Triglyceride Accumulation In 3T3-L1 Adipocytes Research Articles

Exploring the Antidiabetic Mechanism of Brachylaena Ilicifolia Using Target Directed in Vitro Screening

ABSTRACT The potential antidiabetic mechanism of Brachylaena ilicifolia aqueous leaf extract was explored using target-directed in vitro screening. The potential of the extract for cytotoxicity and cell proliferation was evaluated using MTT assay in HepG2 cells and Crystal Violet assay in INS-1 cells. The effects of the extract on glucose uptake in liver cells (HepG2) and muscle cells (L6 myotubes) and the lipid accumulation in 3T3-L1 adipocyte were investigated. The inhibitory effects of the extract on α-amylase, α-glucosidase, pancreatic lipase and dipeptidyl peptidase IV (DPP-IV) were also evaluated. The extract demonstrated a low level of toxicity in HepG2 cells with an IC50 value of 105.3 µg mL−1 while also inducing INS-1 cell proliferation at 25 µg mL−1. The treatment of HepG2 and L6 cells with the extract revealed glucose uptake up to 123% in HepG2 cells and 111% in L6 cells without toxicity to these cells. The plant extract had no effect against triglyceride accumulation in 3T3-L1 adipocytes. The extract had no effect on α-amylase, lipase and DPP-IV; however, it demonstrated weak effect against α-glucosidase activity. The findings observed support the traditional usage of B. ilicifolia in the treatment of diabetes, with possible mechanisms of action linked to the enhancement of glucose uptake and stimulation of pancreatic β cell proliferation.

Effect of Astaxanthin on the Inhibition of Lipid Accumulation in 3T3-L1 Adipocytes via Modulation of Lipogenesis and Fatty Acid Transport Pathways.

Obesity is defined as a condition of excessive fat tissue accumulation. It was the major factor most closely associated with lifestyle-related diseases. In the present study, we investigated the effect of astaxanthin on the inhibition of lipid accumulation in 3T3-L1 adipocytes. 3T3-L1 adipocytes were treated with 0–25 µg/mL of astaxanthin for 0–48 h. The result indicated that astaxanthin significantly decreased the oil Red O stained material (OROSM), intracellular triglyceride accumulation, and glycerol 3-phosphate dehydrogenase (GPDH) activity in 3T3-L1 adipocytes (p < 0.05). At the molecular level, astaxanthin significantly down-regulated the mRNA expression of peroxisome proliferator-activated receptor-γ (PPARγ) in 3T3-L1 adipocytes (p < 0.05). Moreover, target genes of PPARγ on the inhibition of lipogenesis, such as Acetyl-CoA carboxylase (ACC), fatty acid synthase (FAS), fatty acid binding protein (aP2), cluster of differentiation 36 (CD36), and lipoprotein lipase (LPL) in 3T3-L1 adipocytes were significantly down-regulated at a time-dependent manner (p < 0.05). These results suggested that astaxanthin efficiently suppressed lipid accumulation in 3T3-L1 adipocytes and its action is associated with the down-regulation of lipogenesis-related genes and the triglyceride accumulation in 3T3-L1 adipocytes. Therefore, astaxanthin can be developed as a potential nutraceutical ingredient for the prevention of obesity in a niche market.

Chemical Compounds in Natural Medicines That Affect Macropharges and Adipocyte Cells.

Macrophages play major roles in inflammation, immunity and host defense mechanisms. Once activated they produce and release cytokines, oxygen and nitrogen species, and eicosanoids. The best characterized stimuli to induce the transcription of genes encoding pro-inflammatory proteins in macrophages in vitro is bacterial lipopolysaccharide (LPS). LPS could be used alone or in combination with recombinant mouse interferon-γ (IFN-γ). Such stimulation results in cytokine release and the synthesis of enzymes such as cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS). The nitric oxide (NO) radical is known to play a central role in inflammatory and immune reactions for self-protection. However, the excessive production of NO may lead to tissue damage. In inflammatory diseases such as rheumatoid arthritis, excessive NO production by activated macrophages has been observed. Adipose tissue is composed of various cell types such as mature adipocytes, preadipocytes, fibroblasts, endothelial cells, vascular cells, and macrophages. Recent studies indicate that obesity is associated with low-grade chronic inflammation of adipose tissues, and that such inflammation is one of the potential mechanisms leading to the insulin resistance. It has been demonstrated that obese adipose tissue is characterized by the increased infiltration of macrophages. Therefore, we attempted to identify natural anti-inflammatory compounds that not only inhibit the secretion of NO from RAW 264.7 cells, but also inhibit triglyceride accumulation in 3T3-L1 adipocytes. This review describes the NO prpduction inhibitory activity or the TG accumulation inhibitory activity of the compounds obtained from 18 plants and a fungi that have been used as traditional medicines.

Anti-adipogenic effect of oat hull extract containing tricin on 3T3-L1 adipocytes

Oat hull ethyl acetate extract (50μg/μL) significantly inhibited triglyceride accumulation in 3T3-L1 adipocytes without causing cytotoxicity. The major active compound in the extract was isolated and identified as 5,7,4′-trihydroxy-3′,5′-dimethoxyflavone, tricin by mass spectrometry and 1H and 13C NMR analyses. The tricin content in oat hull was approximately 18mg/kg dry weight, and tricin constituted 9.6% of the total phenolic compounds in oat hull extract. The treatment of 3T3-L1 cells either with extract or purified tricin resulted in decreased lipid accumulation. The extract significantly decreased the activity of glycerol-3-phosphate dyhydrogenase, a marker of adipocyte differentiation in a dose dependent manner. The expression of major transcription factors, such as peroxisome proliferator-activated receptor (PPAR)-γ, CCATT/enhancer binding protein-α (CEBP/α), sterol regulatory binding protein 1 (SREBP1) and fatty acid synthase (FAS) was also significantly suppressed by the extract. When commercial purified tricin was added to 3T3-L1 cells the expression of all transcription factors were significantly decreased as low as 1.5μg/μL treatment. These results suggest that oat hull extract containing tricin possesses anti-adipogeneic effect.

Traditional Herbal Formula Oyaksungi-San Inhibits Adipogenesis in 3T3-L1 Adipocytes.

Background. Oyaksungi-san (OYSGS) is a herbal formula that has been used for treating cardiovascular diseases in traditional Asian medicine. Here, we investigated the antiadipogenic effect of OYSGS extract in 3T3-L1 adipose cells. Methods. 3T3-L1 preadipocytes were differentiated into adipocytes with or without OYSGS. After differentiation, we measured Oil Red O staining, glycerol-3-phosphate dehydrogenase (GPDH) activity, leptin production, mRNA, and protein levels of adipogenesis-related factors. Results. OYSGS extract dramatically inhibited intracellular lipid accumulation in the differentiated adipocytes. It also significantly suppressed the (GPDH) activity, triglyceride (TG) content, and leptin production by reducing the expression of adipogenesis-related genes including lipoprotein lipase, fatty acid binding protein 4, CCAAT/enhancer-binding protein-alpha (C/EBP-α), and peroxisome proliferator-activated receptor gamma (PPAR-γ). Furthermore, OYSGS clearly enhanced phosphorylation of AMP-activated protein kinase (AMPK) as well as its substrate acetyl CoA (ACC) carboxylase. Conclusions. Our results demonstrate that OYSGS negatively controls TG accumulation in 3T3-L1 adipocytes. We suggest antiadipogenic activity of OYSGS and its potential benefit in preventing obesity.

In vitro antihyperlipidemic potential of triterpenes from stem bark of Protorhus longifolia.

Two lanostane triterpenes, 3β-hydroxylanosta-9,24-dien-21-oic acid (1) and methyl-3β-hydroxylanosta-9,24-dien-21-oate (2), were isolated from the stem bark of Protorhus longifolia. Their structures were deduced on the basis of spectroscopic analysis (NMR, HRMS, IR). This study investigated the in vitro anti-adipogenic activity of the two triterpenes. Their inhibitory activity was evaluated on selected lipid digestive enzymes (pancreatic lipase and cholesterol esterase). The inhibitory activity of the compounds on hormone-sensitive lipase and their ability to bind bile acids were also evaluated. The effect of the compounds on glucose uptake in C2C12 muscle cells and 3T3-L1 adipocytes, and on triglyceride accumulation in 3T3-L1 adipocytes was investigated. The triterpenes effectively inhibited the activities of the enzymes with IC50 values ranging from 0.04 to 0.31 mg/mL. The compounds showed a high affinity for secondary bile acids. Both compounds stimulated glucose uptake in C2C12 muscle cells and 3T3-L1 adipocytes. Compound 1 significantly reduced triglyceride accumulation in mature differentiated 3T3-L1 adipocytes. It is apparent that these lanostane triterpenes enhance glucose uptake and suppress adipogenesis, which together with their inhibitory effects on lipid digestive enzymes suggests that they have antihyperlipidemic potential.

Pharmacological effects of the water fraction of key components in the traditional Chinese prescription Mai Tong Fang on 3T3-L1 adipocytes and ob/ob diabetic mice.

Mai Tong Fang (MTF), a Chinese herbal combination, has been used for the treatment of diabetic nephropathy in traditional medical clinics in China. However, the anti-adipogenic and anti-hyperglycemic effects of MTF have not been fully elucidated, so this study explored these pharmacological activities in 3T3-L1 adipocytes and ob/ob mice, respectively, of the water fraction of milkvetch root, salviae miltiorrhizae and mulberry as key components of MTF. MTF was found to inhibit adipogenesis and triglyceride accumulation in 3T3-L1 adipocytes. Oral administration of MTF in ob/ob mice for 8 weeks, exhibited positive controls on blood glucose and body weight, and further improved glucose tolerance according to an oral glucose tolerance test. Importantly, MTF extract alleviated fat deposition and ballooning degeneration in liver tissue and blocked the increase of adipocyte size in adipose tissue from treated ob/ob mice. These results indicated that the extract of key components in the traditional Chinese prescription MTF continue a potent anti-adipogenic and glucose-lowering agent.

Sea cucumber and blue mussel: new sources of phospholipid enriched omega-3 fatty acids with a potential role in 3T3-L1 adipocyte metabolism.

Omega (n)-3 polyunsaturated fatty acids (PUFA), namely docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), are known to reduce the risk of insulin resistance and ameliorate obesity-associated disorders. DHA and EPA structured in the phospholipid form possess superior biological effects compared to the triglyceride form available in fish oil. In this study, we have found that sea cucumber (SC) and blue mussel (BM) from Newfoundland and Labrador are rich sources of n-3 PUFA structured in the phospholipid form. Treatment with SC and BM methanolic extracts (250 and 100 μg mL(-1), respectively) significantly (p < 0.01) increased triglyceride accumulation in 3T3-L1 adipocytes, along with an increase in the mRNA expression of the peroxisome proliferator-activated receptor-γ (37 and 39%, respectively) and adiponectin (57 and 56%, respectively) compared with control cells (p < 0.05). Only SC extracts (250 μg mL(-1)) increased the mRNA expression of sterol regulatory element-binding protein-1 (SREBP-1). Treatment with higher concentrations of SC and BM extracts (500 and 750 μg mL(-1), respectively) significantly (p < 0.01) decreased triglyceride accumulation in 3T3-L1 cells as opposed to an increase in triglyceride accumulation at lower concentrations. This was due to inhibition of acetyl-CoA carboxylase-1 and SREBP-1 mRNA expression compared to control cells (p < 0.05). There was no effect of the extracts on the mRNA expression of hormone sensitive lipase or lipolysis, suggesting that the decrease in triglyceride accumulation at higher concentrations is not due to breakdown and release of fat. This is the first report to show that SC and BM are new sources of phospholipid bonded n-3 PUFA, with the potential to target insulin resistance and obesity.

Piromelatine decreases triglyceride accumulation in insulin resistant 3T3-L1 adipocytes: Role of ATGL and HSL

Piromelatine, a novel investigational multimodal sleep medicine, is developed for the treatment of patients with primary and co-morbid insomnia. Piromelatine has been shown to inhibit weight gain and improve insulin sensitivity in high-fat/high-sucrose-fed (HFHS) rats. Considering that piromelatine has also been implicated in lowering of triglyceride levels in HFHS rats, this work elucidated whether this effect involves in the regulation of adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) in triglyceride (TG) metabolism. In this study, we investigated the effects of piromelatine and MT2 receptors inhibition on TG content, insulin-stimulated glucose uptake, and the expressions of ATGL and HSL in 3T3-L1 adipocytes preincubated in high glucose and high insulin (HGI) conditions. Our results showed that culturing 3T3-L1 adipocytes under HGI conditions increased triglyceride accumulation with concomitant decrease of ATGL and HSL expression, inducing insulin resistance in 3T3-L1 adipocytes. We also found that triglyceride accumulation was significantly inhibited and the levels of ATGL/HSL increased after melatonin or piromelatine treatment. The effects of melatonin/piromelatine (10nM) were counteracted by pretreatment with the relatively selective MT2 receptor antagonist luzindole (100nM). In this study, our data demonstrate that piromelatine reverses high glucose and high insulin-induced triglyceride accumulation in 3T3-L1 adipocytes, possibly through up-regulating of ATGL and HSL expression via a melatonin-dependent manner.

Grape skin extract reduces adipogenesis- and lipogenesis-related gene expression in 3T3-L1 adipocytes through the peroxisome proliferator-activated receptor-γ signaling pathway

We previously reported that grape skin ethanol extract (GSE) decreases adipogenic transcription factor gene expression, inhibiting triglyceride accumulation in 3T3-L1 adipocytes. In this study, we hypothesized that GSE may induce differential expression profiles in adipocytes, thus providing protection against obesity. Thirty-five genes involved in the peroxisome proliferator-activated receptor-γ (PPARγ) signaling pathway, lipid metabolism, or adipogenesis were identified through microarray analysis of adipocytes treated with GSE. Expression of the genes involved in PPARγ signaling, Adipoq, Scd1, Nr1h3, Fabp5, Scd2, and Pparg decreased with GSE treatment, whereas expression of Ppargc1a increased. Lipid metabolism-associated genes Mlxp1, Stat5a, Hsl, Plin1, and Vdr were down-regulated. Interestingly, GSE also affected expression of genes related to the mitogen-activated protein kinases pathway. GSE extract treatment decreased expression of aP2, Fas, and Tnfa, known markers of adipogenesis, as measured by real-time polymerase reaction. These findings demonstrate the antiadipogenic effects of GSE on 3T3-L1 adipocytes at the genetic level, primarily on the PPARγ signaling pathway.

Phenolic Compounds from Maté (Ilex paraguariensis) Inhibit Adipogenesis in 3T3-L1 Preadipocytes

Leaves of Ilex paraguariensis are used to prepare a tea known as maté which is a common beverage in several South American countries. The ethanol extract was fractionated to identify the compounds responsible for the anti-adipogenic activity in 3T3-L1 cells. Extracts of both fresh and dried maté leaves were subjected to column chromatography using molecular permeation to obtain the saponin (20% yields) and the polyphenol extracts (40% yields) from the fresh and dried leaves. The phenolic content was determined using high-performance liquid chromatography analysis and the Folin-Ciocalteau method. Also, maté extracts (50μg/ml to 1,000μg/ml) did not display citotoxicity using MTT. The polyphenol extract from the dried leaves was the most effective (50μg/ml) in the inhibition of triglyceride accumulation in 3T3-L1 adipocytes, and rutin (100μg/ml) likely accounted for a large portion of this activity. Additionally, maté extracts had a modulatory effect on the expression of genes related to the adipogenesis as PPARγ2, leptin, TNF-α and C/EBPα.

Nepeta japonica Maximowicz extract from natural products inhibits lipid accumulation

To develop novel and crude anti-obesity drugs from natural products is a promising field to approach the solution to a global health problem such as obesity. The aim of this study was to screen crude anti-obesity drugs from 400 natural products on lipase inhibition activity in vitro. Among the natural products examined, 31 extracts showed significantly inhibition activity against porcine pancreatic lipase (triacylglycerol lipase, EC 3.1.1.3) by using spectrophotometry with 2,4-dinitrophenylbutyrate as a substrate. Furthermore, 31 natural products were investigated with regard to their lipid inhibition in 3T3-L1 cells. Among these, one of most promising was Nepeta japonica Maximowicz extract, which showed inhibition of triglyceride accumulation in 3T3-L1 adipocytes, suggesting anti-obesity activity. Also, the amount of glycerol released from cells into the medium was increased by treatment of Nepeta japonica Maximowicz extract at a concentration of 100 µg mL(-1) . The present study suggests that a promising crude anti-obesity drug screened from 400 natural products might be of therapeutic interest with respect to the treatment of obesity.

Inhibitory Effect of Citrus Peel Extract on Lipid Accumulation of 3T3-L1 Adipocytes

Inhibitory effects of lipid accumulation by citrus peel extract were evaluated. Citrus peel extract (CPE) (500 μg/mL) inhibited lipid and triglyceride accumulation in 3T3-L1 adipocytes by more than 64.5 and 58.7%, respectively and suppressed glycerol-3-phosphate dehydrogenase to the levels of preadipocytes. The expression of perilipin mRNA in 3T3-L1 adipocytes treated with CPE was significantly lower than that in untreated adipocytes (p<0.05), with no accompanying increase in lipolysis-related mRNA. Flavonoid compositions of CPE were hesperidin 13.79, narirutin 7, and naringin 262.5 μg/g, respectively. These findings suggest CPE may indirectly stimulate lipolysis by inhibiting protection of physical barrier on lipid droplet to hinder lipid accumulation in 3T3-L1 adipocytes.

6,7,4′-Trihydroxyisoflavone from Doenjang inhibits lipid accumulation

Doenjang is a unique traditional Korean fermented soybean paste. 6,7,4′-Trihydroxyisoflavone was identified from traditional Korean fermented Doenjang. The four products isolated from 100 g Korean Doenjang using acetonitrile and methanol (70/30, v/v) was identified as daidzein, genistein, glycitein and 6,7,4′-trihydroxyisoflavone. The products were structurally elucidated by GC–MS and 1H NMR analysis. 6,7,4′-Trihydroxyisoflavone was evaluated for antioxidant activity against the free radical 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging and superoxide radical scavenging activity. Among four products isolated, 6,7,4′-trihydroxyisoflavone had remarkable antioxidant activity with IC 50 values of 31.5 μM, 50.0 μM, respectively. Furthermore, 6,7,4′-trihydroxyisoflavone was found to inhibit fat droplet formation triglyceride accumulation in 3T3-L1 adipocytes. Accordingly, 6,7,4′-trihydroxyisoflavone compound is of growing scientific interest due to its attractive biological properties.

Inhibitory effects of flavonoids from Abelmoschus manihot flowers on triglyceride accumulation in 3T3-L1 adipocytes

The 95% EtOH extract from the flowers of Abelmoschus manihot (L.) Medic showed inhibitory activity on TG accumulation in 3T3-L1 preadipocyte. Chemical studies on the active fraction led to the isolation of 14 flavonoids ( 1– 14). To clarify the multi-mechanism of the isolates on preadipocyte differentiation, the levels of TG and FFA and the related role transcription factors (PPARγ, CEBP/α, and ap2) expression were evaluated. At the concentration of 30 μM, compounds 1– 6 and 10– 14 showed inhibitory activity on TG accumulation significantly in mature 3T3-L1 cells. 1, 2, 4– 7, 9, 10, 13, and 14 reduced the level of FFA. At the molecular level, the mRNA expressions of PPARγ, CEBP/α, and ap2 were down-regulated by compounds 1, 5, 9, 12, 13; 1– 8, 10– 14; and 1– 4, 6, 8– 12, 14, respectively. The structure–activity relationships of the 14 flavonoids were also discussed.

Shikonin inhibits fat accumulation in 3T3‐L1 adipocytes

Shikonin, 5,6-dihydroxyflavone-7-glucuronic acid, is the main ingredient of Lithospermum erythrorhizon Sieb. et Zucc, and was reported to have various biological activities including antiinflammatory, anticancer, antimicrobial and others. This study aimed to elucidate, for the first time, the antiobesity activity of shikonin and its mechanism of action. Shikonin was found to inhibit fat droplet formation and triglyceride accumulation in 3T3-L1 adipocytes. The half inhibitory concentration, IC(50), for the inhibition of triglyceride accumulation was found to be 1.1 microM. The expression of genes involved in lipid metabolism, such as FABP4 and LPL, were significantly inhibited following shikonin treatment. Shikonin also inhibited the ability of PPAR gamma and C/EBP alpha, the major transcription factors of adipogenesis, to bind to their target DNA sequences. The expressions of mRNA and protein of PPAR gamma and C/EBPa were significantly down-regulated following shikonin treatment. Among the upstream regulators of adipogenesis, only SREBP1C was found to be down-regulated by shikonin. The results of this study suggest that shikonin down-regulates the expression of SREBP1C and subsequently the expression of PPAR gamma and C/EBP alpha. Together, these changes result in the down-regulation of lipid metabolizing enzymes and reduced fat accumulation.

Ginsenosides Rb1 and Rg1 Suppress Triglyceride Accumulation in 3T3-L1 Adipocytes and Enhance β-Cell Insulin Secretion and Viability in Min6 CellsviaPKA-Dependent Pathways

Ginseng root is known to induce anti-diabetic activity, but the key components involved are unknown. We investigated which major ginsenosides in ginseng enhanced glucose homeostasis by in vitro studies. Rb1 and Rg1 reduced the triglyceride accumulation in 3T3-L1 adipocytes by activating PKA with increased intracellular cAMP. However, the insulin-stimulated glucose uptake was enhanced by Rb1 and Rg1 via activation of phosphatidylinositol-3 kinase. Rb1 and Rg1 promoted glucose-stimulated insulin secretion and cell viability in Min6 cells through PKA which augmented IRS2 expression to enhance insulin/IGF-1 signaling. These results suggest that Rb1 and Rg1 improved glucose homeostasis through the activation of a PKA like glucagon-like peptide-1 receptor agonist.

YM268 increases the glucose uptake, cell differentiation, and mRNA expression of glucose transporter in 3T3-L1 adipocytes.

The purpose of this study was to examine the effects of bis[4-[2,4-dioxo-5-thiazolidinyl)methyl]phenyl]methane (YM-268), a thiazolidinedione derivative, on glucose uptake, adipocyte differentiation through peroxisome proliferator-activated receptor gamma(PPARgamma), and phosphatidylinositol 3-kinase (PI 3-kinase) activity in cultured cells. YM268 and pioglitazone dose-dependently increased the 2-deoxyglucose uptake in 3T3-L1 cells. YM268 facilitated the insulin-stimulated triglyceride accumulation in 3T3-L1 adipocytes and increased the mRNA expression of fatty acid-binding protein. YM268, with and without insulin, increased the mRNA expression of glucose transporter isoforms such as GLUT1 and GLUT4, indicating enhancement of adipocyte differentiation. Additionally, YM268 and pioglitazone showed activity of the PPARgamma ligand, a member of the nuclear receptor superfamily responsible for adipogenesis. To examine the possible involvement of the increased activity of PI 3-kinase in YM268-stimulated glucose uptake, the enzyme activity was estimated by measuring the phosphatidylinositol-3,4,5-trisphosphate (PI-3,4,5-P3) concentration in human monocytic cells. Insulin dose-dependently increased the PI-3,4,5-P3 production but YM268 had no significant effect on the insulin-dependent and -independent PI 3-kinase activation. These results indicate that the mechanism by which YM268 increased glucose uptake, may be accounted for in part by the enhancement of GLUT1 and GLUT4 expression through PPARgamma activation.

β-very low density lipoprotein induces triglyceride accumulation through receptor mediated endocytotic pathway in 3T3-L1 adipocytes

To elucidate the mechanism of triglyceride (TG) accumulation in adipocytes induced by TG-rich lipoproteins, we examined the effect of β-very low density lipoprotein ( β-VLDL) on TG accumulation in 3T3-L1 adipocytes. β-VLDL did not induce TG accumulation in 3T3-L1 preadipocytes but in 3T3-L1 adipocytes. TG accumulation was significantly inhibited by cytochalasin B, an inhibitor of receptor mediated endocytosis. In contrast, cytochalasin B did not inhibit free fatty acid induced TG accumulation in adipocytes. The binding of [ 125I] β-VLDL to preadipocytes was inhibited completely by both β-VLDL and LDL. In sharp contrast, the binding of [ 125I] β-VLDL to adipocytes was inhibited completely by β-VLDL, but partially by LDL. The VLDL receptor mRNA was only expressed in adipocytes. These results suggest that β-VLDL induced TG accumulation in adipocytes may be mediated through the VLDL receptor pathway.