Trigonelline attenuates the adipocyte differentiation and lipid accumulation in 3T3-L1 cells

Trigonelline is a natural alkaloid mainly found in Trigonella Foenum Graecum (fenugreek) Fabaceae and other edible plants with a variety of medicinal applications. Therefore, we investigated the molecular mechanism of trigonelline (TG) on the inhibition of adipocyte differentiation and lipid accumulation in 3T3-L1 cells. Trigonelline suppressed lipid droplet accumulation in a concentration (75 and 100 μM) dependent manner. Treatment of adipocyte with of TG down regulates the peroxisome proliferator-activated receptor (PPARγ) and CCAAT element binding protein (C/EBP-α) mRNA expression, which leads to further down regulation of other gene such as adiponectin, adipogenin, leptin, resistin and adipocyte fatty acid binding protein (aP2) as compared with respective control cells on 5th and 10th day of differentiation. Further, addition of triognelline along with troglitazone to the adipocyte attenuated the troglitazone effects on PPARγ mediated differentiation and lipid accumulation in 3T3-L1 cells. Trigonelline might compete against troglitazone for its binding to the PPARγ. In addition, adipocyte treated with trigonelline and isoproterenol separately. Isoproterenol, a lipolytic agent which inhibits the fatty acid synthase and GLUT-4 transporter expression via cAMP mediated pathway, we found that similar magnitude response of fatty acid synthase and GLUT-4 transporter expression in trigonelline treated adipocyte. These results suggest that the trigonelline inhibits the adipogenesis by its influences on the expression PPARγ, which leads to subsequent down regulation of PPAR-γ mediated pathway during adipogenesis. Our findings provide key approach to the mechanism underlying the anti-adipogenic activity of trigonelline.     

Thiazolidinediones exhibit different effects on preadipocytes isolated from rat mesenteric fat tissue and cell line 3T3-L1 cells derived from mice

The effects of PPAR-gamma agonists, thiazolidinediones (TZDs), on preadipocytes isolated from rat mesenteric adipose tissue and murine cell line 3T3-L1 were compared using an in vitro cell culture system. After each cell formed a confluent monolayer under appropriate medial conditions, pioglitazone or troglitazone was applied at 10 microM to each medium for cell maturation. We observed morphological changes in each cell, especially the accumulation of lipid droplets in the cytoplasm, during the culture periods. At the end of culture, DNA content, triglyceride (TG) content and glycerol-3-phosphate dehydrogenase (GPDH) activity were determined. Adiponectin concentrations in each culture medium were also measured during appropriate experimental periods. Application of TZDs increased the DNA content, TG accumulation and GPDH activity in the 3T3-L1 cells but not in the mesenteric adipocytes. Although TG accumulation was unchanged, the number of lipid particles was decreased and the size of lipid particles in the mesenteric adipocytes was increased by TZD application. Although the TZDs increased adiponectin release from the 3T3-L1 cells, adiponectin release from mesenteric adipocytes was suppressed (P<0.05). Thus, the effects of TZDs differed between the primary culture of mesenteric adipose cells and the line cell culture of 3T3-L1 cells. The source of adipocytes is an important factor in determining the action of TZDs in vitro, and particular attention should be paid when evaluating the effect of PPAR-gamma agonists on adipose tissues.     

Kibizu concentrated liquid suppresses the accumulation of lipid droplets in 3T3-L1 cells

Adipocyte size is closely related to the occurrence of diabetes, metabolic syndrome, and insulin resistance. Thus, researchers are searching for active substances that function to reduce adipocyte size. In the present study, we focused on sugar cane vinegar, Kibizu, and evaluated the function of Kibizu to reduce adipocyte size by using an in vitro model system, because people in Amami Oshima famous for longevity regularly consume Kibizu. Results showed that Kibizu treatment significantly reduced the size and number of lipid droplets in 3T3-L1 cells, relative to treatment with Kurozu, another traditional vinegar. Results of an extraction experiment suggest that the active components in Kibizu are lipophilic and hydrophobic. In addition, an in vivo experiment on rats treated with Kibizu showed that the active components were contained in large vein blood. Results of an additional in vivo experiment suggest that metabolites generated by Kibizu-treated rats are primarily contained or modified specifically in the large vein blood.     

Effect of sterols and fatty acids on growth and triglyceride accumulation in 3T3-L1 cells

Epidemiologic studies suggest a role of dietary fat in the development of obesity. Populations that consume Western diets have a higher incidence of obesity than do those that consume a vegetarian type diet such as Asians. Because dietary fats are made up mostly of triglyceride with minor lipids such as sterols, the objective of this study was to examine the effect of different fatty acids, the main component of triglycerides, and sterols on cell growth and triglyceride accumulation in 3T3-L1 cells. These cells are being used as an in vitro model for studying obesity because upon differentiation in culture they accumulate triglycerides. Cells were seeded at 5,000 cells/cm(2) and supplemented with 0, 3, 10, or 30 microM of oleic acid, elaidic acid, or docosahexaenoic acid (DHA). Similarly, cells were supplemented with 0, 2, 8, or 16 microM of cholesterol, beta-sitosterol (SIT), or campesterol. Cell growth was measured by cell counting. Cellular triglycerides were measured by the Oil Red O method. In some experiments, fatty acids were combined with sterols and growth and triglyceride content were assessed as described. Both DHA and SIT had inhibitory effects on 3T3-L1 cell growth. However, SIT was more potent than DHA in this regard. The combination of SIT and oleic acid was the most potent in inhibiting cell growth and increasing cellular triglyceride content. It is concluded that cell growth and triglyceride accumulation in 3T3-L1 cells is influenced by fatty acid and sterols. When used alone, DHA and SIT inhibit cell growth. SIT was more effective in this process than was DHA. There was an interaction between fatty acids and sterols. The most effective combination inhibiting cell growth and triglyceride concentration was the combination of SIT and oleic acid. This combination reduced cell growth and increased triglyceride accumulation. These data suggest that diets rich in both monounsaturated fatty acids and phytosterols may play a role in controlling obesity.     

Regulation of lipid accumulation in 3T3-L1 cells: insulin-independent and combined effects of fatty acids and insulin

The insulin-independent and combined effects of fatty acids (FA; linoleic and oleic acids) and insulin in modulating lipid accumulation and adipogenesis in 3T3-L1 cells was investigated using a novel protocol avoiding the effects of a complex hormone 'induction' mixture. 3T3-L1 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) plus serum (control) or in DMEM plus either 0.3 mmol/l linoleic or oleic acids with 0.3 mmol/l FA-free bovine serum albumin in the presence or absence of insulin. Cells were cultured for 4 to 8 days and cell number, lipid accumulation, peroxisome proliferator-activated receptor-gamma (PPAR-γ) and glucose transporter 4 (GLUT-4) protein expression were determined. Cell number appeared to be decreased in comparison with control cultures. In both oleic acid and linoleic acid-treated cells, notably in the absence (and presence) of insulin, oil-red O stain-positive cells showed abundant lipid. The percentage of cells showing lipid accumulation was greater in FA-treated cultures compared with control cells grown in DMEM plus serum (P < 0.001). Treatment with both linoleic and oleic acid-containing media evoked higher levels of PPAR-γ than observed in control cultures (P < 0.05). GLUT-4 protein also increased in response to treatment with both linoleic and oleic acid-containing media (P < 0.001). Lipid accumulation in 3T3-L1 cells occurs in response to either oleic or linoleic acids independently of the presence of insulin. Both PPAR-γ and GLUT-4 protein expression were stimulated. Both proteins are considered markers of adipogenesis, and these observations suggest that these cells had entered the physiological state broadly accepted as differentiated. Furthermore, 3T3-L1 cells can be induced to accumulate lipid in a serum-free medium supplemented with FA, without the use of induction protocols using complex hormone mixtures. We have demonstrated a novel model for the study of lipid accumulation that will improve the understanding of adipogenesis in adipocyte lineage cells.     

Adipogenetic effects of retrofractamide A derivatives in 3T3-L1 cells

In a previous study, retrofractamide A from the fruit of Piper chaba was shown to promote adipogenesis in 3T3-L1 cells. In the present study, retrofractamide A and its derivatives were synthesized, and their adipogenetic effects in 3T3-L1 cells were examined. Among the tested compounds, an amide composed of 9-(3′,4′-methylenedioxyphenyl)-nona-2E,4E,8E-trienoic acid and an n-butyl or n-pentyl amine showed strongest activity. Moreover, the amide with the n-pentyl amine moiety significantly increased the uptake of 2-deoxyglucose into the cells, and also increased the mRNA levels of adiponectin, peroxisome proliferator-activated receptor γ2 (PPARγ2), glucose transporter 4 (GLUT4), fatty acid-binding protein (aP2), and CCAAT/enhancer-binding protein (C/EBP) α and β in a similar manner as the PPARγ agonist troglitazone, although it had less agonistic activity against PPARγ.     

Anti-obesity effects of kaempferol by inhibiting adipogenesis and increasing lipolysis in 3T3-L1 cells

Journal of Physiology and Biochemistry - Kaempferol is a natural flavonoid widely found in fruits, vegetables, and tea. Kaempferol possesses beneficial biological properties such as...     

Stimulative effects of insulin on Toxoplasma gondii replication in 3T3-L1 cells

The influence of insulin and 2-deoxy-glucose (D-glucose) on the intracellular protozoan Toxoplasma gondii replication in 3T3-L1 cells was investigated. Insulin and D-glucose had a dose-responsive mitogenic effect on intracellular T. gondii replication and development in 3T3-L1 cells. Insulin concentrations between 10(-2) and 10(-1) microg/ml combination of 4.5 g/l D-glucose in DMEM medium gave maximum stimulus to T. gondii replication. The number of tachyzoites increased rapidly, with the growth peaking typically on day 3 or 4 of culture, and then declining quickly. However, insulin, in the absence of d-glucose, had comparably less effect on T. gondii growth than two of their combination. d-glucose concentrations significantly affected the tachyzoite replication and appear to be indispensable for maintaining the host 3T3-L1 cells.     

Inhibitory effects of thioethers on fatty acid synthase and 3T3-L1 cells

Thioethers are the main flavor compounds found in Liliaceae Allium vegetables and have been shown to have beneficial effects against several diseases correlated with metabolic syndrome. The inhibitory effects of six thioethers on fatty acid synthase (FAS) were investigated. Dose-dependent and time-dependent inhibitions of FAS by one trisulfide and two disulfides were revealed. Diallyl trisulfide (DATS, IC₅₀ = 8.37 μM) was the most active of these thioethers. Inhibition kinetics, substrate protection analysis, and stoichiometric assay revealed that DATS interacted with both essential sulfhydryl groups on the acyl-carrier protein and β-ketoacyl synthase domain of FAS to inactivate the enzyme. The inactivation by DATS represented affinity-labeling kinetics. The active thioethers also inhibited the differentiation and lipid accumulation of 3T3-L1 preadipocytes, and the effect was related to their inhibition of FAS. It is suggested that the inhibition on FAS by thioethers and Allium vegetables is an important factor for their effects against metabolic syndrome.     

27-Hydroxycholesterol suppresses lipid accumulation by down-regulating lipogenic and adipogenic gene expression in 3T3-L1 cells

Cholesterol oxidation products (oxycholesterols) are produced from cholesterol by automatic and/or enzymatic oxidation of the steroidal backbone and side-chain. Oxycholesterols are present in plasma and serum, suggesting that oxycholesterols are related to the development and progression of various diseases. However, limited information is available about the absolute amounts of oxycholesterols in organs and tissues, and the physiological significance of oxycholesterols in the body. In the present study, we quantified the levels of 13 oxycholesterols in white adipose tissue (WAT) of mice and then evaluated correlations between each oxycholesterol level and WAT weight. The sum of the levels of 13 oxycholesterols in WAT (white adipose tissue) was 15.9 ± 3.4 μg/g of WAT weight and approximately 1 % of cholesterol level. Among oxycholesterols, the levels of 27-hydroxycholesterol (27-OH), an endogenous oxycholesterol produced by enzymatic oxidation, and the relative WAT weights were significantly negatively correlated. Next, we evaluated the effects of 27-OH on lipogenesis and adipogenesis in 3T3-L1 cells. TO901317 (TO), a potent and selective agonist for LXRα, significantly increased intracellular TAG contents, while 27-OH significantly reduced the contents to half when compared with control (DMSO) and completely abolished the effect of TO. In addition, 27-OH significantly reduced the mRNA levels of lipogenic (LXRα and FAS) and adipogenic genes (PPARγ and aP2) during adipocyte maturation of 3T3-L1 cells. In conclusion, our results indicate that 27-OH suppresses lipid accumulation by down-regulating lipogenic and adipogenic gene expression in 3T3-L1 cells.     

Adenovirus transduction of 3T3-L1 cells

3T3-L1 cells offer an excellent model system for studies of differentiation and biochemistry of fat cells. However, these cells are limited in their utility by the low efficiency with which DNA can be introduced by transfection. Gene delivery by viral vectors, particularly adenovirus, has proven a powerful means for introduction of genes into certain cell types. Furthermore, adenovirus transduction has been used to study mechanisms involved in the differentiation of 3T3-L1 cells into mature fat cells. We show in this study that 3T3-L1 cells are inefficiently transduced by adenovirus. The potential advantages offered by adenovirus transduction led us to examine methods designed to enhance transduction of 3T3-L1 cells by adenovirus. Of these methods, polylysine-mediated enhancement demonstrates considerable promise because it permits up to 100% of cells to be transduced and because it does not inhibit differentiation of 3T3-L1 cells. -- Orlicky D. J., and J. Schaack. Adenovirus transduction of 3T3-L1 cells. J. Lipid Res. 2001. 42: 460--466.     

Alpha-lipoic acid attenuates adipocyte differentiation and lipid accumulation in 3T3-L1 cells via AMPK-dependent autophagy

Aims

During the adipocyte differentiation, some intracellular organelles are degraded and instead lipid droplets are gradually accumulated in the cytoplasm for energy storage. Autophagy, a self-eating process, has been implicated in the removal of intracellular components in adipogenesis, but its mechanism is poorly understood. In this work we examined how α-lipoic acid modulates the autophagic process during the adipocyte differentiation.

Main methods

3T3-L1 pre-adipocytes were differentiated in the medium containing insulin, dexamethasone, and 1-methyl-3-isobutylxanthine. Lipid contents in adipocytes were determined by Oil-Red O staining. Autophagy was evaluated by Western blotting, accumulation of acidic vacuoles in cells.

Key findings

We observed that formation of LC3-II, an indicative marker for autophagy, was greatly down-regulated at the beginning stage of differentiation, but it was gradually increased with respect to earlier differentiation time. In addition, ATG5-12 conjugates were similarly produced, and acidic autophagic vacuoles were greatly elevated at the earlier stages of differentiation. Furthermore, α-lipoic acid deteriorated the intracellular accumulation of lipid droplets by blocking the production of acidic autophagic vacuoles, LC3-II, and other autophagy-related proteins during the adipocyte differentiation and influenced expression of adipocyte-stimulating factors. It also specifically suppressed activation of AMPK, an essential modulator for autophagy, at the earlier step of adipocyte differentiation.

Significance

These data suggest that α-lipoic acid significantly attenuates adipocyte differentiation via the direct modulation of intracellular degradation process and consequently decrease intracellular fat deposit of adipocytes.     

Differential expression and accumulation of 14-3-3 paralogs in 3T3-L1 preadipocytes and differentiated cells

The 14-3-3 protein family interacts with more than 2000 different proteins in mammals, as a result of its specific phospho-serine/phospho-threonine binding activity. Seven paralogs are strictly conserved in mammalian species. Here, we show that during adipogenic differentiation of 3T3-L1 preadipocytes, the level of each 14-3-3 protein paralog is regulated independently. For instance 14-3-3β, γ, and η protein levels are increased compared to untreated cells. In contrast, 14-3-3ε protein levels decreased after differentiation while others remained constant. In silico analysis of the promoter region of each gene showed differences that explain the results obtained at mRNA and protein levels.     

Stimulation of FA and phosphatase-1 activities by insulin in 3T3-L1 cells

The phosphatase-1 activator FA and phosphatase-1 were assayed in 3T3-L1 cells exposed to insulin. The cytosolic FA activity was transiently stimulated (7-8-fold) 1 and 2 min after exposure to 10(-8) M insulin and returned to control values within 5-10 min. Cytosolic phosphatase-1 (assayed after trypsin treatment) was activated (120-140% of controls) between 2 and 5 min and returned to control values within 10 min. Insulin effects were dose-dependent, with maximum stimulation of both activities at 10(-8) M insulin. The possibility that FA and other kinases mediate phosphatase activation by insulin is discussed.