Differentiation Of 3T3-L1 Fibroblasts Research Articles

Enhanced 3T3-L1 Differentiation into Adipocytes by Pioglitazone Pharmacological Activation of Peroxisome Proliferator Activated Receptor-Gamma (PPAR-γ).

Simple SummaryPioglitazone is a potent activator of PPAR-γ, a transcriptional factor that is involved in insulin sensibilization and adipocyte differentiation. Here, we propose an optimized methodology for adipocyte differentiation that is critical for secretome release. To achieve this goal, different concentrations of pioglitazone (0–10 µM) were tested and the adipocyte lipidic accumulation was studied. The secretome was then incubated with prostatic cells and macrophages, and the aggressiveness and expression of the inflammatory cytokines were evaluated. We concluded that pioglitazone enhanced adipocyte differentiation and secretome production, making this secretome an excellent adiposity study model.Despite the primary function of pioglitazone in antidiabetic treatment, this drug is a potent inducer of PPAR-γ, a crucial receptor that is involved in adipocyte differentiation. In this work, we propose an optimized methodology to enhance the differentiation of 3T3-L1 fibroblasts into adipocytes. This process is crucial for adipocyte secretome release, which is fundamental for understanding the molecular mechanisms that are involved in obesity for in vitro studies. To achieve this, a pioglitazone dose-response assay was determined over a range varying from 0 to 10 µM. Lipid accumulation was evaluated using Oil-Red-O. The results showed that 10 µM pioglitazone enhanced differentiation and increased secretome production. This secretome was then added into two cell lines: PC3 and RAW264.7. In the PC3 cells, an increase of aggressiveness was observed in terms of viability and proliferation, with the increase of anti-inflammatory cytokines. Conversely, in RAW264.7 cells, a reduction of viability and proliferation was observed, with a decrease in the overexpression of pro-inflammatory cytokines. Overall, the present work constitutes an improved method for adipocyte secretome production that is suitable for experimental biology studies and that could help with our understanding of the molecular mechanisms underlying adiposity influence in other cells.

Alendronate inhibits triglyceride accumulation and oxidative stress in adipocytes and the inflammatory response of macrophages which are associated with adipose tissue dysfunction.

Alendronate, a bisphosphonate used to prevent osteoporosis, stimulates osteogenesis but impairs adipogenesis. Different clinical trials suggest that the incidence of diabetes may be lower in patients treated with alendronate. Taking into account the importance of adipocytes and macrophages of adipose tissue in insulin resistance and type 2 diabetes, it is necessary to evaluate the effect of alendronate in both cell types. In this paper, we investigated the effect of alendronate on the differentiation to adipocytes of 3T3-L1 fibroblasts, the cell line most used to study adipogenesis, and also its effect on lipid content and oxidative stress in mature adipocytes as well as on the inflammatory response of macrophages. We found that alendronate inhibits differentiation of 3T3-L1 fibroblasts to adipocytes in keeping with reports in other cell lines. On the other hand, treatment of 3T3-L1 adipocytes with alendronate was able to decrease triglyceride content and to prevent H2O2-induced lipid peroxidation which was evaluated as an indicator of oxidative stress. In addition, it was found that activation of RAW 264.7 macrophages to a pro-inflammatory M1 type is inhibited by this bisphosphonate. These results suggest that alendronate may contribute to prevent adipocyte excessive enlargement and the induction of oxidative stress in 3T3-L1 adipocytes as well as the activation of macrophages to a pro-inflammatory M1 type, which are events associated with adipose tissue dysfunction and insulin resistance. In this study, we unraveled the underlying mechanisms of events that were previously observed in clinical trials.

MCAM knockdown impairs PPARγ expression and 3T3-L1 fibroblasts differentiation to adipocytes.

We investigated for the first time the expression of melanoma cell adhesion molecule (MCAM) and its involvement in the differentiation of 3T3-L1 fibroblasts to adipocytes. We found that MCAM mRNA increased subsequent to the activation of the master regulator of adipogenesis, PPARγ, and this increase was maintained in the mature adipocytes. On the other hand, MCAM knockdown impaired differentiation and induction of PPARγ as well as expression of genes activated by PPARγ. However, events that precede and are necessary for early PPARγ activation, such as C/EBPβ induction, β-catenin downregulation, and ERK activation, were not affected in the MCAM knockdown cells. In keeping with this, the increase in PPARγ mRNA that precedes MCAM induction was not altered in the knockdown cells. In conclusion, our findings suggest that MCAM is a gene upregulated and involved in maintaining PPARγ induction in the late but not in the early stages of 3T3-L1 fibroblasts adipogenesis.

Ayurvedic anti-diabetic formulation Lodhrasavam inhibits alpha-amylase, alpha-glucosidase and suppresses adipogenic activity in vitro

BackgroundThe patho-physiological cross-talk between diabetes and obesity is well established. However, the choices of drugs suitable for combined treatment of diabetes and obesity are limited. Integration of complementary and alternative medicines (CAMs), like Ayurveda, with modern medicine would be a promising strategy to fill this gap. The diagnostic principles of Ayurveda define obesity as one of the predisposing factors of Madhumeha (correlated as diabetes) and recommends specific formulations for managing obese-diabetes. Lodhrasavam is one such poly-herbal formulation prescribed for obese-diabetic patients. ObjectivesThe present study is an attempt to demonstrate the possible modes of action of Lodhrasavam, built on the hypothesis that the formulation can exert both anti-diabetic and anti-obesity actions. Materials and methodsLodhrasavam, following simulated gastro-intestinal digestion, was monitored for inhibition of α-amylase, α-glucosidase (key digestive enzyme targets of anti-diabetic drugs) and adipogenesis using standard in vitro model systems. ResultsLodhrasavam digest inhibited α-amylase (90%) and α-glucosidase (78%) activity as well as reduced the differentiation of 3T3-L1 fibroblasts to adipocytes. Upon fractionation, the enzyme inhibitory activity and anti-adipogenic activity of the digest were found distributed in different solvent fractions. This partly indicates a potential pharmacological networking of chemically and functionally diverse bioactive molecules in Lodhrasavam. ConclusionThe study provides a possible mode of action and an experimental support for the Ayurvedic use of Lodhrasavam for managing obese-diabetes. Generating scientific evidences and understanding the modes of action, in contemporary scientific language, would essentially help in expanding global acceptance of potentials of CAMs in the management of life style disorders.

Lead enhancement of 3T3-L1 fibroblasts differentiation to adipocytes involves ERK, C/EBPβ and PPARγ activation.

Lead (Pb) is an environmental and industrial contaminant that still represents a public health problem. Elevated Pb exposure has been inversely correlated with femoral bone density and associated with osteoporosis. In the last years, it has been shown that inhibition of osteogenesis from mesenchymal stem cells activates adipogenesis and vice versa. In this paper, we investigated the effect of Pb on the differentiation of 3T3-L1 fibroblasts to adipocytes which is the cell model most used to study adipogenesis. After induction of differentiation, 2days post-confluent cells re-enter the cell cycle and undergo mitotic clonal expansion (MCE) followed by expression of genes that produce the adipocyte phenotype. The presence of concentrations of Pb up to 10μM during differentiation of 3T3-L1 fibroblasts did not interfere with MCE but enhanced the accumulation of cytosolic lipids that occur during adipogenesis, as well as, the induction of PPARγ, the master gene in adipogenesis. It is known that PPARγ upregulation is subsequent to induction of C/EBPβ and ERK activation, which are early events in adipogenesis. We found that both events were enhanced by Pb treatment. Our results support a stimulatory effect of Pb on adipogenesis which involves ERK activation and C/EBPβ upregulation prior to PPARγ and adipogenesis activation.

Formononetin, an isoflavone, activates AMP-activated protein kinase/β-catenin signalling to inhibit adipogenesis and rescues C57BL/6 mice from high-fat diet-induced obesity and bone loss.

Balance between adipocyte and osteoblast differentiation is the key link of disease progression in obesity and osteoporosis. We have previously reported that formononetin (FNT), an isoflavone extracted from Butea monosperma, stimulates osteoblast formation and protects against postmenopausal bone loss. The inverse relationship between osteoblasts and adipocytes prompted us to analyse the effect of FNT on adipogenesis and in vivo bone loss, triggered by high-fat diet (HFD)-induced obesity. The anti-obesity effect and mechanism of action of FNT was determined in 3T3-L1 cells and HFD-induced obese male mice. Our findings show that FNT suppresses the adipogenic differentiation of 3T3-L1 fibroblasts, through down-regulation of key adipogenic markers such as PPARγ, CCAAT/enhancer-binding protein alpha (C/EBPα) and sterol regulatory element-binding protein (SREBP) and inhibits intracellular TAG accumulation. Increased intracellular reactive oxygen species levels and AMP-activated protein kinase (AMPK) activation accompanied by stabilisation of β-catenin were attributed to the anti-adipogenic action of FNT. In vivo, 12 weeks of FNT treatment inhibited the development of obesity in mice by attenuating HFD-induced body weight gain and visceral fat accumulation. The anti-obesity effect of FNT results from increased energy expenditure. FNT also protects against HFD-induced dyslipidaemia and rescues deterioration of trabecular bone volume by increasing bone formation and decreasing bone resorbtion caused by HFD. FNT's rescuing action against obesity-induced osteoporosis commenced at the level of progenitors, as bone marrow progenitor cells, obtained from the HFD mice group supplemented with FNT, showed increased osteogenic and decreased adipogenic potentials. Our findings suggest that FNT inhibits adipogenesis through AMPK/β-catenin signal transduction pathways and protects against HFD-induced obesity and bone loss.

Rosiglitazone drives cavin-2/SDPR expression in adipocytes in a CEBPα-dependent manner.

Caveolae are abundant adipocyte surface domains involved in insulin signaling, membrane trafficking and lipid homeostasis. Transcriptional control mechanisms for caveolins and cavins, the building blocks of caveolae, are thus arguably important for adipocyte biology and studies in this area may give insight into insulin resistance and diabetes. Here we addressed the hypothesis that one of the less characterized caveolar components, cavin-2 (SDPR), is controlled by CCAAT/Enhancer Binding Protein (CEBPα) and Peroxisome Proliferator-Activated Receptor Gamma (PPARG). Using human mRNA expression data we found that SDPR correlated with PPARG in several tissues. This was also observed during differentiation of 3T3-L1 fibroblasts into adipocytes. Treatment of 3T3-L1-derived adipocytes with the PPARγ-activator Rosiglitazone increased SDPR and CEBPα expression at both the mRNA and protein levels. Silencing of CEBPα antagonized these effects. Further, adenoviral expression of PPARγ/CEBPα or Rosiglitazone-treatment increased SDPR expression in primary rat adipocytes. The myocardin family coactivator MKL1 was recently shown to regulate SDPR expression in human coronary artery smooth muscle cells. However, we found that actin depolymerization, known to inhibit MKL1 and MKL2, was without effect on SDPR mRNA levels in adipocytes, even though overexpression of MKL1 and MKL2 had the capacity to increase caveolins and cavins and to repress PPARγ/CEBPα. Altogether, this work demonstrates that CEBPα expression and PPARγ-activity promote SDPR transcription and further supports the emerging notion that PPARγ/CEBPα and MKL1/MKL2 are antagonistic in adipocytes.

Data from proteomic characterization of the role of Snail1 in murine mesenchymal stem cells and 3T3-L1 fibroblasts differentiation.

The transcription factor (TF) Snail1 is a major inducer of the epithelial–mesenchymal transition (EMT) during embryonic development and cancer progression. Ectopic expression of Snail in murine mesenchymal stem cells (mMSC) abrogated their differentiation to osteoblasts or adipocytes. We used either stable isotopic metabolic labeling (SILAC) for 3T3-L1 cells or isobaric labeling with tandem mass tags (TMT) for mMSC stably transfected cells with Snail1 or control. We carried out a proteomic analysis on the nuclear fraction since Snail is a nuclear TF that mediates its effects mainly through the regulation of other TFs. Proteomics data have been deposited in ProteomeXchange via the PRIDE partner repository with the dataset identifiers PXD001529 and PXD002157 (Vizcaino et al., 2014) [1]. Data are associated with a research article published in Molecular and Cellular Proteomics (Pelaez-Garcia et al., 2015) [2].

Design, synthesis and characterization of novel binary V(V)-Schiff base materials linked with insulin-mimetic vanadium-induced differentiation of 3T3-L1 fibroblasts to adipocytes. Structure–function correlations at the molecular level

Among the various roles of vanadium in the regulation of intracellular signaling, energy metabolism and insulin mimesis, its exogenous activity stands as a contemporary challenge currently under investigation and a goal to pursue as a metallodrug against Diabetes mellitus II. In this regard, the lipogenic activity of vanadium linked to the development of well-defined anti-diabetic vanadodrugs has been investigated through: a) specifically designing and synthesizing Schiff base organic ligands L, bearing a variable number of terminal alcohols, b) a series of well-defined soluble binary V(V)-L compounds synthesized and physicochemically characterized, c) a study of their cytotoxic effect and establishment of adipogenic activity in 3T3-L1 fibroblasts toward mature adipocytes, and d) biomarker examination of a closely-linked molecular target involving or influenced by the specific V(V) forms, cumulatively delineating factors involved in potential pathways linked to V(V)-induced insulin-like activity. Collectively, the results a) project the importance of specific structural features in Schiff ligands bound to V(V), thereby influencing the emergence of its (a)toxicity and for the first time its insulin-like activity in pre-adipocyte differentiation, b) contribute to the discovery of molecular targets influenced by the specific vanadoforms seeking to induce glucose uptake, and c) indicate an interplay of V(V) structural speciation and cell-differentiation biological activity, thereby gaining insight into vanadium's potential as a future metallodrug in Diabetes mellitus.

3,4-Oxo-isopropylidene-shikimic acid promotes adiopkine expression during murine 3T3-L1 fibroblast differentiation into adipocytes

Objective3,4-Oxo-isopropylidene-shikimic acid (ISA), a derivative of shikimic acid, has exhibited ameliorative effect on cognitive impairment in experimental animal models of dementia. This study investigated the effect of ISA on lipid accumulation and adipokine secretion during differentiation of 3T3-L1 fibroblasts to adipocytes. Methods3T3-L1 cells were cultured and treated with ISA (50–800 μM) from days 3–8. Lipid accumulation and triglyceride content were measured. Gene expression of adipokines (adiponectin, leptin, and resistin), CCAAT/enhancer binding protein (C/EBP) β, C/EBP α and peroxisome proliferator-activated receptor γ (PPAR γ) and PPAR target genes, including adipocyte fatty acid binding protein (aP2) and fatty acid synthase (FAS) were investigated. ResultsISA promoted 3T3-L1 fibroblast differentiation to adipocytes and increased triglyceride content by 26%. On mechanistic levels, ISA increased expressions of C/EBP β, PPAR γ, C/EBP α, aP2 and FAS. Moreover, ISA stimulated expressions of adipokines secreted by adipocytes, including adiponectin, leptin, and resistin. ConclusionsThese findings demonstrated that ISA promoted adipogenesis by up-regulating expressions of C/EBP β, PPAR γ, C/EBP α, aP2 and FAS, and also stimulated adipokines during adipocyte differentiation. Further study should clarify the relationship between stimulation of adipokines and cognitive enhancing effect of ISA.

Anti-adipogenic and Pro-osteoblastogenic Activities of Spergularia marina Extract.

For decades, Spergularia marina, a local food that is popular in South Korea, has been regarded as a nutritious source of amino acids, vitamins, and minerals. While several halophytes are reported to possess distinct bioactivities, S. marina has yet to be promoted as a natural source of bioactives. In this study, the effects of S. marina on the adipogenic differentiation of 3T3-L1 fibroblasts and the osteoblastic differentiation of MC3T3-E1 pre-osteoblasts and C2C12 myoblast cells were evaluated. The anti-adipogenic effect of S. marina was assessed by measuring lipid accumulation and adipogenic differentiation marker expression. S. marina treatment significantly reduced lipid accumulation and notably decreased the gene levels of peroxisome proliferator-activated receptor γ, CCAAT/enhancer-binding protein α, and sterol regulatory element binding protein 1c. In addition, S. marina enhanced osteoblast differentiation, as indicated by increased alkaline phosphatase activity and increased levels of osteoblastogenesis indicators, namely bone morphogenetic protein-2, osteocalcin, and type I collagen. In conclusion, S. marina could be a source of functional food ingredients that improve osteoporosis and obesity. Further studies, including activity-based fractionation, will elucidate the mechanism of action and active ingredients of S. marina, which would provide researchers with a better understanding of the nutraceutical and therapeutic applications of S. marina.

Exchange protein activated by cyclic AMP is involved in the regulation of adipogenic genes during 3T3-L1 fibroblasts differentiation

Adipogenesis is stimulated in 3T3-L1 fibroblasts by a combination of insulin, dexamethasone and isobutylmethylxanthine, IBMX, (I+D+M). Two transcription factors are important for the acquisition of the adipocyte phenotype, C/EBP beta (CCAT enhancer-binding protein beta) and PPAR gamma (peroxisome proliferator-activated receptor gamma). IBMX increases cAMP content, which can activate protein kinase A (PKA) and/or EPAC (exchange protein activated by cAMP). To investigate the importance of IBMX in the differentiation mixture, we first evaluated the effect of the addition of IBMX on the increase of C/EBP beta and PPAR gamma and found an enhancement of the amount of both proteins. IBMX addition (I+D+M) or its replacement with a cAMP analogue, dibutyryl-cAMP or 8-(4-chlorophenylthio)-2-O'-methyl-cAMP (8CPT-2-Me-cAMP), the latter activates EPAC and not PKA, remarkably increased PPAR gamma mRNA. However, neither I+D nor any of the inducers alone, increased PPAR gamma mRNA to a similar extent, suggesting the importance of the presence of both IBMX and I+D. It was also found that the addition of IBMX or 8CPT-2-Me-cAMP was able to increase the content of C/EBP beta with respect to I+D. In agreement with these findings, a microarray analysis showed that the presence of either 8CPT-2-Me-cAMP or IBMX in the differentiation mixture was able to upregulate PPAR gamma and PPAR gamma-activated genes as well as other genes involved in lipid metabolism. Our results prove the involvement of IBMX-cAMP-EPAC in the regulation of adipogenic genes during differentiation of 3T3-L1 fibroblasts and therfore contributes to elucidate the role of cyclic AMP in this process.

Fructose promotes the differentiation of 3T3-L1 adipocytes and accelerates lipid metabolism

Excessive fructose consumption and elevated glucocorticoids contribute to metabolic syndrome. We show that fructose as the only carbohydrate source is sufficient for the differentiation of 3T3-L1 fibroblasts into adipocytes. Differentiation of cells in fructose containing medium resulted in increased 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1) expression and activity. Experiments with transfected HEK-293 cells suggested more efficient NADPH generation by fructose compared with glucose in the endoplasmic reticulum (ER). Adipocytes differentiated in the presence of fructose showed increased FABP4 expression, C/EBPα to C/EBPβ ratio and lipolysis. Thus, excessive fructose may cause adverse metabolic effects by enhancing 11β-HSD1 activity and increasing lipolysis in adipocytes.

Sorting of GLUT4 into its insulin-sensitive store requires the Sec1/Munc18 protein mVps45.

Insulin stimulates glucose transport in fat and muscle cells by regulating delivery of the facilitative glucose transporter, glucose transporter isoform 4 (GLUT4), to the plasma membrane. In the absence of insulin, GLUT4 is sequestered away from the general recycling endosomal pathway into specialized vesicles, referred to as GLUT4-storage vesicles. Understanding the sorting of GLUT4 into this store is a major challenge. Here we examine the role of the Sec1/Munc18 protein mVps45 in GLUT4 trafficking. We show that mVps45 is up-regulated upon differentiation of 3T3-L1 fibroblasts into adipocytes and is expressed at stoichiometric levels with its cognate target-soluble N-ethylmaleimide-sensitive factor attachment protein receptor, syntaxin 16. Depletion of mVps45 in 3T3-L1 adipocytes results in decreased GLUT4 levels and impaired insulin-stimulated glucose transport. Using sub-cellular fractionation and an in vitro assay for GLUT4-storage vesicle formation, we show that mVps45 is required to correctly traffic GLUT4 into this compartment. Collectively our data reveal a crucial role for mVps45 in the delivery of GLUT4 into its specialized, insulin-regulated compartment.

The role of FSH in castration-induced adipogenesis: Highlighting differences between orchiectomy, GNRH agonists, and antagonists.

e16011 Background: Androgen deprivation therapy (ADT) results in weight gain and development of the metabolic syndrome. Inspired by the observation that adiposity accompanies the incremental increase in serum FSH levels in menopause, we hypothesized that GNRH antagonist which lower FSH levels will associate with reduced adiposity as compared to GNRH analogues and orchiectomy. Methods: 3T3-L1 fibroblasts differentiation into adipocytes and intracellular lipid accumulation were examined in the presence of escalating FSH doses (0-1000 IU/L) in charcoal stripped FCS supplemented medium to simulate castration. Cellular lipogenesis was assessed by image analysis (oil red staining) and by immunoblotting for fatty acid synthase (FAS) expression. In vivo, 6-week old C57Bl/6 male mice (n=30) were divided to receive either: orchiectomy + vehicle, sham procedure + vehicle, orchiectomy + GNRH antagonist (degarelix 50 mg/kg in vehicle), sham + degarelix, orchiectomy + GNRH analogue (enantone 2 mg/kg in vehicle), or sham + enantone. Serum testosterone, LH and FSH levels and differences in animal weight, visceral fat mass (VFm) and liver lipogenesis (oil red staining) were determined following sacrifice at 6 weeks. BMI was measured as weight (gr) divided by the distance between the tail root and the lower incisors (cm2). Results: The mean lowest and highest serum FSH levels were recorded in mice treated with Degarelix vs. orchiectomy (0.43 and 0.935 mIU/mL resp,) and the mean lowest and highest serum testosterone levels were in mice treated with orchiectomy+degarelix vs. sham control (0.12 and 13.1 ng/ml resp). Mice treated with degarelix had a significantly lower BMI compared to enantone (p=0.02). VFm (perirenal fat weight) was significantly lower (p=0.035) in mice castrated by degarelix (mean 0.38gr) as compared to enantone(mean 0.47gr)). Addition of degarelix or enanton to orchiectomy increased the differences in VFm and BMI between the two groups (0.12gr vs. 0.47gr and 3.73 vs. 4.29 gr/cm2 resp.) In vitro, FSH increased lipid accumulation and FAS expression in 3T3-L1 cells. Conclusions: FSH level elevation following castration promotes fat accumulation and weight gain in preclinical models.

Assessing the Safety of a Replacement Chemical: Nongenomic Activity of Bisphenol S

Suspected links between bisphenol A (BPA) and adverse human health effects have spurred a search for replacement chemicals for use in common applications such as polycarbonate plastics, food can linings, and thermal papers. Bisphenol S (BPS) has been adopted for some uses. A new study now shows that low doses of BPS can disrupt nongenomic signaling pathways in cultured pituitary cells [EHP 121(3):352–358; Vinas and Watson]. Nearly 93% of U.S. residents over age 6 carry measurable amounts of BPA in their bodies. The compound is suspected to contribute to the development of diseases such as diabetes, asthma, and cancer, and animal studies have shown it adversely affects reproduction. In contrast to genomic regulation, which involves receptors in the cell nucleus, nongenomic regulation involves receptors in the cell membrane. Since BPA can interact with cell-membrane estrogen receptors, researchers explored whether structurally similar BPS might likewise influence nongenomic pathways. Treatments included various low doses of BPS alone and paired with estradiol, an endogenous estrogen. Measured responses included activation of ERK and JNK, mitogen-activated protein kinases involved in cell growth, proliferation, and apoptosis (programmed cell death); activation of caspases 8 and 9, enzymes that also are involved in apoptosis; and release of prolactin, a hormone that helps regulate hundreds of biological functions, including metabolism, reproduction, and lactation. BPS has been adopted as a replacement for BPA in applications such as thermal receipt paper. BPS appeared to interact predominantly with the cell-membrane estrogen receptor ER〈. Estradiol and BPS each increased cell proliferation, but following exposure to both compounds there were fewer cells than in control cultures. This can result from decreased proliferation or increased cell death, which is why the investigators also looked at caspase 8 and caspase 9. The former was activated by BPS alone and when combined with estradiol, but caspase 9 was less affected, consistent with an increase in apoptosis. The lowest concentrations of BPS triggered the strongest ERK response; however, ERK activation was not as strong in the presence of both compounds. BPS alone did not activate JNK, but in combination with estradiol it increased JNK activation more than the estrogen alone. The quality and timing of ERK and JNK responses to activation by estradiol were often altered by BPS. By itself, BPS did not affect prolactin secretion, but it significantly suppressed estradiol-induced secretion at most concentrations. Based on these results, low concentrations of BPS appear to affect nongenomic signaling in estrogen-responsive cells, with potential consequences for cell function. The results emphasize the need to screen compounds for estrogenic activity prior to their use in manufacturing.

A commercial formulation of glyphosate inhibits proliferation and differentiation to adipocytes and induces apoptosis in 3T3-L1 fibroblasts

Glyphosate-based herbicides are extensively used for weed control all over the world. Therefore, it is important to investigate the putative toxic effects of these formulations which include not only glyphosate itself but also surfactants that may also be toxic. 3T3-L1 fibroblasts are a useful tool to study adipocyte differentiation, this cell line can be induced to differentiate by addition of a differentiation mixture containing insulin, dexamethasone and 3-isobutyl-1-methylxanthine. We used this cell line to investigate the effect of a commercial formulation of glyphosate (GF) on proliferation, survival and differentiation. It was found that treatment of exponentially growing cells with GF for 48h inhibited proliferation in a dose-dependent manner. In addition, treatment with GF dilution 1:2000 during 24 or 48h inhibited proliferation and increased cell death, as evaluated by trypan blue-exclusion, in a time-dependent manner. We showed that treatment of 3T3-L1 fibroblasts with GF increased caspase-3 like activity and annexin-V positive cells as evaluated by flow cytometric analysis, which are both indicative of induction of apoptosis. It was also found that after the removal of GF, remaining cells were able to restore proliferation. On the other hand, GF treatment severely inhibited the differentiation of 3T3-L1 fibroblasts to adipocytes. According to our results, a glyphosate-based herbicide inhibits proliferation and differentiation in this mammalian cell line and induces apoptosis suggesting GF-mediated cellular damage. Thus, GF is a potential risk factor for human health and the environment.

Synthesis, Characterization and Biological Evaluation of Ureidofibrate-Like Derivatives Endowed with Peroxisome Proliferator-Activated Receptor Activity

A series of ureidofibrate-like derivatives was prepared and assayed for their PPAR functional activity. A calorimetric approach was used to characterize PPARγ-ligand interactions, and docking experiments and X-ray studies were performed to explain the observed potency and efficacy. R-1 and S-1 were selected to evaluate several aspects of their biological activity. In an adipogenic assay, both enantiomers increased the expression of PPARγ target genes and promoted the differentiation of 3T3-L1 fibroblasts to adipocytes. In vivo administration of these compounds to insulin resistant C57Bl/6J mice fed a high fat diet reduced visceral fat content and body weight. Examination of different metabolic parameters showed that R-1 and S-1 are insulin sensitizers. Notably, they also enhanced the expression of hepatic PPARα target genes indicating that their in vivo effects stemmed from an activation of both PPARα and γ. Finally, the capability of R-1 and S-1 to inhibit cellular proliferation in colon cancer cell lines was also evaluated.

Adipocytes Promote B16BL6 Melanoma Cell Invasion and the Epithelial-to-Mesenchymal Transition

Metastatic melanoma is one of the most deadly and evasive types of cancer. On average, cancer patients with metastatic melanoma survive only 6-9months after diagnosis. Epidemiological and animal studies suggest that obesity increases the metastatic ability of malignant melanoma, though the mechanism is not known. In the present studies, we assessed the ability of 3T3L1 adipocytes to modulate B16BL6 melanoma cell invasion and the Epithelial-to-Mesenchymal Transition (EMT). For this purpose, we induced the differentiation of 3T3L1 fibroblasts to adipocytes. Then, we collected the cell culture media from both fibroblasts and adipocytes and determined their effect on the invasive ability and EMT gene expression of B16BL6 melanoma cells. Results show that adipocyte media increased that ability of B16BL6 cells to invade. The higher invasive ability of B16BL6 melanoma cells was associated with increased expression of EMT genes such as Snai1, MMP9, Twist, and Vimentin. Additionally, the expression of the cell-to-cell adhesion protein E-cadherin and the metastasis suppressor gene Kiss1 were down-regulated in these B16BL6 cells. Also, adipocytes had high levels of the pro-inflammatory cytokine Interleukin 6 (IL-6). Treatment of B16BL6 cells with IL-6 elicited similar effects as the adipocyte media; IL-6 promoted the invasive ability of B16BL6 melanoma cells, increased the expression of Snai1, and decreased Kiss1 expression. IL-6 neutralization, however, did not have a visible effect on adipocyte media-induced invasion and snai1 staining. In summary, adipocytes may increase the invasive ability of B16BL6 melanoma cells by promoting EMT and decreasing the expression of genes such as E-cadherin and Kiss1.

Sequential Synthesis and Methylation of Phosphatidylethanolamine Promote Lipid Droplet Biosynthesis and Stability in Tissue Culture and in Vivo

Triacylglycerols are stored in eukaryotic cells within lipid droplets (LD). The LD core is enwrapped by a phospholipid monolayer with phosphatidylcholine (PC), the major phospholipid, and phosphatidylethanolamine (PE), a minor component. We demonstrate that the onset of LD formation is characterized by a change in cellular PC, PE, and phosphatidylserine (PS). With induction of differentiation of 3T3-L1 fibroblasts into adipocytes, the cellular PC/PE ratio decreased concomitant with LD formation, with the most pronounced decline between confluency and day 5. The mRNA for PS synthase-1 (forms PS from PC) and PS decarboxylase (forms PE from PS) increased after day 5. Activity and protein of PE N-methyltransferase (PEMT), which produces PC by methylation of PE, are absent in 3T3-L1 fibroblasts but were induced at day 5. High fat challenge induced PEMT expression in mouse adipose tissue. PE, produced via PS decarboxylase, was the preferred substrate for methylation to PC. A PEMT-GFP fusion protein decorated the periphery of LD. PEMT knockdown in 3T3-L1 adipocytes correlated with increased basal triacylglycerol hydrolysis. Pemt(-/-) mice developed desensitization against adenosine-mediated inhibition of basal hydrolysis in adipose tissue, and adipocyte hypotrophy was observed in Pemt(-/-) animals on a high fat diet. Knock-out of PEMT in adipose tissue down-regulated PS synthase-1 mRNA, suggesting coordination between PE supply and converting pathways during LD biosynthesis. We conclude that two consecutive processes not previously related to LD biogenesis, (i) PE production via PS and (ii) PE conversion via PEMT, are implicated in LD formation and stability.