Intracellular mediators of procyanidin-induced lipolysis in 3T3-L1 adipocytes

We have previously reported that grape seed procyanidins stimulate long-term lipolysis on 3T3-L1 fully differentiated adipocytes. To unravel the molecular mechanism by which procyanidins exert this effect, we checked the involvement of two main cellular targets in adipose cells: protein kinase A (PKA) and peroxisome proliferator-activated receptor-gamma (PPAR-gamma). Procyanidin treatment increased intracellular cAMP levels in 3T3-L1 adipocytes, and their lipolytic effect was inhibited by simultaneous treatment with H89, a PKA specific inhibitor. BRL49653, a very highly specific ligand of PPAR-gamma, totally abolished the lipolytic effect of procyanidins. Simultaneous to this long-term lipolytic effect, the mRNA levels of some differentiation adipocyte markers decreased, although there were no changes in the triglyceride content of the cells. BRL49653 did not antagonize the decrements of differentiation markers. These results support a mediation of PPAR-gamma and PKA on the lipolytic effects of procyanidins on 3T3-L1 adipocytes.     

Effects of flavonoids and phenolic acids on the inhibition of adipogenesis in 3T3-L1 adipocytes

Obesity has become a global epidemic in both developed and developing countries, and it is a significant risk factor for various diseases such as diabetes, cancer, heart disease, and hypertension. In the present study, the effect of naturally occurring antioxidants (flavonoids and phenolic acids) on the inhibition of adipogenesis in 3T3-L1 adipocytes was investigated. The results showed that o-coumaric acid and rutin had the highest inhibition on intracellular triglyceride (61.3 and 83.0%, respectively) among 15 phenolic acids and 6 flavonoids tested. However, the oil red o stained material (OROSM) showed that cell number in 3T3-L1 adipocytes was not influenced by those compounds. For glycerol-3-phosphate dehydrogenase (GPDH) activity, the data indicated that o-coumaric acid and rutin had the highest inhibition on GPDH activity (54.2 and 66.8%, respectively) among the compounds tested. o-Coumaric acid and rutin also inhibited the expression of PPARgamma, C/EBPalpha and leptin and then up-regulated expression of adiponectin at the protein level. Some naturally occurring antioxidants efficiently suppressed adipogenesis in 3T3-L1 adipocytes. These results suggest that o-coumaric acid and rutin targeted for adipocyte functions could be effective in improving the symptoms of metabolic syndrome.     

Beta-conglycinin embeds active peptides that inhibit lipid accumulation in 3T3-L1 adipocytes in vitro

Obesity is a worldwide health concern because it is a well-recognized predictor of premature mortality. The objective was to identify soybean varieties that have improved potential to inhibit fat accumulation in adipocytes by testing the effects of soy hydrolysates having a range of protein subunit compositions on lipid accumulation and adiponectin expression in 3T3-L1 adipocytes. The results showed that differences in the protein distribution of 15 soy genotypes led to different potentials for the reduction of fat accumulation. The inhibition of lipid accumulation of soy alcalase hydrolysates in 3T3-L1 adipocytes ranged from 29 to 46%. Soy hydrolysates made from genotypes with 45.3 +/- 3.3% of total protein as beta-conglycinin, on average, showed significantly higher inhibition of lipid accumulation compared to those with 24.7 +/- 1.5% of extracted total protein as beta-conglycinin. Moreover, after in vitro simulated digestion with pepsin-pancreatin of the soy alcalase hydrolysates, 86% of the original activity remained. Adiponectin expression was induced in 3T3-L1 adipocytes treated with 15 soy hydrolysates up to 2.49- and 2.63-fold for high and low molecular weight adiponectin, respectively. The inhibition of lipid accumulation calculated from a partial least squares (PLS) analysis model correlated well with experimental data (R(2) = 0.91). In conclusion, it was feasible to differentiate soy varieties on the basis of the potential of their proteins to reduce fat accumulation using a statistical model and a cell-based assay in vitro. Furthermore, beta-conglycinin embeds more peptides than glycinin subunits that inhibit lipid accumulation and induce adiponectin in 3T3-L1 adipocytes. Therefore, soy ingredients containing beta-conglycinin may be important food components for the control of lipid accumulation in adipose tissue.     

Identification of benzophenone C-glucosides from mango tree leaves and their inhibitory effect on triglyceride accumulation in 3T3-L1 adipocytes

A 70% ethanol-water extract from the leaves of Mangifera indica L. (Anacardiaceae) inhibited triglyceride (TG) accumulation in 3T3-L1 cells. From the active fraction, seven new benzophenone C-glycosides, foliamangiferosides A (1), A(1) (2), A(2) (3), B (4), C(1) (5), C(2) (6), and C(3) (7), together with five known compounds were isolated and the structures were elucidated on the basis of chemical and physicochemical evidence. The effects of these compounds on TG and the free fatty acid level in 3T3-L1 cells were determined, and the structure-activity relationship was discussed. On the basis of the AMPK signaling pathway, several compounds were found to increase the AMPK enzyme expression and down-regulate lipogenic enzyme gene expression such as SREBP1c, FAS, and HSL.     

Metabolic fate of glucose on 3T3-L1 adipocytes treated with grape seed-derived procyanidin extract (GSPE). Comparison with the effects of insulin

In this paper we investigate the effects of a grape seed procyanidin extract (GSPE) on the metabolic fate of glucose in adipocytes. Differentiated 3T3-L1 cells were treated with 140 mg/L GSPE or 100 nM insulin for a short period (1 h, acute treatment) or for a long period (15 h, chronic treatment). 2-Deoxy-[1-(3)H]glucose uptake and [1-(14)C]glucose incorporation into cells, glycogen, and lipid were measured. We found that GSPE mimicked the anabolic effects of insulin but there were several important differences. GSPE stimulated glycogen synthesis less than insulin. After chronic exposure, GSPE induced a higher incorporation of glucose into lipid, mainly due to the increase in glucose directed to glycerol synthesis. Our main conclusions, therefore, are that GSPE has insulinomimetic properties and activates glycogen and lipid synthesis. However, the differences between the effects of GSPE and the effects of insulin indicate that GSPE uses mechanisms complementary to those of insulin signaling pathways to bring about these effects.     

Gallic acid induces apoptosis in 3T3-L1 pre-adipocytes via a Fas- and mitochondrial-mediated pathway

Gallic acid (3,4,5-trihydroxybenzoic acid) is a naturally abundant plant phenolic compound. Our previous studies have shown that some phenolic acids such as gallic acid inhibit cell growth and induce apoptosis in 3T3-L1 pre-adipocytes. However, the molecular mechanism of gallic acid in the induction of cell apoptosis is still unclear. In this study, we investigated the effect of gallic acid on the apoptotic pathway in 3T3-L1 pre-adipocytes. Western blot data revealed that gallic acid stimulated an increase in the protein expression of Fas, FasL, and p53. The ratio of expression levels of pro- and anti-apoptotic Bcl-2 family members was changed by gallic acid treatment. Gallic acid released mitochondrial cytochrome c into the cytosol and subsequently induced the activation of caspase-9 and caspase-3, which were followed by the cleavage of poly(ADP-ribose) polymerase. Pretreatment with a general caspase-9 inhibitor (Z-LEHD-FMK) and caspase-3 inhibitor (Z-DEVD-FMK) prevented gallic acid from inhibiting cell viability in 3T3-L1 pre-adipocytes. The data also indicated that treatment with gallic acid inhibited histone deacetylase activity in 3T3-L1 pre-adipocytes. These results demonstrate that gallic acid induces apoptosis in 3T3-L1 pre-adipocytes through the Fas and mitochondrial pathway. The induction of cell apoptosis by gallic acid may prove to be a pivotal mechanism for decreased pre-adipocyte proliferation.     

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.     

miR-196a-1在3T3-L1脂肪细胞分化中的表达及作用

为研究miR-196a-1在脂肪形成中的作用,从3T3-L1细胞基因组中扩增miR-196a-1前体序列,构建miR-196a-1的表达载体,获得稳定表达miR-196a-1的3T3-L1细胞株,成脂诱导后检测脂肪细胞分化关键转录因子的mRNA表达和脂滴累积情况。结果表明,miR-196a-1在3T3-L1细胞分化过程中表达上调,在诱导分化的第2天达到高峰,并在之后的分化过程中恢复到正常水平;稳定表达miR-196a-1的3T3-L1细胞株中miR-196a-1的持续高水平表达导致脂肪形成关键基因过氧化物酶体增殖物活化受体γ(PPARγ)、CCAAT/增强子结合蛋白α(C/EBPα)和脂蛋白脂酶(LPL)的mRNA水平及脂滴累积明显增加。这些结果指明,miR-196a-1促进3T3-L1脂肪细胞分化,利用所构建的稳定表达miR-196a-1的细胞株可进一步研究miR-196a-1调节脂肪形成的分子机制。     

Antiadipogenic effect of dietary apigenin through activation of AMPK in 3T3-L1 cells

Adipocyte differentiation (adipogenesis) is a complex process including the coordinated changes in hormone sensitivity and gene expression in response to various stimuli. Natural compounds are known to be involved in the regulation of this process. Here we investigated the effects of dietary apigenin, a plant flavonoid, on adipogenesis. Apigenin suppressed adipocyte differentiation of mouse adipocytic 3T3-L1 cells and reduced the accumulation of intracellular lipids. Quantitative PCR and Western blot analyses revealed that apigenin decreased the levels of peroxisome proliferator-activated receptor γ and its target genes such as fatty acid binding protein 4 (aP2) and stearoyl-CoA desaturase. Apigenin decreased or had no effect on the expression of lipolytic genes such as adipose triglyceride lipase, hormone sensitive lipase, and monoacyl glyceride lipase, thereby reducing glycerol release from adipocytes. Noteworthily, apigenin activated 5'-adenosine monophosphate-activated protein kinase (AMPK) in an apigenin dose-dependent manner, which activation is known to suppress adipogenesis. These results provide a novel insight into the molecular mechanism involved in the action of apigenin: the apigenin-induced activation of AMPK leads to decreased expression of adipogenic and lipolytic genes, thus suppressing adipogenesis in 3T3-L1 cells. Thus, dietary apigenin may contribute to lower body-fat content and body-weight gain through the activation of AMPK.     

Insulin-releasing properties of a series of cinnamic acid derivatives in vitro and in vivo

Cinnamic acid derivatives are naturally occurring substances found in fruits, vegetables, and flowers and are consumed as dietary phenolic compounds. In the present study, cinnamic acid and its derivatives were evaluated for insulin secreting activity in perfused rat pancreas and pancreatic beta-cells (INS-1) as well as an increase in [Ca(2+)]i in vitro. The presence of m-hydroxy or p-methoxy residues on cinnamic acid was a significantly important substituent as an effective insulin releasing agent. The introduction of p-hydroxy and m-methoxy-substituted groups in cinnamic acid structure (ferulic acid) displayed the most potent insulin secreting agent among those of cinnamic acid derivatives. In particular, the stimulatory insulin secreting activities of test compounds were associated with a rise of [Ca(2+)]i in INS-1. In perfused rat pancreas, m-hydroxycinnamic acid, p-methoxycinnamic acid, and ferulic acid (100 microM) significantly stimulated insulin secretion during 10 min of administration. The onset time of insulin secretion of those compounds was less than 1 min and reached its peak at 4 min that was about 2.8-, 3.3-, and 3.4-fold of the baseline level, respectively. Intravenous administration of p-methoxycinnamic acid and ferulic acid (5 mg/kg) significantly decreased plasma glucose and increased insulin concentration in normal rats and maintained its level for 15 min until the end of experiment. Meanwhile, m-hydroxycinnamic acid induced a significant lowering of plasma glucose after 6 min, but the effects were transient with plasma glucose concentration, rapidly returning to basal levels. Our findings suggested that p-methoxycinnamic acid and ferulic acid may be beneficial for the treatment of diabetes mellitus because they regulated blood glucose level by stimulating insulin secretion from pancreatic beta-cells.     

The apoptotic effect of green tea (-)-epigallocatechin gallate on 3T3-L1 preadipocytes depends on the Cdk2 pathway

This study was designed to investigate the effect of green tea catechins, especially (-)-epigallocatechin gallate (EGCG), on the apoptosis of 3T3-L1 preadipocytes. Preadipocyte apoptosis as indicated by formation of DNA fragments was induced by EGCG in dose-dependent manners. While EGCG was demonstrated to decrease Cdk2 expression and activity and increase caspase-3 activity, overexpression of Cdk2 and treatment with the caspase-3 inhibitor respectively prevented preadipocytes from induction of DNA fragmentation and caspase-3 activity by doses of 100-400 muM of EGCG. This suggests the Cdk2- and caspase-3-dependent apoptotic effects of EGCG. Moreover, EGCG was more effective than EC, ECG, and EGC in changing the apoptotic signals. Results of this study may relate to the mechanism by which EGCG modulates body weight.     

Inhibitory effect of phenolic acids on the proliferation of 3T3-L1 preadipocytes in relation to their antioxidant activity

Obesity is an important topic in the world of public health and preventive medicine. Inhibition of preadipocyte proliferation plays an important role in the mechanisms of proposed antiobesity. In this in vitro study, the inhibitory effect of phenolic acids on 3T3-L1 preadipocytes was evaluated, and a relationship analysis was then conducted. The results showed that the addition of phenolic acids to the growth medium decreased the cell population growth of 3T3-L1 preadipocytes. The IC50 values of chlorogenic acid, gallic acid, o-coumaric acid and m-coumaric acid on 3T3-L1 preadipocytes were 72.3, 43.3, 48.2, and 49.2 microM, respectively. A relationship analysis indicated that there is a significant linear correlation between the influence of phenolic acids on cell population growth and their antioxidant activity (r = 0.77, p < 0.01). The cell cycle assay indicated that the treatment of 3T3-L1 preadipocytes with chlorogenic acid, o-coumaric acid, and m-coumaric acid caused cell cycle arrest in the G1 phase. Gallic acid did not affect the cell cycle profile; however, it increased the number of apoptotic cells (sub-G1 phase) in a time- and dose-dependent manner. Annexin V-fluorescein isothiocyanate (FITC)-propidium iodide (PI) apoptosis flow cytometric assay showed that gallic acid increased the number of early apoptotic (annexin V-FITC+/PI-) and late apoptotic cells (annexin V-FITC+/PI+) but not necrotic cells (annexin V-FITC-/PI+). The treatment of cells with gallic acid caused the loss of mitochondrial membrane potential (delta psi(m)). These results indicate that the inhibition of preadipocyte population growth by some phenolic acids might have further implication in in vivo antiobesity effects.     

Inhibitory effect of natural phenolic lipids upon NAD-dependent dehydrogenases and on triglyceride accumulation in 3T3-L1 cells in culture

Alkylresorcinols are phenolic lipids present at levels of 0.03-0.15% in wheat and rye grains and almost 10 times higher in respective bran products. Despite numerous studies on the influence of dietary fibers on the regulation of energy metabolism, this issue still remains controversial. The objective of our current studies was to investigate whether 5-n-alk(en)ylresorcinols, natural phenolic components of high fiber human diets, may be considered as natural regulators of excessive fat accumulation. Our studies revealed that 5-n-alk(en)ylresorcinols isolated from wheat and rye bran inhibit glycerol-3-phosphate dehydrogenase, the key enzyme in triglyceride synthesis in adipocytes, specifically and effectively. Further in vitro studies showed that these compounds also prevent triglyceride accumulation in 3T3-L1 cells. The most effective compound in both systems was 5-n-heneicosylresorcinol. The results indicate that the potential to prevent triglyceride accumulation increases with the hydrophobicity of the phenolic inhibitor.     

Effect of cinnamic acid on soil microbial characteristics in the cucumber rhizosphere

In order to elucidate the effect of allelochemicals on soil microbial characteristics in the cucumber rhizosphere, the soil microbial biomass and respiration, community functional diversity and RAPD marker diversity as affected by exogenous cinnamic acid were studied. Exogenous cinnamic acid increased soil microbial respiration and the metabolic quotient, but decreased soil microbial biomass-C. Soil microbial community functional diversity and genetic diversity (as indicated by RAPD markers) were also significantly altered by exogenous cinnamic acid. These results suggest that allelochemicals can change soil microbial genetic diversity, biological activity and microbial metabolic activity, which alter soil microbial ecology and accordingly affect the growth of cucumber with accumulation in the soil of allelochemicals.     

T3x须家河组砂岩饱水作用下水岩相互作用规律研究

为分析研究三峡库区水位升降作用对库岸边坡稳定性的影响作用,通过研究三峡库区内分布广泛的须家河组(T3x)砂岩在天然状态、饱水1,2,5,10,20,30 d等不同饱水时间下的物理力学性质变化规律性,测定其不同饱水时间的含水率、吸水率及抗剪强度,预测分析砂岩抗剪强度、砂岩含水率、砂岩吸水率随不同饱水时间的变化规律以及砂岩抗剪强度随含水率的变化规律等.试验结果表明,砂岩C,Φ值与饱水时间分别呈对数和指数关系,砂岩含水率ω、吸水率ωa与饱水时间呈对数关系.应用这一规律,对于在评价库水位升降作用对岸坡稳定性的影响作用时进行岸坡岩土体的物理力学参数取值有十分重要的理论和实际意义.     

A Laboratory Study of the Interaction of NH3 and NO2 with Sea Salt Particles

It is known from the literature that the reaction 2 NO₂(aq) + Cl-(aq) → NO₃-(aq) + ClNO(g) takes place in the presence of NO₂ and sea salt aerosol. This work indicates that the reaction is first order in Cl- and second order in NO₂(aq) and the rate coefficient was determined to be 8.9 ± 2.1) × 10¹⁰ M-² s-¹ at 293 K. The study shows that the formation rate of nitrate in liquid sea salt aerosol is not influenced by pH in the range between 5.1 and 7.5. The uptake of ammonia in liquid sea salt particles in the present experimental system is governed by the rate of the reaction CO₂(aq) + H₂O → H₂CO₃. The addition of NH₃ to NO₂ and dry sea salt particles does not change the extent of nitrate formation in the particles. The dry aerosol shows an unexpected content of ammonium which increases with decreasing relative humidity. The nitrate formation in frozen sea salt particles at 266 K exposed to only NO₂, is about 5 times lower than for the corresponding droplets at 293 K.     

添加有机酸对土壤镉形态转化及苋菜镉积累的影响

植物根系分泌的低分子量有机酸能够与土壤中的镉形成镉–有机酸复合体,从而影响根际镉的移动性。本文通过添加有机酸对土壤镉形态转化的研究,阐明有机酸与镉生物积累的关系。采用盆栽试验及土壤培养等方法,研究了添加苹果酸、柠檬酸对赤红壤和黄棕壤中镉的形态转化以及超积累型苋菜天星米镉生物积累的影响。结果表明,与Cd 25 mg/kg处理比较,Cd 25 mg/kg＋苹果酸、Cd 25mg/kg＋柠檬酸处理对苋菜生物量未产生影响,但显著增加苋菜根系及地上部镉含量;添加苹果酸、柠檬酸处理显著降低土壤专性吸附态Cd含量,却显著增加了交换态Cd、碳酸盐结合态Cd和有机结合态Cd含量。说明添加苹果酸、柠檬酸还能够通过影响土壤镉形态转化而促进苋菜对镉的积累。