3T3-L1 Mouse Fibroblasts Research Articles

Novel Pyrazole-Based Benzofuran Derivatives as Anticancer Agents: Synthesis, Biological Evaluation, and Molecular Docking Investigations.

In this work, the design, synthesis, and mechanistic studies of novel pyrazole-based benzofuran derivatives 1-8 as anticancer agents were discussed. Cytotoxic potency of the title compounds was evaluated against the lung carcinoma A-549, human-derived colorectal adenocarcinoma HT-29, breast adenocarcinoma MCF-7 cells as well as mouse fibroblast 3T3-L1 cells using XTT assay. Anticancer mechanistic studies were carried out with flow cytometry. XTT results revealed that all compounds exhibited dose-dependent anti-proliferative activity against the tested cancer cells, and especially compound 2 showed the strongest anti-proliferative activity with an IC50 value of 7.31 μM and the highest selectivity (15.74) on MCF-7 cells. Flow cytometry results confirmed that the cytotoxic power of compound 2 on MCF-7 cells is closely related to mitochondrial membrane damage, caspase activation, and apoptosis orientation. Finally, molecular docking studies were applied to determine the interactions between compound 2 and caspase-3 via in-silico approaches. By molecular docking studies, free binding energy (ΔGBind), docking score, Glide score values as well as amino acid residues in the active binding site were determined. Consequently, these results constitute preliminary data for in vivo anticancer studies and have the potential as a chemotherapeutic agent.

S-13-3: AN APPROACH TO IDENTIFY THE UNDETERMINED ADIPOCYTE-DERIVED FACTOR(S) THAT STIMULATE ALDOSTERONE SYNTHASE GENE (CYP11B2) EXPRESSION

Objectives: Hypertension is a frequently observed complication among obese patients. Recently, undetermined adipocyte-derived factor(s) have been recognized as one of the causes of the obesity-related hypertension independent of angiotensin (A) II. In this study, we aim to identify the undetermined adipocyte-derived factor(s) that stimulate aldosterone synthase gene (CYP11B2) expression aldosterone secretion that may induce hypertension. Methods: We first differentiated mouse fibroblast 3T3-L1 cells into adipocytes and collected their supernatants. The supernatants were incubated with human adrenocortical carcinoma H295R cells, and their CYP11B2 mRNA expression was measured by quantitative PCR with reverse transcription (qRT-PCR). Thereafter, the supernatants were fractionated by ammonium sulfate precipitation to purify adipocyte-derived factor(s) that stimulate CYP11B2 expression. The precipitates were collected in 30%, 60% and 90% saturated concentration of ammonium sulfate. Each of precipitates was concentrated using 100 kDa ultrafiltration membrane and incubated with H295R cells. CYP11B2 mRNA expression in H295R cells was measured by qRT-PCR. Then, we purified the precipitation of the supernatants that obtained in 60% ammonium sulfate by anion-exchange chromatography. Each of the fractions was incubated with human adrenocortical carcinoma H295R cells, and their CYP11B2 mRNA expression was measured by qRT-PCR. Results: The supernatants obtained from 3T3-L1 adipocytes were demonstrated to stimulate CYP11B2 expression. Fractionation of the supernatants by ammonium sulfate precipitation demonstrated that the ability to stimulate CYP11B2 expression was detected in fractions precipitated in 60% saturated concentration. Fractionation of the supernatants by anion-exchange chromatography demonstrated that the ability to stimulate CYP11B2 expression was detected in two specific fractions. Specific fractions were analyzed by mass spectrometry, resulting in the identification of 39 specific proteins. We are now investigating that the relationship between CYP11B2 gene expression and 39 proteins.

Isolation and cytotoxic activities of undescribed iridoid and xanthone glycosides from Centaurium erythraea Rafn. (Gentianaceae)

Centaurium erythraea Rafn. (Gentianaceae) is used in internal traditional therapy as an anthelmintic, hypotensive, antipyretic, and antidiabetic. It is used externally for the treatment of wounds. Ursolic acid, maslinic acid, secologanin, secologanin dimethyl acetal, centauroside A, erythraeaxanthone I, erythraeaxanthone II, and demethyleustomin were isolated from aerial parts of Centaurium erythraea and were identified using spectroscopic methods, including NMR and mass spectrometry. The cytotoxic potency of undescribed compounds was evaluated by the XTT assay against human breast cancer MCF-7, MDA-MB-453 and mouse fibroblast 3T3-L1 cell lines. Erythraeaxanthone II was found to have the most potent cytotoxic activity.

A bis indole analog molecule fluorescent probe for living cell staining

In this study, the fluorescent probe property of a bis indole compound (5,14-dimethyl-5,6,7,8,9,14-hexahydroindolo [2 ', 3′: 3,4] cycloocta [1,2-b] indole), a Caulerpin derivative, which is a member of the indole family, was investigated. For this purpose, firstly, the fluorescence character of the molecule was investigated by absorption and fluorescence spectroscopy techniques in both solvent and surfactant environments. Afterwards, the cytotoxicity of the molecule was tested on the human colon cancer HT-29 cells and breast cancer MCF-7 cells as well as mouse fibroblast 3T3-L1 cells. After determining that the molecule has no cytotoxic effect against all the tested cell lines by 100 µM, the cell staining feature of the probe was examined. The obtained data showed us that the molecule, whose confocal microscopy images were taken at 360 nm excitation wavelength, stained the cell cytoplasm. In addition, optical absorption spectra, oscillator strength (f), transition configuration and transition characteristics of the Ars-In in different solvents were investigated at the theoretical level using Time Dependent-Density Functional Theory (TD-DFT). In conclusion, these results confirm that this indole derivative molecule could be evaluated as an effective fluorescence probe for biological systems.

AGMO Inhibitor Reduces 3T3-L1 Adipogenesis.

Alkylglycerol monooxygenase (AGMO) is a tetrahydrobiopterin (BH4)-dependent enzyme with major expression in the liver and white adipose tissue that cleaves alkyl ether glycerolipids. The present study describes the disclosure and biological characterization of a candidate compound (Cp6), which inhibits AGMO with an IC50 of 30–100 µM and 5–20-fold preference of AGMO relative to other BH4-dependent enzymes, i.e., phenylalanine-hydroxylase and nitric oxide synthase. The viability and metabolic activity of mouse 3T3-L1 fibroblasts, HepG2 human hepatocytes and mouse RAW264.7 macrophages were not affected up to 10-fold of the IC50. However, Cp6 reversibly inhibited the differentiation of 3T3-L1 cells towards adipocytes, in which AGMO expression was upregulated upon differentiation. Cp6 reduced the accumulation of lipid droplets in adipocytes upon differentiation and in HepG2 cells exposed to free fatty acids. Cp6 also inhibited IL-4-driven differentiation of RAW264.7 macrophages towards M2-like macrophages, which serve as adipocyte progenitors in adipose tissue. Collectively, the data suggest that pharmacologic AGMO inhibition may affect lipid storage.

In Vitro Anticancer Activity of Novel Co(II) and Ni(II) Complexes of Non-steroidal Anti-inflammatory Drug Niflumic Acid Against Human Breast Adenocarcinoma MCF-7 Cells.

Herein, we report the synthesis, characterization and anticancer activity of six novel complexes of non-steroidal anti-inflammatory drug niflumic acid with Co(II) and Ni(II). In vitro cytotoxicity screening in MCF-7, HepG2 and HT-29 cancer cell lines showed that the complex 3 [Co(nif)2(met)(4-pic)] and complex 6 [Ni(nif)2(met)(4-pic)] among all the complexes exhibited the highest cytotoxicity against MCF-7 cells with IC50 values of 11.14 µM and, 41.47 µM, respectively. Besides, all the complexes exhibited significantly higher selectivity towards mouse fibroblast 3T3L1 cells. Further mechanistic studies with both complexes on MCF-7 cells revealed their cytotoxic action through the mitochondrial-dependent apoptotic pathway causing an increase oxidative/nitrosative stress, decrease in mitochondrial membrane potential (ΔΨm), inducing the multicaspase activation and arresting the cell cycle at S phase. q-PCR analysis resulted in an increase in the expression of the apoptotic marker proteins bax, p53 and caspase-3 and -8 in MCF-7 cells, but a decrease in the expression of antiapoptotic bcl-2 gene. Moreover, both complexes induced the apoptosis through the inhibition of PI3K/Akt signaling pathway by decreasing the expression of PI3K and increasing dephosphorylation form of Akt protein. These results provide a significant contribution to the explanation of the anticancer mechanisms of these complexes in MCF-7 cells.

Dictyostelium Differentiation-Inducing Factor-1 Promotes Glucose Uptake, at Least in Part, via an AMPK-Dependent Pathway in Mouse 3T3-L1 Cells.

Differentiation-inducing factor-1 (DIF-1) is a chlorinated alkylphenone (a polyketide) found in the cellular slime mold Dictyostelium discoideum. DIF-1 and its derivative, DIF-1(3M) promote glucose consumption in vitro in mammalian cells and in vivo in diabetic rats; they are expected to be the leading antiobesity and antidiabetes compounds. In this study, we investigated the mechanisms underlying the actions of DIF-1 and DIF-1(3M). In isolated mouse liver mitochondria, these compounds at 2–20 μM promoted oxygen consumption in a dose-dependent manner, suggesting that they act as mitochondrial uncouplers, whereas CP-DIF-1 (another derivative of DIF-1) at 10–20 μM had no effect. In confluent mouse 3T3-L1 fibroblasts, DIF-1 and DIF-1(3M) but not CP-DIF-1 induced phosphorylation (and therefore activation) of AMP kinase (AMPK) and promoted glucose consumption and metabolism. The DIF-induced glucose consumption was reduced by compound C (an AMPK inhibitor) or AMPK knock down. These data suggest that DIF-1 and DIF-1(3M) promote glucose uptake, at least in part, via an AMPK-dependent pathway in 3T3-L1 cells, whereas cellular metabolome analysis revealed that DIF-1 and DIF-1(3M) may act differently at least in part.

Anti-Adhesion Effect of Composite Film Materials Based on Glycoluril-Modified Sodium Carboxymethyl Cellulose.

The aim of the study was to develop composite film materials derived from modified sodium carboxymethyl cellulose and to evaluate their anti-adhesive effects.Materials and Methods.The modified film materials were obtained by dissolving sodium carboxymethyl cellulose (Na-CMC) in an aqueous solution of a modifier (glycoluril) with subsequent homogenization and drying in a vacuum drying oven at room temperature. Physicomechanical parameters of the obtained films were determined using the Instron 3369 universal electromechanical testing machine (Great Britain) equipped with a climatic chamber (300–523 K), improved video extensometer, and the MKC-25 micrometer (Russia). Cytotoxicity of glycoluril-modified film materials derived from Na-CMC was studied by incubating cell cultures of 3T3-L1 mouse fibroblasts directly with extracts from films under study using a colorimetric test. Their anti-adhesion effect was investigated on 40 female Wistar rats by modeling a flat adhesion by inducing abrasion of the cecum and suturing the deserosed surface of the abdominal wall anatomically opposite the abrasion area. The presence of adhesions was assessed on day 8 after the operation. Commercial membrane Seprafilm (USA) was used as a reference sample.Results.It was found that extracts obtained from film materials derived from Na-CMC modified with glycoluril at a concentration of 0.01 and 0.05 wt. % had no cytotoxic effect on the cell culture of mouse fibroblasts 3T3-L1. Flat adhesions were not detected when using Seprafilm. When film materials under study were placed in the abdominal cavity between the injured areas, formation of flat adhesions was not observed or observed in one case out of ten.Conclusion.The obtained films are promising for preventing adhesions as a barrier-type agent. Modifying Na-CMC with glycoluril made it possible to create films that prevent formation of flat adhesions, have improved physical and mechanical properties and no cytotoxic effect.

Citral, a Monoterpene Inhibits Adipogenesis Through Modulation of Adipogenic Transcription Factors in 3T3-L1 Cells

Obesity is considered as a major global human health problem which significantly increases the risk for development of type 2 diabetes. Citral, a bioactive compound widely found in a variety of foods that are consumed daily. In this study, we investigated the inhibitory effect of citral against adipogenic genes in 3T3-L1 cells. The mouse fibroblast 3T3-L1 pre-adipocytes were differentiated into adipocytes using adipogenic cocktail (5g/ml insulin, 0.5mM isobutylmethylxanthine and 10M dexamethasone). Differentiation of adipocytes was evaluated by assessing triglyceride accumulation assay and cell viability by MTT assay. The PI3K/AKT signaling, adipogenic specific transcription factors (PPARγ, SREBP-1c, FAS and CPD) and inflammatory genes (TNF-α, IL-6 and MCP-1) were analyzed by western blotting and reverse transcriptase PCR in differentiated 3T3-L1 cell lines. In this study, triglyceride accumulation was increased in adipogenic cocktail induced 3T3-L1 cells, whereas treatment of citral significantly decreased levels of triglyceride accumulation in concentration dependent manner. Further, MTT assay shows that there was no reduction of cell viability during the differentiation of 3T3-L1 cells. The differentiated 3T3-L1 cell significantly increases the expression of PI3K/AKT, adipogenic transcription factors (PPARγ, SREBP-1c, FAS and CPD) and inflammatory biomarkers (TNF-α, IL-6 and MCP-1). Conversely, cells were treated with citral significantly suppress the expression of PI3K/AKT, PPARγ, SREBP-1c, FAS, CPD, TNF-α, IL-6 and MCP-1 in dose dependent manner. Thus, citral exhibits beneficial effects to inhibit adipogenesis in 3T3-L1 adipocytes through the modulation of adipogenic transcription factors and inflammatory markers.

Fabrication, characterization and application of laccase-nylon 6,6/Fe3+ composite nanofibrous membrane for 3,3'-dimethoxybenzidine detoxification.

In this study, laccase was immobilized on nylon 6,6/Fe3+ composite (NFC) nanofibrous membrane and used for the detoxification of 3,3'-dimethoxybenzidine (DMOB). The average size and tensile strength of the NFC membrane were found to be 60-80nm (diameter) and 2.70MPa, respectively. The FTIR results confirm that the amine (N-H) group of laccase was attached with Fe3+ particles and the carbonyl (C=O) group of NFC membrane via hydrogen bonding. The half-life of the laccase-NFC membrane storage stability was increased from 6 to 11weeks and the reusability was significantly extended up to 43 cycles against ABTS oxidation. Enhanced electro-oxidation of DMOB by laccase was observed at 0.33V and the catalytic current was found to be 30µA. The DMOB-treated mouse fibroblast 3T3-L1 preadipocytes showed maximum (97%) cell inhibition at 75µML-1 within 24h. The cytotoxicity of DMOB was significantly decreased to 78% after laccase treatment. This study suggests that laccase-NFC membrane might be a good candidate for emerging pollutant detoxification.

Mitochondrial metabolites extend lifespan.

Disruption of mitochondrial respiration in the nematode Caenorhabditis elegans can extend lifespan. We previously showed that long-lived respiratory mutants generate elevated amounts of α-ketoacids. These compounds are structurally related to α-ketoglutarate, suggesting they may be biologically relevant. Here, we show that provision of several such metabolites to wild-type worms is sufficient to extend their life. At least one mode of action is through stabilization of hypoxia-inducible factor-1 (HIF-1). We also find that an α-ketoglutarate mimetic, 2,4-pyridinedicarboxylic acid (2,4-PDA), is alone sufficient to increase the lifespan of wild-type worms and this effect is blocked by removal of HIF-1. HIF-1 is constitutively active in isp-1(qm150) Mit mutants, and accordingly, 2,4-PDA does not further increase their lifespan. Incubation of mouse 3T3-L1 fibroblasts with life-prolonging α-ketoacids also results in HIF-1α stabilization. We propose that metabolites that build up following mitochondrial respiratory dysfunction form a novel mode of cell signaling that acts to regulate lifespan.

2G11 電気インピーダンスによる脂肪細胞分化の検出に関する基礎的検討

Obesity and metabolic syndrome which are currently social problems, are thought to be caused by the accumulation of lipid droplets in the body. It is important to develop the screening system for functional foods to treat the obesity or metabolic syndrome. Since fat tissue has a high impedance values, it is considered that the amount of lipid droplets affect the impedance value. Therefore, culturing adipocytes and monitoring their electric impedance simultaneously would enable to evaluate the amount of lipid droplets. In this study, mouse fibroblasts 3T3-L1 were cultured on comb-shaped indium-tin oxide electrodes and the electric impedance measurements were performed to evaluate adipogenesis and lipid accumulation. Briefly, the fibroblasts were cultured under two conditions (with and without adipogenesis) and the real and imaginary parts of impedance were measured. As the results, the imaginary part of impedance of fibroblasts under adipogenesis condition showed different response compared to that under without adipogenesis condition.

Lipid Abundance in Zebrafish Embryos Is Regulated by Complementary Actions of the Endocannabinoid System and Retinoic Acid Pathway.

The endocannabinoid system (ECS) and retinoic acid (RA) signaling have been associated with influencing lipid metabolism. We hypothesized that modulation of these pathways could modify lipid abundance in developing vertebrates and that these pathways could have a combinatorial effect on lipid levels. Zebrafish embryos were exposed to chemical treatments altering the activity of the ECS and RA pathway. Embryos were stained with the neutral lipid dye Oil-Red-O (ORO) and underwent whole-mount in situ hybridization (WISH). Mouse 3T3-L1 fibroblasts were differentiated under exposure to RA-modulating chemicals and subsequently stained with ORO and analyzed for gene expression by qRT-PCR. ECS activation and RA exposure increased lipid abundance and the expression of lipoprotein lipase. In addition, RA treatment increased expression of CCAAT/enhancer-binding protein alpha. Both ECS receptors and RA receptor subtypes were separately involved in modulating lipid abundance. Finally, increased ECS or RA activity ameliorated the reduced lipid abundance caused by peroxisome proliferator-activated receptor gamma (PPARγ) inhibition. Therefore, the ECS and RA pathway influence lipid abundance in zebrafish embryos and have an additive effect when treated simultaneously. Furthermore, we demonstrated that these pathways act downstream or independently of PPARγ to influence lipid levels. Our study shows for the first time that the RA and ECS pathways have additive function in lipid abundance during vertebrate development.

Derivatives of Dictyostelium differentiation-inducing factors inhibit lysophosphatidic acid–stimulated migration of murine osteosarcoma LM8 cells

Osteosarcoma is a common metastatic bone cancer that predominantly develops in children and adolescents. Metastatic osteosarcoma remains associated with a poor prognosis; therefore, more effective anti-metastatic drugs are needed. Differentiation-inducing factor-1 (DIF-1), −2, and −3 are novel lead anti-tumor agents that were originally isolated from the cellular slime mold Dictyostelium discoideum. Here we investigated the effects of a panel of DIF derivatives on lysophosphatidic acid (LPA)-induced migration of mouse osteosarcoma LM8 cells by using a Boyden chamber assay. Some DIF derivatives such as Br-DIF-1, DIF-3(+2), and Bu-DIF-3 (5–20 μM) dose-dependently suppressed LPA-induced cell migration with associated IC50 values of 5.5, 4.6, and 4.2 μM, respectively. On the other hand, the IC50 values of Br-DIF-1, DIF-3(+2), and Bu-DIF-3 versus cell proliferation were 18.5, 7.2, and 2.0 μM, respectively, in LM8 cells, and >20, 14.8, and 4.3 μM, respectively, in mouse 3T3-L1 fibroblasts (non-transformed). Together, our results demonstrate that Br-DIF-1 in particular may be a valuable tool for the analysis of cancer cell migration, and that DIF derivatives such as DIF-3(+2) and Bu-DIF-3 are promising lead anti-tumor agents for the development of therapies that suppress osteosarcoma cell proliferation, migration, and metastasis.

Pathways commonly dysregulated in mouse and human obese adipose tissue: FAT/CD36 modulates differentiation and lipogenesis

Obesity is linked to adipose tissue hypertrophy (increased adipocyte cell size) and hyperplasia (increased cell number). Comparative analyses of gene datasets allowed us to identify 1426 genes which may represent common adipose phenotype in humans and mice. Among them we identified several adipocyte-specific genes dysregulated in obese adipose tissue, involved in either fatty acid storage (acyl CoA synthase ACSL1, hormone-sensitive lipase LIPE, aquaporin 7 AQP7, perilipin PLIN) or cell adhesion (fibronectin FN1, collagens COL1A1, COL1A3, metalloprotein MMP9, or both (scavenger receptor FAT/CD36). Using real-time analysis of cell surface occupancy on xCELLigence system we developed a new method to study lipid uptake and differentiation of mouse 3T3L1 fibroblasts and human adipose stem cells. Both processes are regulated by insulin and fatty acids such as oleic acid. We showed that fatty acid addition to culture media increased the differentiation rate and was required for full differentiation into unilocular adipocytes. Significant activation of lipogenesis, i.e. lipid accumulation, by either insulin or oleic acid was monitored in times ranging from 1 to 24 h, depending on differentiation state, whereas significant effects on adipogenesis, i.e., surperimposed lipid accumulation and gene transcriptional regulations were measured after 3 to 4 d. Combination of selected times for analysis of lipid contents, cell counts, size fractionations, and gene transcriptional regulations showed that FAT/CD36 specific inhibitor AP5258 significantly increased cell survival of oleic acid-treated mouse and human adipocytes, and partially restored the transcriptional response to oleic acid in the presence of insulin through JNK pathway. Taken together, these data open new perspectives to study the molecular mechanisms commonly dysregulated in mouse and human obesity at the level of lipogenesis linked to hypertrophy and adipogenesis linked to hyperplasia.

Laccase mediated diclofenac transformation and cytotoxicity assessment on mouse fibroblast 3T3-L1 preadipocytes

Diclofenac is recently considered as one of the most devastating environmental pollutants, because of its biomagnification in the food chain which leads to potential harmful effects on non-targeted organisms. This study describes the optimized laccase mediated diclofenac transformation using response surface methodology (RSM) and cytotoxicity testing on mouse fibroblast 3T3-L1 preadipocytes. Three factors (laccase, syringaldehyde, reaction time) were used to optimize the diclofenac transformation. The optimum level of laccase, syringaldehyde and reaction time was found to be 1.91 U mL−1, 187 μg and 51 min for diclofenac transformation (20 mg L−1). The cytotoxicity assessment on mouse fibroblast 3T3-L1 preadipocytes showed that a maximum of 67.9% cell death occurred at 72 h treatment with diclofenac (200 μg mL−1), while the cells treated with laccase treated diclofenac (LTD) showed less toxicity on the cells. These findings can be addressed for the removal of diclofenac toxicity.

SCREENING OF ANTIBACTERIAL AND CYTOTOXIC ACTIVITY OF EXTRACTS FROM EPIDERMIS AND EPIDERMAL MUCUS OF BARBONYMUS SCHWANENFELDII (TINFOIL BARB FISH)

Aim: The main objective of this work is to increase the utilization of by-products like mucus and skin from Tinfoil barb fish in order to isolate antibacterial protein against gram positive and gram negative microorganism. Materials and Methods: The fish epidermis and epidermal mucus samples were extracted with crude, ethanol and dichloromethane to identify potential antimicrobial agents including the ethanol and dichloromethane soluble compounds. Preliminary screening for antimicrobial activity of the extracts were tested against two Gram-positive bacteria Bacillus cereus (MTCC 1306)and Staphylococcus aureus(MTCC 3160) and two Gram-negative bacteria Shigellaboydii and E.coli(MTCC 1303) using standardized disc susceptibility test method recommended by the British Society of Antimicrobial Chemotherapy(BSAC). The activity was measured in terms of zone of inhibition in mm. Also Mouse fibroblast 3T3 L1 cell lines were used to measure cytotoxic activity. Minimum cell viability was shown by sample of 10 mg/ml concentration of the epidermal mucus. Result: The ethanol extract exhibited a bactericidal activity and inhibited the growth of Bacillus cereus, Staphylococcus aureus, Shigellaboydii and E.coli. The dichloromethane exhibited activity against Bacillus cereus, Staphylococcus aureus, E.coli and no activity against Shigellaboydii. The aqueous showed no bactericidal activity for any of the human pathogen tested. These results show the presence of antimicrobial agent and the role of fish byproducts like mucus and epidermis in antimicrobial protection. Conclusion: the present result suggested that the mucus and epidermal extract of Barbonymus schwanenfeldii (Tinfoil barb fish) may be a potent source of antimicrobial agent for human pathogen.

Study of lactoferrin gene expression in human and mouse adipose tissue, human preadipocytes and mouse 3T3-L1 fibroblasts. Association with adipogenic and inflammatory markers

Lactoferrin is considered an epithelial protein present in different gland secretions. Administration of exogenous lactoferrin is also known to modulate adipogenesis and insulin action in human adipocytes. Here, we aimed to investigate lactoferrin gene expression (real-time polymerase chain reaction) and protein (enzyme-linked immunosorbent assay) levels in human (n=143) and mice adipose tissue samples, in adipose tissue fractions and during human preadipocyte and 3T3-L1 cell line differentiation, evaluating the effects of inducers (rosiglitazone) and disruptors (inflammatory factors) of adipocyte differentiation. Lactoferrin (LTF) gene and protein were detectable at relatively high levels in whole adipose tissue and isolated adipocytes in direct association with low-density lipoprotein-related protein 1 (LRP1, its putative receptor). Obese subjects with type 2 diabetes and increased triglycerides had the lowest levels of LTF gene expression in subcutaneous adipose tissue. Specifically, LTF gene expression was significantly increased in adipocytes, mainly from lean subjects, increasing during differentiation in parallel to adipogenic genes and gene markers of lipid droplets. The induction or disruption of adipogenesis led to concomitant changes (increase and decrease, respectively) of lactoferrin levels during adipocyte differentiation also in parallel to gene markers of adipogenesis and lipid droplet development. The administration of lactoferrin led to autopotentiated increased expression of the LTF gene. The decreased lactoferrin mRNA levels in association with obesity and diabetes were replicated in mice adipose tissue. In conclusion, this is the first observation, to our knowledge, of lactoferrin gene expression in whole adipose tissue and isolated adipocytes, increasing during adipogenesis and suggesting a possible contribution in adipose tissue physiology through LRP1.

Peanut sprout ethanol extract inhibits the adipocyte proliferation, differentiation, and matrix metalloproteinases activities in mouse fibroblast 3T3-L1 preadipocytes

3T3-L1 preadipocyte were differentiated to adipocytes, and then treated with 0, 10, 20, and 40 µg/mL of peanut sprout ethanol extract (PSEE). The main component of PSEE is resveratrol which contained 5.55 mg/mL of resveratrol. The MTT assay, Oil-Red O staining, glycerol-3-phosphate dehydrogenase (GPDH) activity, and the triglyceride concentration were determined in 3T3-L1 cells. MMP-2 and MMP-9 activities as well as mRNA expressions of C/EBP β and C/EBP α were also investigated. As the concentration of PSEE in adipocytes increased, the cell proliferation was decreased in a dose-dependent manner from 4 days of incubation (P < 0.05). The GDPH activity (P < 0.05) and the triglyceride concentration (P < 0.05) were decreased as the PSEE treatment concentration increased. The mRNA expression of C/EBPβ in 3T3-L1 cells was significantly low in groups of PSEE-treated, compared with control group (P < 0.05). The MMP-9 (P < 0.05) and MMP-2 (P < 0.05) activities were decreased in a dose-dependent manner as the PSEE concentration increased from 20 µg/mL. In conclusion, it was found that PSEE has an effect on restricting proliferation and differentiation of adipocytes.

Development of a Novel Polygalacturonic Acid-Gelatin Blend Scaffold Fabrication and Biocompatibility Studies for Tissue-Engineering Applications

In this study, we report the enhanced osteoblast differentiation of mouse embryonic stem cell (mESC) on a novel polygalacturonic acid (PGA)/gelatin scaffold. The matrices of various PGA/geletin ratios were fabricated, biophysically characterized, and optimized for cell culture applications. Blended 2% PGA/gelatin scaffolds were highly porous and were robust with enhanced mechanical strength. Swelling studies showed they had high swelling capacity (six-fold higher than only gelatin scaffold) along with complete degradation in the presence of phosphate-buffered saline. Cytocompatibility of the matrices was evaluated using mouse fibroblast 3T3-L1 cell line showing normal spreading and proliferation as assessed by scanning electron microscopy (SEM) and MTT assay. Among different blends of PGA/gelatin, 2% PGA/gelatin (2PG) scaffold showed the optimum physical and biological compatibility for use in cell culture and differentiation of mESC, especially for osteoblast differentiation. The scaffold, as reported in this study, presents a promising tool for tissue engineering applications.