Anti-adipogenic effect of dioxinodehydroeckol via AMPK activation in 3T3-L1 adipocytes

Dioxinodehydroeckol (DHE) isolated from Ecklonia cava, has previously been investigated for its inhibition of the differentiation of 3T3-L1 preadipocytes into adipocytes. Levels of lipid accumulation were measured, along with changes in the expression of genes and proteins associated with adipogenesis and lipolysis. Confluent 3T3-L1 preadipocytes in medium with or without different concentrations of DHE for 7 days were differentiated into adipocytes. Lipid accumulation was quantified by measuring direct triglyceride contents and Oil-Red O staining. The expression of genes and proteins associated with adipogenesis and lipolysis was measured using RT-PCR, quantitative real-time RT-PCR and Western blotting analysis. It was found that the presence of DHE significantly reduced lipid accumulation and down-regulated the expression of peroxisome proliferator-activated receptor-γ (PPARγ), sterol regulatory element-binding protein 1 (SREBP1) and CCAAT/enhancer-binding proteins (C/EBPα) in a dose-dependent manner. Moreover, DHE suppressed regulation of the adipocyte-specific gene promoters such as fatty acid binding protein (FABP4), fatty acid transport protein (FATP1), fatty acid synthase (FAS), lipoprotein lipase (LPL), acyl-CoA synthetase 1 (ACS1), leptin, perilipin and HSL compared to control adipocytes. The specific mechanism mediating the effects of DHE was confirmed by activation of phosphorylated AMP-activated protein kinase (pAMPK). Therefore, these results suggest that DHE exerts anti-adipogenic effect on adipocyte differentiation through the activation and modulation of the AMPK signaling pathway.     

Biophysical Assessment of Human Aquaporin-7 as a Water and Glycerol Channel in 3T3-L1 Adipocytes

The plasma membrane aquaporin-7 (AQP7) has been shown to be expressed in adipose tissue and its role in glycerol release/uptake in adipocytes has been postulated and correlated with obesity onset. However, some studies have contradicted this view. Based on this situation, we have re-assessed the precise localization of AQP7 in adipose tissue and analyzed its function as a water and/or glycerol channel in adipose cells. Fractionation of mice adipose tissue revealed that AQP7 is located in both adipose and stromal vascular fractions. Moreover, AQP7 was the only aquaglyceroporin expressed in adipose tissue and in 3T3-L1 adipocytes. By overexpressing the human AQP7 in 3T3-L1 adipocytes it was possible to ascertain its role as a water and glycerol channel in a gain-of-function scenario. AQP7 expression had no effect in equilibrium cell volume but AQP7 loss of function correlated with higher triglyceride content. Furthermore it is also reported for the first time a negative correlation between water permeability and the cell non-osmotic volume supporting the observation that AQP7 depleted cells are more prone to lipid accumulation. Additionally, the strong positive correlation between the rates of water and glycerol transport highlights the role of AQP7 as both a water and a glycerol channel and reflects its expression levels in cells. In all, our results clearly document a direct involvement of AQP7 in water and glycerol transport, as well as in triglyceride content in adipocytes.     

2-(2-Bromophenyl)-formononetin and 2-heptyl-formononetin are PPARγ partial agonists and reduce lipid accumulation in 3T3-L1 adipocytes

Isoflavones are bioactive compounds that have been shown to decrease lipid accumulation in vitro. However, the knowledge of the isoflavone formononetin is limited. The aim of the study was to assess the effects of formononetin and its two synthetic analogues, 2-(2-bromophenyl)-formononetin and 2-heptyl-formononetin, on lipid accumulation in 3T3-L1 adipocytes and investigate possible mechanisms. Formononetin and the two analogues were added day 0–8 or day 8–10 of the differentiation period, and lipid accumulation, glycerol release and gene expression were measured. Additionally, competitive peroxisome proliferator-activated receptor (PPAR)-γ binding assay, PPARγ transactivation assay and Western blot for phosphorylated AMP-activated protein kinase (AMPK) were performed. Chronic treatment (day 0–8) with formononetin increased lipid accumulation, whereas the two analogues decreased lipid accumulation partly due to decreased differentiation. The two analogues, but not formononetin, also decreased lipid content in mature adipocytes. 2-Heptyl-formononetin increased glycerol release and lipolytic gene expression and decreased lipogenic gene expression. Formononetin did not bind to or activate PPARγ whereas both analogues bound to the receptor and behaved as PPARγ partial agonists in the transactivation assay. Neither of the compounds affected phosphorylation of AMPK. In conclusion, the analogues of formononetin decreased lipid accumulation possibly in part by acting as PPARγ partial agonists.     

MicroRNA-425 controls lipogenesis and lipolysis in adipocytes

An increasing number of studies have demonstrated that some microRNAs participate in the regulation of growth and development of adipocytes. The present study shows that microRNA-425-5p (miR-425) is a novel strong regulator of adipogenesis and adipolysis in adipocytes. Forced expression of miR-425 in mice promoted body fat accumulation and the development of obesity due to high-fat intake, whereas silencing miR-425 prevented mice from being obese. Mechanically, the expression of miR-425 is controlled by PPARγ during the adipogenesis process in adipocytes. MiR-425 overexpression resulted in a reduction in the proliferation of 3t3-L1 pre-adipocytes but significantly accelerated cellular adipogenic differentiation. Mapk14, a negative regulator of adipogenesis, was predicted and confirmed as a real target gene of miR-425. Moreover, knocking down miR-425 remarkably intensified intracellular lipolysis and promoted lipid oxidation, which is related to the activation of AMPK, a monitor for intracellular energy balance. MiR-425 activated AMPK not only by decreasing cellular ATP concentrations but also by targeting the gene of Cab39, which is an upstream co-activator of AMPK. The findings of the present study suggest that miR-425 could control adipogenesis and adipolysis in adipocytes by simultaneously triggering multidirectional targets.     

Euphorbiasteroid,a component of Euphorbia lathyris L., inhibits adipogenesis of 3T3‐L1 cells via activation of AMP‐activated protein kinase

The purpose of this study is to investigate the effects of euphorbiasteroid, a component of Euphorbia lathyris L., on adipogenesis of 3T3‐L1 pre‐adipocytes and its underlying mechanisms. Euphorbiasteroid decreased differentiation of 3T3‐L1 cells via reduction of intracellular triglyceride (TG) accumulation at concentrations of 25 and 50 μM. In addition, euphorbiasteroid altered the key regulator proteins of adipogenesis in the early stage of adipocyte differentiation by increasing the phosphorylation of AMP‐activated protein kinase (AMPK) and acetyl‐CoA carboxylase. Subsequently, levels of adipogenic proteins, including fatty acid synthase, peroxisome proliferator‐activated receptor‐γ and CCAAT/enhancer‐binding protein α, were decreased by euphorbiasteroid treatment at the late stage of adipocyte differentiation. The anti‐adipogenic effect of euphorbiasteroid may be derived from inhibition of early stage of adipocyte differentiation. Taken together, euphorbiasteroid inhibits adipogenesis of 3T3‐L1 cells through activation of the AMPK pathway. Therefore, euphorbiasteroid and its source plant, E. lathyris L., could possibly be one of the fascinating anti‐obesity agent. Copyright © 2015 John Wiley & Sons, Ltd.