARγ) and CCAAT/enhancer-binding protein alpha (C/EBPα); adipogenic genes - fatty acid-binding protein 4 (FABP4/aP2) and fatty acid translocase (FAT/CD36); and the lipogenic gene - perilipin by using quantitative RT-PCR. Conclusion Our data suggest that serum-free differentiation can significantly enhance the free fatty acid accumulation as well as adipogenic gene expression in both NIH-3T3-L1 and ST2 pre-adipocyte cells. Given the shortcomings of FBS, this method may provide advantages to the serum-inclusive protocols described previously.

Genistein Combined Polysaccharide (GCP) Can Inhibit Intracrine Androgen Synthesis in Prostate Cancer Cells.

Our group and others have previously shown that genistein combined polysaccharide (GCP), an aglycone isoflavone-rich extract with high bioavailability and low toxicity, can inhibit prostate cancer (CaP) cell growth and survival as well as androgen receptor (AR) activity. We now elucidate the mechanism by which this may occur using LNCaP and PC-346C CaP cell lines; GCP can inhibit intracrine androgen synthesis in CaP cells. UPLC-MS/MS and qPCR analyses demonstrated that GCP can mediate a ~3-fold decrease in testosterone levels (p < 0.001) and cause decreased expression of intracrine androgen synthesis pathway enzymes (~2.5-fold decrease of 3ßHSD (p < 0.001), 17ßHSD (p < 0.001), CYP17A (p < 0.01), SRB1 (p < 0.0001), and StAR (p < 0.01)), respectively. Reverse-phase HPLC fractionation and bioassay identified three active GCP fractions. Subsequent NMR and LC-MS analysis of the fraction with the highest level of activity, fraction 40, identified genistein as the primary active component of GCP responsible for its anti-proliferative, pro-apoptotic, and anti-AR activity. GCP, fraction 40, and genistein all mediated at least a ~2-fold change in these biological activities relative to vehicle control (p < 0.001). Genistein caused similar decreases in the expression of 17ßHSD and CYP17A (2.5-fold (p < 0.001) and 1.5-fold decrease (p < 0.01), respectively) compared to GCP, however it did not cause altered expression of the other intracrine androgen synthesis pathway enzymes; 3ßHSD, SRB1, and StAR. Our combined data indicate that GCP and/or genistein may have clinical utility and that further pre-clinical studies are warranted.