Anti-adipogenic Properties Research Articles

Carnosic acid (CA) prevents lipid accumulation in hepatocytes through the EGFR/MAPK pathway

Carnosic acid (CA), found in rosemary, has been reported to have antioxidant and anti-adipogenic properties. We recently demonstrated that CA protects against steatosis in ob/ob mice. In the present report, we investigated the molecular mechanism by which CA inhibits lipids accumulation both in vivo and in vitro. In the in vivo study, ob/ob mice were fed a standard chow diet with or without CA for 5 weeks, then their hepatocyte lipid accumulation was determined. The serum concentrations of cytokines, the levels of lipid regulatory mediators, and the hepatic metabolic and signaling molecules were also evaluated. In the in vitro study, HepG2 cells were used to further clarify the effects of CA on cellular lipid accumulation and to confirm the signaling pathways involved in these effects. CA significantly reduced hepatocyte lipid accumulation. This effect was associated with repressed levels of hepatic PPARγ, reduced expression of inflammatory cytokines such as IL-1β, IL-12, IL-17, IFN-γ, MCP-1, and MIP-1β, and increased ATP, acetyl CoA, NAD(P)(+), and NAD(P)H. Other signaling molecules, such as EGFR, MAPK, AMPK, and ACC, which regulate lipid metabolism, were activated in mice fed the CA diet. CA inhibited palmitate-induced cellular lipid accumulation and stimulated the phosphorylation of both EGFR and MAPK. Pretreatment with either the EGFR inhibitor AG1478 or the MEK-specific inhibitor U0126 abolished the effects of CA on cellular lipid accumulation and decreased both the protein expression and activity of PPARγ. EGFR/MAPK signaling plays an important role in the inhibitory effect of CA on hepatocyte lipid accumulation.

A potential role of IL-6 in the chito-oligosaccharide-mediated inhibition of adipogenesis

Recent studies have suggested that chito-oligosaccharides can have anti-adipogenic properties. The objectives of the present study were to evaluate the anti-adipogenic potential of four different chito-oligosaccharides (molecular weight (MW) < 1000, 1000-3000, 3000-5000 and 5000-10,000 Da) and to identify molecular mechanisms underlying the chito-oligosaccharide-mediated inhibition of adipogenesis. Mouse 3T3-L1 cells were allowed to differentiate in the presence of chito-oligosaccharide. At day 8 post-induction of differentiation, lipid accumulation, free glycerol release and the quantitative expression of adipogenic marker genes were evaluated. Chito-oligosaccharides had concentration- and MW-dependent inhibitory effects on lipid accumulation (P < 0·001 and < 0·05, respectively), as well as a concentration-dependent effect (P < 0·001) on free glycerol release and the expression of adipogenic marker genes. The 5000-10,000 Da chito-oligosaccharide was selected for subsequent molecular studies. A panel of forty-four lipid metabolic pathway-specific genes was analysed by quantitative real-time PCR. Chito-oligosaccharide-mediated inhibition of adipogenesis was associated with the up-regulation of the IL-6 gene at all concentrations of chito-oligosaccharide examined and the PG-endoperoxide synthase 2 (PTGS2) gene at higher concentrations of chito-oligosaccharide. The effect of chito-oligosaccharide on gene expression was validated by measuring IL-6 protein concentrations in the media. Finally, an IL-6 promoter assay was developed to characterise the effect of chito-oligosaccharide on the transcriptional activity of the IL-6 promoter, which was increased in a concentration-dependent manner (P < 0·001). We conclude that IL-6 is a candidate signalling molecule in the chito-oligosaccharide-mediated inhibition of adipogenesis in 3T3-L1 cells.

Modulation of intracellular glutathione affects adipogenesis in 3T3‐L1 cells

Impairment of redox homeostasis has been extensively associated with obesity, as a consequence of the chronic inflammatory state present in overweight subjects. Deregulation of glutathione (GSH), the most important non-enzymatic intracellular anti-oxidant, induces insulin resistance in mature adipocytes, but data are lacking about its effects on adipogenesis. In this report we demonstrate that during adipogenesis of 3T3-L1 cells the GSH/GSSG ratio decreases, shifting redox status towards oxidizing conditions. Moreover, we demonstrate that inhibition of GSH synthesis, obtained by treatment with L-buthionine-sulfoximine (BSO), enhances C/EBPβ LAP/LIP ratio and PPARγ expression during mitotic clonal expansion (MCE) stimulating adipogenesis. On the contrary, GSH ethyl ester (GSHest) supplementation completely abrogates this process also in the presence of BSO. GSH decrement during the first 24 h of adipogenesis is sufficient to induce higher triglyceride accumulation in differentiated adipocytes with respect to control, whereas GSHest treatment inhibits lipid droplets formation. We further demonstrate that Resveratrol (RV) could exert anti-adipogenic properties also by increasing GSH content through γ-glutamyl-cysteine ligase (GCL) induction. Overall data indicate that in pre-adipocytes the decrease of GSH accelerates adipogenesis, suggesting that the use of agents able to maintain GSH redox status in adipose tissue, such as RV, could be promising in stopping the lipogenic loop of obesity.

Modification of Curcumin with Polyethylene Glycol Enhances the Delivery of Curcumin in Preadipocytes and Its Antiadipogenic Property

Conjugation of curcumin (CCM) by polyethylene glycol (PEG) has been previously developed to improve water solubility of the natural form of CCM and its antiproliferative role in some human cancer cell lines. This study examined the cellular uptake kinetics of the natural form of CCM and CCM-PEG. Their cytotoxic effect in proliferating preadipocytes and antiadipogenic property in differentiating preadipocytes had also been investigated. CCM and CCM-PEG were found to be differently absorbed in 3T3-L1 preadipocytes and adipocytes with a limited amount of CCM-PEG absorption in the cell. The improved water solubility of CCM-PEG was correlated with increased cellular retention of CCM in 3T3-L1 cells, particularly in preadipocytes. Consequently, CCM-PEG treatment sensitized proliferating preadipocytes to CCM-induced cell toxicity. Furthermore, incubation of differentiating 3T3-L1 cells with CCM-PEG resulted in improvement of the inhibitory role of CCM in adipocyte differentiation with no toxic effect. These results suggest that pegylation-improved water solubility and cellular retention of CCM may be uniquely useful for improving the delivery of CCM in preadipocytes and its antiadipogenic ability.

Abstract 3557: Pterostilbene, a natural polyphenol with anticancer activity, induces tumor cell death through autophagy activation

Abstract Polyphenols (PFs) conform a large class of plants/fruits-derived molecules with potential benefits for human health. Resveratrol (Resv) (3,4′,5-trihydroxy-trans-stilbene), has been extensively studied for its anti-diabetic, neuroprotective, anti-adipogenic, cardioprotective and anti-tumoral properties. However its low bioavailability (half-life in blood was 10.2 minutes after i.v. adm. of 20 mg/kg to mice) strongly limits Resv potential in vivo. Thus, compounds that may mimic Resv effects, but showing more bioavailability, may improve health benefits and should be investigated. Pterostilbene (Pter) (3,5-dimethoxy-4′-hydroxystilbene, another natural phytoalexin), has shown similar or even more potent antitumor activities than Resv. However, Pter's half-life in mouse blood (77.9 min) is approx. 7.5 times higher. Pter causes cancer cell death in vitro at bioavailable concentrations, and decreases tumor growth in mice. The aim of our present study was a) to determine whether Pter causes cytotoxicity in human tumors at concentrations that are reliable under in vivo conditions; and b) identify which death-related molecular mechanisms are activated by Pter. For these purposes we used a panel of different human tumors: melanoma, breast cancer, lung cancer, and colon cancer. Resv and Pter were assayed in a μM range, between 10 μM (below the IC50 for all cell lines) and 200 μM (an unachievable in vivo concentration). Cell cycle and death induction were determined by flow cytometry. The effects observed in the presence of Resv or Pter, under in vitro conditions, were concentration and exposure time dependent. In fact, an increase in PF concentration switched the type tumor cell death. Resv or Pter induced inhibition of tumor cell division and autophagy activation, although no caspase 3 activation was detected within a 24 h-period in the presence of either PF. On the other hand by further increasing Resv or Pter concentration, apoptosis and necrosis were progressively activated as shown by flow cytometry, caspase 3 activity and lactate dehydrogenase activity released to the culture medium. The main difference between Resv and Pter is the effective concentration, much lower for Pter and thus close to bioavailable levels. Whereas bioavailable levels of Resv do not cause tumor cell death. Different molecular events associated Pter with autophagy activation: a) an increase of LC3-II form, indicating the processing of LC3 protein to its lipidated form; b) an increase in P62 bands and GFP-LC3 punctation were observed thus indicating P62 accumulation and translocation of LC3 to autophagic membranes, respectively. Besides low levels of Pter, in addition of activating autophagy, also cause a rapid loss of tubulin organization as detected by immunochemistry. Therefore our results demonstrate that Pter induces tumor cell cycle arrest and autophagy activation at bioavailable concentrations. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3557.

Sodium selenate inhibits adipogenesis in vitro

Adipogenesis refers to the differentiation of predadipocytes into mature adipocytes. This process is a potential target to treat or prevent obesity. Adipogenesis not only increases fat accumulation, but also elevates inflammation and oxidative stress. Selenium, an essential micromineral, has been shown to have potent anti‐oxidative and anti‐inflammatory properties. We therefore hypothesized that selenium can serve as a potential anti‐adipogenic agent. Sodium selenate was chosen over sodium selenite due to its relatively low toxicity in 3T3‐L1 cells. We found that selenate significantly inhibits adipogenesis in vitro in a dose dependent manner. Complete inhibition was observed at 50 microM, at which there were no negative effects on cell viability. We revealed that selenate's inhibitory action is limited to the early stage (day 0 to 2) of adipogenesis. Furthermore, we found that selenate reduced mRNA expression levels of adipogenic transcription factors, such as, peroxisome proliferator‐activated receptor‐gamma (PPAR‐gamma). More importantly, selenate was likely to maintain preadipocyte factor‐1 (Pref‐1), a preadipocyte marker gene expression, during adipogenesis. Taken together, our results support selenate's anti‐adipogenic properties in vitro. These effects are likely due to selenate‐mediated alteration of the adipogenic transcriptional program in the early stage of differentiation. This project is partially funded by the USDA‐AFRI grant and The Showalter Trust Fund Award.Grant Funding Source: USDA‐AFRI and The Showalter Trust Fund Award

Inhibitiory role of curcumin in adipogenesis

Adipogenesis has been suggested to be a target mechanism to treat and prevent obesity. Induction of inflammatory response and oxidative stress has been observed in the process of adipogenesis. Curcumin having anti‐oxidative and anti‐inflammatory properties brought us to test the hypothesis that curcumin inhibits adipogenesis. We found that curcumin significantly inhibits 3T3‐L1 preadipocyte adipogenesis in vitro in a dose‐dependent manner and a complete inhibition of adipogenesis was observed at 30 µM. Interestingly, curcumin inhibits cell viability of proliferating 3T3‐L1 preadipocytes in a dose‐dependent manner but not in differentiating 3T3‐L1 cells. We also revealed that the inhibitory effect of curcumin on 3T3‐L1 preadipocyte differentiation is limited to the early stage (Day 0 to 2) of adipogenesis. Image analysis of cellular uptake of curcumin revealed the presence of curcumin in the differentiating 3T3‐L1 adipocytes. Furthermore, we found that curcumin significantly reduced mRNA expression levels of adipogenic transcription factors, including peroxisome proliferator‐activated receptorγ (PPARγ) and CAATT/enhancer binding proteinα (C/EBPα), but not C/EBPβ. Taken together, our results support that curcumin has anti‐adipogenic property in vitro and this is likely due to inhibition of mRNA expression of PPARγ and C/EBPα in the early stage of adipogenesis.Grant Funding SourceStart‐up research fund

Compartmentalization of stearoyl-coenzyme A desaturase 1 activity in HepG2 cells

Stearoyl-coenzyme A desaturase 1 (SCD1) catalyzes the conversion of stearate (18:0) to oleate (18:1n-9) and of palmitate (16:0) to palmitoleate (16:1), which are key steps in triglyceride synthesis in the fatty acid metabolic network. This study investigated the role of SCD1 in fatty acid metabolism in HepG2 cells using SCD1 inhibitors and stable isotope tracers. HepG2 cells were cultured with [U-(13)C]stearate, [U-(13)C]palmitate, or [1,2-(13)C]acetate and (1) DMSO, (2) compound CGX0168 or CGX0290, or (3) trans-10,cis-12 conjugated linoleic acid (CLA). (13)C incorporation into fatty acids was determined by GC-MS and desaturation indices calculated from the respective ion chromatograms. FAS, SCD1, peroxisome proliferator-activated receptor alpha, and peroxisome proliferator-activated receptor gamma mRNA levels were assessed by semiquantitative RT-PCR. The addition of CGX0168 and CGX0290 decreased the stearate and palmitate desaturation indices in HepG2 cells. CLA led to a decrease in the desaturation of stearate only, but not palmitate. Comparison of desaturation indices based on isotope enrichment ratios differed, depending on the origin of saturated fatty acid. SCD1 gene expression was not affected in any group. In conclusion, the differential effects of SCD1 inhibitors and CLA on SCD1 activity combined with the dependence of desaturation indices on the source of saturated fatty acid strongly support the compartmentalization of desaturation systems. The effects of SCD1 inhibition on fatty acid composition in HepG2 cells occurred through changes in the dynamics of the fatty acid metabolic network and not through transcriptional regulatory mechanisms.

Antidiabetes and Anti‐Obesity Activity of Lagerstroemia speciosa

The leaves of Lagerstroemia speciosa (Lythraceae), a Southeast Asian tree more commonly known as banaba, have been traditionally consumed in various forms by Philippinos for treatment of diabetes and kidney related diseases. In the 1990s, the popularity of this herbal medicine began to attract the attention of scientists worldwide. Since then, researchers have conducted numerous in vitro and in vivo studies that consistently confirmed the antidiabetic activity of banaba. Scientists have identified different components of banaba to be responsible for its activity. Using tumor cells as a cell model, corosolic acid was isolated from the methanol extract of banaba and shown to be an active compound. More recently, a different cell model and the focus on the water soluble fraction of the extract led to the discovery of other compounds. The ellagitannin Lagerstroemin was identified as an effective component of the banaba extract responsible for the activity. In a different approach, using 3T3-L1 adipocytes as a cell model and a glucose uptake assay as the functional screening method, Chen et al. showed that the banaba water extract exhibited an insulin-like glucose transport inducing activity. Coupling HPLC fractionation with a glucose uptake assay, gallotannins were identified in the banaba extract as components responsible for the activity, not corosolic acid. Penta-O-galloyl-glucopyranose (PGG) was identified as the most potent gallotannin. A comparison of published data with results obtained for PGG indicates that PGG has a significantly higher glucose transport stimulatory activity than Lagerstroemin. Chen et al. have also shown that PGG exhibits anti-adipogenic properties in addition to stimulating the glucose uptake in adipocytes. The combination of glucose uptake and anti-adipogenesis activity is not found in the current insulin mimetic drugs and may indicate a great therapeutic potential of PGG.

Up-regulation of a thermogenesis-related gene (UCP1) and down-regulation of PPARγ and aP2 genes in adipose tissue: possible features of the antiobesity effects of a β3-adrenergic agonist

A number of experiments have demonstrated the antiobesity effects of β3-adrenergic receptor stimulation by promoting thermogenesis and/or lipolysis. While many studies have been performed in order to develop β3-adrenergic agonists as a novel strategy in the management of obesity, more information is needed about the mechanisms involved in thermogenesis and the actions of these drugs on adipocyte differentiation. To address this, the possible thermogenic and antiadipogenic properties of Tertatolol, a β3-adrenergic agonist, in a diet-induced obesity model has been tested. Animals fed on a high-fat diet gained more weight and fat mass as compared with control and high-fat fed animals treated with Tertatolol. A RT-PCR was carried out in white adipose tissue specific genes involved in thermogenesis such as uncoupling proteins (UCPs) and adipogenesis such as peroxisome proliferator-activated receptor (PPARγ2), retinoid receptors (RXRα/RARα), and fatty acid binding protein (aP2). Levels of UCP1 mRNA were augmented in the Tertatolol-treated group as compared to non-treated high-fat fed animals, while the β3-adrenergic agonist treatment significantly decreased the expression levels of aP2 and transcription factors such as PPARγ2 and the ratio RXRα/RARα as compared to obese rats. Altogether these data suggest that the antiobesity effects of β3-adrenergic agonists are not limited to the promotion of thermogenesis and/or lipolysis and support the implication that these β3-adrenergic agonists also affect fat deposition by impairing adipogenesis in white adipose tissue (WAT).

Antiadipogenic properties of retinol in primary cultured differentiating human adipocyte precursor cells

The aim of this study was to investigate the effect of retinol on the human adipose conversion process using primary cultured human adipocyte precursor cells. When these cells were seeded in a medium containing retinol (concentrations ranging from 3.5 nM to 3.5 muM), cell proliferation was slightly inhibited by high concentrations of retinol, as demonstrated by cell counting and [(3)H]-thymidine incorporation. Moreover, the differentiation capacities of these cells were markedly and dose-dependently inhibited by retinol, as shown by the reduced expression of the lipogenic enzyme glycerol-3-phosphate dehydrogenase and by microscopic morphological analysis. These results strongly suggest that retinol, by inhibiting the ability of human preadipocytes to convert into mature adipocytes, could be of potential interest in the prevention of human adipose tissue development in general and of cellulitis in particular.

The role of TNF-alpha in human adipose tissue: prevention of weight gain at the expense of insulin resistance?

Since evidence has appeared that tumor necrosis factor-alpha (TNF) is involved in the loss of body fat in the course of wasting diseases, a large number of studies have investigated the physiological role of this cytokine in adipose tissue. TNF treatment of several in vitro models of adipogenesis clearly showed that TNF is a potent inhibitor of adipose differentiation. This antiadipogenic property is accompanied by suppression of developmental and metabolic markers of fat cell differentiation, such as peroxisome proliferator-activated receptor (PPAR)-gamma2, lipoprotein lipase (LPL), glycerol-3-phosphate dehydrogenase (GPDH) and GLUT4. Moreover, TNF promotes lipolysis in mature adipocytes and, subsequently, a reversion of the adipocyte phenotype. Recent studies demonstrated that TNF directly interferes with the insulin signaling cascade at early steps and, thus, impairs insulin-stimulated glucose transport. Further progress in understanding the role of TNF in adipose tissue was made when endogenous TNF mRNA expression was demonstrated in adipose tissue. Obesity was found to represent a state of overexpression of the TNF system. Such findings support the hypothesis that TNF is a mediator of obesity-linked insulin resistance. However, this concept is mainly based on animal data and is so far only partially supported by studies in humans. Taken together, the results of a variety of experimental and clinical studies suggest that TNF may act as an important auto/paracrine regulator of fat cell function which serves to limit adipose tissue expansion, probably by inducing insulin resistance which may in turn cause metabolic disturbances. Elucidation of the molecular mechanisms of TNF production and action in adipose tissue may help to find new approaches for the treatment of insulin resistance in humans.