Lipid Accumulation In 3T3-L1 Preadipocytes Research Articles

Enzymatic Process for High-Yield Turanose Production and Its Potential Property as an Adipogenesis Regulator.

Turanose is a sucrose isomer naturally existing in honey and a promising functional sweetener due to its low glycemic response. In this study, the extrinsic fructose effect on turanose productivity was examined in Neisseria amylosucrase reaction. Turanose was produced, by increasing the amount of extrinsic fructose as a reaction modulator, with high concentration of sucrose substrate, which resulted in 73.7% of production yield. In physiological functionality test, lipid accumulation in 3T3-L1 preadipocytes in the presence of high amounts of pure glucose was attenuated by turanose substitution in a dose-dependent manner. Turanose treatments at concentrations representing 50%, 75%, and 100% of total glucose concentration in cell media significantly reduced lipid accumulation by 18%, 35%, and 72%, respectively, as compared to controls. This result suggested that turanose had a positive role in controlling adipogenesis, and enzymatic process of turanose production has a potential to develop a functional food ingredient for controlling obesity and related chronic diseases.

Abstract 911: Dietary oleuropein (OL) improves high-fat diet-induced metabolic stress and suppresses solid tumor growth and lymph node (LN) metastasis in the B16F10 melanoma allograft model

Abstract Epidemiological studies indicate that obesity-associated metabolic stress increases the risk of developing several cancers. We previously reported that a high-fat diet (HFD)-induced obesity stimulates tumor growth and metastasis of B16F10 melanoma cancer cells in C57BL/6 mice. OL, the most abundant phenolic compound in olives, was reported to exert several physiological activities, including antioxidative, anticancer, and antimicrobial activities. In the present study, we examined whether OL inhibits HFD-induced melanoma progression in the B16F10 allograft model. Four-week old, male C57BL/6N mice were fed a diet [control diet (10 kcal% fat), HFD (60 kcal% fat), HFD + 0.02% OL, or HFD + 0.04% OL]. Twelve weeks after the initiation of feeding, fasting blood glucose, plasma insulin, and HOMA-IR were increased in HFD-fed mice, which was suppressed by dietary OL. Dual-energy X-ray absorptiometry results revealed that the proportion of lean body mass was decreased, whereas that of fat mass was increased in HFD-fed mice, and these changes were suppressed by dietary OL. After 16 weeks of feeding, B16F10-luc cells were subcutaneously injected into the animals’ left flank; bioluminescence imaging was conducted; and the primary tumor was resected 3 weeks later. OL suppressed HFD-induced solid tumor growth; HFD-induced increases in the expression of Ki67, cyclin D1, VEGF-A, VEGF-C, VEGF-D, CD31 (platelet endothelial cell adhesion molecule-1) and LYVE (lymphatic vessel endothelial receptor)-1; and HFD-induced decreases in TUNEL-positive apoptotic cells and cleaved PARP levels in the tumor tissues. OL also suppressed HFD-induced changes in the levels of many cytokines/chemokines in tumor tissues. All animals were killed 3 weeks after the tumor resection. OL suppressed HFD-induced increases in the size of LN; LN metastasis; and expression of VEGF-A and VEGF-C in the LN. OL suppresses HFD-induced increases in the size of adipocytes and accumulation of F4/80+ macrophages in adipose tissues surrounding the LN. In vitro culture results revealed that OL inhibited lipid accumulation in 3T3-L1 preadipocytes and the migration and viability of B16F10 cells. The present results demonstrated that dietary OL suppresses tumor growth and LN metastasis in HFD-fed mice. These effects are probably mediated, at least in part, via the inhibition of lipid accumulation, thereby reducing obesity-associated metabolic stress. Additionally, in vitro results indicate that OL may directly inhibit melanoma cell growth and migration, which may be one of the mechanisms by which dietary OL suppress melanoma progression in these mice. Citation Format: Hyerim Song, Jae In Jung, Gyoo Taik Kwon, So Young Park, Han Jin Cho, Jung Han Yoon Park. Dietary oleuropein (OL) improves high-fat diet-induced metabolic stress and suppresses solid tumor growth and lymph node (LN) metastasis in the B16F10 melanoma allograft model. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 911. doi:10.1158/1538-7445.AM2015-911

Antiadipogenic Effects of Salvia plebeia R. Br. Extracts by Extraction Conditions in 3T3-L1 Preadipocytes

*YD Life Science Research Institutes, YD Life Science Co. Ltd., Seongnam 462-807, Korea.**Department of Food and Nutrition, Kongju National University, Yesan 340-802, Korea.ABSTRACT : This study was carried out to investigate the effects of Salvia plebeia R. Br. ethanolic extract with differentaspects (stem/leaf and whole plant) on differentiation and lipid accumulation in 3T3-L1 preadipocytes. The morphologicalchanges and the degrees of lipid accumulation in 3T3-L1 cells were measured by Oil Red O staining and intra-cellular trig-lyceride (TG) assay. The mRNA expressions of special peroxisome proliferation activated receptor- genes (PPAR), CCAAT/enhancer-binding protein (C/EBPα), fatty acid synthase (FAS) and lipoprotein lipase (LPL) were detected by reverse tran-scriptase polymerase chain reaction (RT-PCR). The 50% ethanolic extracts (100μg/mL) of stem and leaf (SALE) and 30%ethanolic extracts (100 g/mL) of whole plant (SAE) from Salvia plebeia R. Br. were significantly attenuated lipid accumula-tion during adipogenesis in 3T3-L1 cells. Ethyl acetate-soluble fractions (50μg/mL) significantly inhibited lipid dropletaccumulation in 3T3-L1 cells. In addition, SALE induced down-regulation of specific adipogenic transcriptional factors (C/EBPα and PPARγ) and target genes (FAS and LPL) during adipogenesis. Salvia plebeia R. Br. may be used as a safe and effi-cient natural substance to manage obesity.Key Words : Salvia plebeia, 3T3-L1, Adipogenesis, C/EBP α, PPARγ, FAS, LPL

β-Caryophyllene potently inhibits solid tumor growth and lymph node metastasis of B16F10 melanoma cells in high-fat diet–induced obese C57BL/6N mice

We reported previously that high-fat diet (HFD) feeding stimulated solid tumor growth and lymph node (LN) metastasis in C57BL/6N mice injected with B16F10 melanoma cells. β-caryophyllene (BCP) is a natural bicyclic sesquiterpene found in many essential oils and has been shown to exert anti-inflammatory activities. To examine whether BCP inhibits HFD-induced melanoma progression, 4-weeks old, male C57BL/6N mice were fed a control diet (CD, 10 kcal% fat) or HFD (60 kcal% fat + 0, 0.15 or 0.3% BCP) for the entire experimental period. After 16 weeks of feeding, B16F10s were subcutaneously injected into mice. Three weeks later, tumors were resected, and mice were killed 2 weeks post-resection. Although HFD feeding increased body weight gain, fasting blood glucose levels, solid tumor growth, LN metastasis, tumor cell proliferation, angiogenesis and lymphangiogenesis, it decreased apoptotic cells, all of which were suppressed by dietary BCP. HFD feeding increased the number of lipid vacuoles and F4/80+ macrophage (MΦ) and macrophage mannose receptor (MMR)+ M2-MΦs in tumor tissues and adipose tissues surrounding the LN, which was suppressed by BCP. HFD feeding increased the levels of CCL19 and CCL21 in the LN and the expression of CCR7 in the tumor; these changes were blocked by dietary BCP. In vitro culture results revealed that BCP inhibited lipid accumulation in 3T3-L1 preadipocytes; monocyte migration and monocyte chemoattractant protein-1 secretion by B16F10s, adipocytes and M2-MΦs; angiogenesis and lymphangiogenesis. The suppression of adipocyte and M2-cell accumulation and the inhibition of CCL19/21-CCR7 axis may be a part of mechanisms for the BCP suppression of HFD-stimulated melanoma progression.

The effect of myostatin on proliferation and lipid accumulation in 3T3-L1 preadipocytes.

Myostatin is a critical negative regulator of skeletal muscle development, and has been reported to be involved in the progression of obesity and diabetes. In the present study, we explored the effects of myostatin on the proliferation and differentiation of 3T3-L1 preadipocytes by using 3-[4,5-dimethylthiazol-2-yl] 2,5-diphenyl tetrazolium bromide spectrophotometry, intracellular triglyceride (TG) assays, and real-time quantitative RT-PCR methods. The results indicated that recombinant myostatin significantly promoted the proliferation of 3T3-L1 preadipocytes and the expression of proliferation-related genes, including Cyclin B2, Cyclin D1, Cyclin E1, Pcna, and c-Myc, and IGF1 levels in the medium of 3T3-L1 were notably upregulated by 35.2, 30.5, 20.5, 33.4, 51.2, and 179% respectively (all P<0.01) in myostatin-treated 3T3-L1 cells. Meanwhile, the intracellular lipid content of myostatin-treated cells was notably reduced as compared with the non-treated cells. Additionally, the mRNA levels of Pparγ, Cebpα, Gpdh, Dgat, Acs1, Atgl, and Hsl were significantly downregulated by 22-76% in fully differentiated myostatin-treated adipocytes. Finally, myostatin regulated the mRNA levels and secretion of adipokines, including Adiponectin, Resistin, Visfatin, and plasminogen activator inhibitor-1 (PAI-1) in 3T3-L1 adipocytes (all P<0.001). Above all, myostatin promoted 3T3-L1 proliferation by increasing the expression of cell-proliferation-related genes and by stimulating IGF1 secretion. Myostatin inhibited 3T3-L1 adipocyte differentiation by suppressing Pparγ and Cebpα expression, which consequently deceased lipid accumulation in 3T3-L1 cells by inhibiting the expression of critical lipogenic enzymes and by promoting the expression of lipolytic enzymes. Finally, myostatin modulated the expression and secretion of adipokines in fully differentiated 3T3-L1 adipocytes.

Chemical constituents of Triticum aestivum and their effects on adipogenic differentiation of 3T3-L1 preadipocytes.

In this report, we investigated the anti-obesity effect of wheat sprouts and their component compounds. Twenty compounds (1-20) were isolated from Triticum aestivum. Among them, glycolipids 1-5 were determined for the first time from T. aestivum and its sprouts. The HPLC analysis demonstrated that compounds 1-3, 5, 8, 12, and 14 were major peak in the HPLC chromatogram of the active fraction. The effects of the compounds on lipid accumulation were assessed at concentrations ranging from 1.0 to 100 μM. At concentration of 10.0 μM, compounds 1-7, 10-15, and 17-19 significantly decreased lipid accumulation in 3T3-L1 preadipocytes. Glycolipids 1, 2, and phenolic 17 significantly reduced lipid accumulation in the differentiated adipocytes in a concentration-dependent manner. Quantitative analysis based on measurement of the optical density of Oil Red O indicated that, at 100 μM, compounds 1, 2, and 17 reduced lipid accumulation by 41, 37, and 48%, respectively, compared with the positive control.

Anti-adipogenic activity of the edible brown alga Ecklonia stolonifera and its constituent fucosterol in 3T3-L1 adipocytes.

Fucosterol is a sterol metabolite of brown algae and regulates genes involved with cholesterol homeostasis. As a part of our continuous search for anti-obesity agents from natural marine sources, the anti-adipogenic activities of Ecklonia stolonifera and its sterol, fucosterol, were evaluated for the inhibition of adipocyte differentiation and lipid formation. Oil Red O staining was used to evaluate triglyceride contents in 3T3-L1 pre-adipocytes primed by differentiation medium (DM) I and DM II. The methanolic extract of E. stolonifera showed strong anti-adipogenic activity, and was thus fractionated with several solvents. Among the tested fractions, the dichloromethane (CH2Cl2) fraction was found to be the most active fraction, with significant inhibition (40.5 %) of intracellular lipid accumulation at a non-toxic concentration, followed by the ethyl acetate fraction (30.2 %) at the same concentration, while the n-butanol and water fractions did not show inhibitory activity within the tested concentrations. The strong anti-adipogenic CH2Cl2-soluble fraction was further purified by a repeated chromatography to yield fucosterol. Fucosterol reduced lipid contents in a concentration-dependent manner without showing any cytotoxicity. Fucosterol treatment also yielded a decrease in the expression of the adipocyte marker proteins peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer-binding protein α (C/EBPα) in a concentration-dependent manner. Taken together, these results suggest that fucosterol inhibits expression of PPARγ and C/EBPα, resulting in a decrease of lipid accumulation in 3T3-L1 pre-adipocytes, indicating that the potential use of E. stolonifera and its bioactive fucosterol as an anti-obesity agent.

Cocoa polyphenols suppress adipogenesis in vitro and obesity in vivo by targeting insulin receptor

To investigate the inhibitory effect of cocoa polyphenol extract (CPE) on adipogenesis and obesity along with its mechanism of action. 3T3-L1 preadipocytes were cultured with isobutylmethylxanthine, dexamethasone and insulin (MDI), and male C57BL/6N mice (N=44) were fed a high-fat diet (HFD) for 5 weeks with or without CPE. CPE at 100 or 200 μg ml(-1) inhibited MDI-induced lipid accumulation without diminishing cell viability. In particular, CPE reduced the protein expression levels of PPARγ and CEBPα, and blocked mitotic clonal expansion (MCE) of preadipocytes by reducing proliferating signaling pathways. This in turn attenuates lipid accumulation during the differentiation of 3T3-L1 preadipocytes. CPE effectively suppressed MDI-induced phosphorylation of extracellular signal-regulated kinase (ERK) and Akt, and their downstream signals. We then examined whether CPE regulates insulin receptor (IR), a common upstream regulator of ERK and Akt. We found that although CPE does not affect the protein expression level of IR, it significantly inhibits the activity of IR kinase via direct binding. Collectively, the results suggested that CPE, a direct inhibitor of IR kinase activity, inhibits cellular differentiation and lipid accumulation in 3T3-L1 preadipocytes. Consistently, CPE attenuated HFD-induced body weight gain and fat accumulation in obese mice fed with a HFD. We also found that HFD-induced increased fasting glucose levels remained unaffected by CPE. This study demonstrates that CPE inhibits IR kinase activity and its proliferative downstream signaling markers, such as ERK and Akt, in 3T3-L1 preadipocytes, and also prevents the development of obesity in mice fed with a HFD.

Highly hydroxylated fullerene localizes at the cytoskeleton and inhibits oxidative stress in adipocytes and a subcutaneous adipose-tissue equivalent

Adipose tissue is a crucial site for pathologic changes in obesity/metabolic syndrome-related diseases. Interaction between adipogenesis and reactive oxygen species (ROS) in adipose tissue involving chronic low-grade inflammation is postulated to be causal in the development of insulin resistance and other metabolic consequences. We used different culture systems to investigate the relationship between ROS and adipogenesis at three levels: within adipocytes, during adipocyte–monocyte interactions, and in a subcutaneous adipose tissue model. The effects of highly hydroxylated fullerene (HHF; C60(OH)36) on adipogenesis-accompanying oxidative stress and inflammatory changes were examined using these three systems. We demonstrated that H2O2 stimulates lipid accumulation in 3T3-L1 preadipocytes, and lipid uptake causes ROS generation in OP9 preadipocytes, both of which were then markedly suppressed with HHF treatment. HHF significantly inhibited the adipogenic stimulant insulin-rich serum replacement (SR)-induced triacylglycerol accumulation, ROS production, and macrophage activation in cultured OP9 cells and an OP9–U937 monocyte-like cell coculture system. H2O2-induced intracellular ROS production in OP9 adipocytes was also notably inhibited by HHF. We developed a three-dimensional subcutaneous adipose-tissue equivalent (SATE) consisting of air-exposed cultures of HaCaT keratinocytes on an OP9 adipocyte-populated collagen gel in a culture insert. With SR stimulation and under suitable conditions, fat accumulation, ROS generation, and macrophage infiltration were observed in the SATE and significantly inhibited by HHF. By western blotting, we demonstrated that HHF localized at the cytoskeleton, which controls the transport of lipids. In conclusion, HHF is able to inhibit oxidative stress in adipocytes and adipogenesis-related macrophage activation in adipose tissues through its antioxidation.

Effects of Capsaicin on Induction of Apoptosis and Inhibition of Adipogenesis in 3T3-L1 Cells

Currently, at the beginning of the 21st century, obesity has become the leading metabolic disease in the world. It is a serious health problem in industrialized countries. Previous research has suggested that decreased preadipocyte differentiation and proliferation and decreased lipogenesis are mechanisms to reduce obesity. In the present study, the effects of capsaicin on the induction of apoptosis and inhibition of lipid accumulation in 3T3-L1 preadipocytes and adipocytes were investigated. The results demonstrated that capsaicin decreased cell population growth of 3T3-L1 preadipocytes, assessed with the MTT assay. Flow cytometric analysis of 3T3-L1 preadipocytes exposed to capsaicin showed that apoptotic cells increased in a time- and dose-dependent manner. Treatment with capsaicin decreased the number of normal cells and increased the number of early apoptotic and late apoptotic cells in a dose-dependent manner. The treatment of cells with capsaicin caused the loss of mitochondria membrane potential (delta psi m). The induction of apoptosis in 3T3-L1 preadipocytes by capsaicin was mediated through the activation of caspase-3, Bax, and Bak, and then through the cleavage of PARP and the down-regulation of Bcl-2. Moreover, capsaicin significantly decreased the amount of intracellular triglycerides and glycerol-3-phosphate dehydrogenase (GPDH) activity in 3T3-L1 adipocytes. Capsaicin also inhibited the expression of PPARgamma, C/EBPalpha, and leptin, but induced up-regulation of adiponectin at the protein level. These results demonstrate that capsaicin efficiently induces apoptosis and inhibits adipogenesis in 3T3-L1 preadipocytes and adipocytes.

Cytokines Promote Wnt Signaling and Inflammation and Impair the Normal Differentiation and Lipid Accumulation in 3T3-L1 Preadipocytes

Obesity with enlarged fat cells is associated with high local concentrations of interleukin-6 (IL-6) and tumor necrosis factor alpha (TNFalpha) in the adipose tissue. We examined the effects of this inflammatory state on 3T3-L1 preadipocyte development and differentiation to mature adipose cells. Both IL-6 and TNFalpha impaired the normal differentiation pattern and lipid accumulation. However, IL-6 allowed a normal early induction of differentiation with inhibition of Wnt10b and Pref-1, whereas expression of CCAAT/enhancer-binding protein alpha, in contrast to peroxisome proliferator-activated receptor gamma, was markedly reduced. TNFalpha also allowed a normal early induction of differentiation, whereas the terminal differentiation to adipose cells was completely prevented. However, both cytokines induced an inflammatory phenotype of the cells but with different profiles. Remarkably, both IL-6 and TNFalpha maintained and augmented the canonical Wnt signaling associated with low axin and high low density lipoprotein receptor-related protein (LRD), Dishevelled, and beta-catenin levels. TNFalpha, but not IL-6, activated Wnt10b expression, whereas IL-6 increased the apparent phosphorylation of Dishevelled. Thus, both IL-6 and TNFalpha prevent the normal development of preadipocytes to fully differentiated adipose cells and, instead, promote an inflammatory phenotype of the adipocytes. These results provide an explanation as to why obesity and diabetes are associated with both local and systemic inflammation, insulin resistance, and ectopic lipid accumulation.