Polyamine metabolism is involved in adipogenesis of 3T3-L1 cells

Polyamines spermidine and spermine are known to be required for mammalian cell proliferation and for embryonic development. Alpha-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase (ODC) a limiting enzyme of polyamine biosynthesis, depleted the cellular polyamines and prevented triglyceride accumulation and differentiation in 3T3-L1 cells. In this study, to explore the function of polyamines in adipogenesis, we examined the effect of polyamine biosynthesis inhibitors on adipocyte differentiation and lipid accumulation of 3T3-L1 cells. The spermidine synthase inhibitor trans-4-methylcyclohexylamine (MCHA) increased spermine/spermidine ratios, whereas the spermine synthase inhibitor N-(3-aminopropyl)-cyclohexylamine (APCHA) decreased the ratios in the cells. MCHA was found to decrease lipid accumulation and GPDH activity during differentiation, while APCHA increased lipid accumulation and GPDH activity indicating the enhancement of differentiation. The polyamine-acetylating enzyme, spermidine/spermine N ¹-acetyltransferase (SSAT) activity was increased within a few hours after stimulus for differentiation, and was found to be elevated by APCHA. In mature adipocytes APCHA decreased lipid accumulation while MCHA had the opposite effect. An acetylpolyamine oxidase and spermine oxidase inhibitor MDL72527 or an antioxidant N-acetylcysteine prevented the promoting effect of APCHA on adipogenesis. These results suggest that not only spermine/spermidine ratios but also polyamine catabolic enzyme activity may contribute to adipogenesis.     

Ginsenoside Rb1 promotes adipogenesis in 3T3-L1 cells by enhancing PPARgamma2 and C/EBPalpha gene expression

Evidence has accumulated that ginseng and its main active constituents, ginsenosides, possess anti-diabetic and insulin-sensitizing properties which may be partly realized by regulating adipocyte development and functions. In the present study, we explored the effect of ginsenoside Rb(1), the most abundant ginsenoside in ginseng root, on adipogenesis of 3T3-L1 cells. We found that with standard differentiation inducers, ginsenoside Rb(1) facilitated adipogenesis of 3T3-L1 preadipocytes in a dose-dependent manner; 10 microM Rb(1) increased lipid accumulation by about 56%. Treatment of differentiating adipocytes with 10 microM Rb(1) increased the expression of mRNA and protein of PPARgamma(2) and C/EBPalpha, as well as mRNA of ap2, one of their target genes. After the treatment of differentiating adipocytes with Rb(1), basal and insulin-mediated glucose uptake was significantly augmented, accompanied by the up-regulation of mRNA and protein level of GLUT4, but not of GLUT1. In addition, ginsenoside Rb(1) also inhibited the proliferation of preconfluent 3T3-L1 preadipocytes. Our data indicate that anti-diabetic and insulin-sensitizing activities of ginsenosides, at least in part, are involved in the enhancing effect on PPARgamma2 and C/EBPalpha expression, hence promoting adipogenesis.     

Tannins present in Cichorium intybus enhance glucose uptake and inhibit adipogenesis in 3T3-L1 adipocytes through PTP1B inhibition

Insulin resistance is a fundamental aspect for the etiology of non-insulin dependent diabetes mellitus (NIDDM) and has links with a wide array of secondary disorders including weight gain and obesity. The present study analyzes the effect of Cichorium intybus methanolic (CME) extract on glucose transport and adipocyte differentiation in 3T3-L1 cells by studying the radiolabelled glucose uptake and lipid accumulation assays, respectively. By performing detannification (CME/DT), the role of tannins present in CME on both the activities was evaluated. CME and CME/DT exhibited significant glucose uptake in 3T3-L1 adipocytes with a dose-dependent response. Glucose uptake profile in the presence of PI3K and IRTK inhibitors (Wortmannin and Genistein) substantiates the mechanism used by both the extracts. CME inhibited the differentiation of 3T3-L1 preadipocytes but failed to show glucose uptake in inhibitor treated cells. The activity exhibited by CME/DT is exactly vice versa to CME. Furthermore, the findings from PTP1B inhibition assay, mRNA and protein expression analysis revealed the unique behavior of CME and CME/DT. The duality exhibited by C. intybus through adipogenesis inhibition and PPARgamma up regulation is of interest. Current observation concludes that the activities possessed by C. intybus are highly desirable for the treatment of NIDDM because it reduces blood glucose levels without inducing adipogenesis in 3T3-L1 adipocytes.     

Fenoxycarb promotes adipogenesis in 3T3‐L1 preadipocytes

Lipophilic insect hormones and their analogs affect mammalian physiology by regulating the expression of metabolic genes. Therefore, we determined the effect of fenoxycarb, a juvenile hormone analog, on lipid metabolism in adipocytes. Here, we demonstrated that fenoxycarb dose‐dependently promoted lipid accumulation in 3T3‐L1 adipocytes during adipocyte differentiation and that its lipogenic effect was comparable to that of rosiglitazone, a well‐known ligand for peroxisome proliferator‐activated receptor gamma (PPARγ). Furthermore, fenoxycarb stimulated PPARγ activity without affecting other nuclear receptors, such as liver X receptor (LXR), farnesoid X‐activated receptor (FXR) and Nur77. In addition, fenoxycarb treatment increased the expression of PPARγ and fatty acid transporter protein 1 (FATP1) in 3T3‐L1 adipocytes, suggesting that fenoxycarb may facilitate adipocyte differentiation by enhancing PPARγ signaling, the master regulator of adipogenesis. Together, our results suggest that fenoxycarb promoted lipid accumulation in adipocytes, in part, by stimulating PPARγ.     

Adipogenesis and lipid production in adipocytes subjected to sustained tensile deformations and elevated glucose concentration: a living cell-scale model system of diabesity

Adipocyte fate commitment is characterized by morphological changes of fibroblastic pre-adipocyte cells, and specifically by accumulation of lipid droplets (LDs) as part of the adipogenesis metabolism. Formation of LDs indicates the production of triglycerides from glucose through an insulin-regulated glucose internalization process. In obesity, adipocytes typically become insulin resistant, and glucose transport into the cells is impaired, resulting in type 2 diabetes. In the present study, we monitored the adipogenesis in 3T3-L1 cultured cells exposed to high (450 mg/dL hyperglycemia) and low (100 mg/dL physiological) glucose concentrations, in a novel cell culture model system of diabesity. In addition to glucose conditions, cells were concurrently exposed to different substrate tensile strains (12% and control) based on our prior work which revealed that adipogenesis is accelerated in cultures subjected to static, chronic substrate tensile deformations. Phase-contrast images were taken throughout the adipogenesis process (3 weeks) and were analyzed by an image processing algorithm which quantitatively monitors cell differentiation and lipid accumulation (number of LDs per cell and their radius as well as cell size and shape). The results indicated that high glucose concentrations and substrate tensile strains delivered to adipocytes accelerated lipid production by 1.7- and 1.4-fold, respectively. In addition, significant changes in average cell projected area and in other morphological attributes were observed during the differentiation process. The importance of this study is in characterizing the adipogenesis parameters as potential read-outs that can predict the occurrence of insulin resistance in the development of diabesity.     

Prostaglandin F(2)alpha enhances glucose consumption through neither adipocyte differentiation nor GLUT1 expression in 3T3-L1 cells

Arachidonic acid (AA) at 0.2 mM enhances glucose uptake through increased levels of glucose transporter (GLUT) 1 protein in 3T3-L1 adipocytes. Since AA is a precursor of prostaglandins (PGs), we investigated the effect of PGs on glucose consumption in 3T3-L1 cells. Among several PGs, only prostaglandin F(2)alpha (PGF(2)alpha) enhanced glucose consumption in 3T3-L1 cells treated with dexamethasone (DEX), 3-isobutyl-1-methyl-xanthine (IBMX), and insulin. To study the mechanism of PGF(2)alpha-enhanced glucose consumption, we investigated the effect of PGF(2)alpha on glycerol-3-phosphate dehydrogenase (GPDH) activity, triglycerides (TGs) content, and the expression of GLUT1 protein. PGF(2)alpha suppressed GPDH activity and did not increase the expression of GLUT1 protein in 3T3-L1 cells treated with DEX, IBMX, and insulin. These results suggest that AA-stimulated glucose uptake is not through the effect of PGF(2)alpha. Our results indicate that PGF(2)alpha is a unique regulator of adipocyte differentiation (suppression) and glucose consumption (enhancement) in 3T3-L1 cells.     

GULP1 deficiency reduces adipogenesis and glucose uptake via downregulation of PPAR signaling and disturbing of insulin/ERK signaling in 3T3-L1 cells

Obesity and metabolic disorders caused by alterations in lipid metabolism are major health issues in developed, affluent societies. Adipose tissue is the only organ that stores lipids and prevents lipotoxicity in other organs. Mature adipocytes can affect themselves and distant metabolism-related tissues by producing various adipokines, including adiponectin and leptin. The engulfment adaptor phosphotyrosine-binding domain-containing 1 (GULP1) regulates intracellular trafficking of glycosphingolipids and cholesterol, suggesting its close association with lipid metabolism. However, the role of GULP1 in adipocytes remains unknown. Therefore, this study aimed to investigate the function of GULP1 in adipogenesis, glucose uptake, and the insulin signaling pathway in adipocytes. A 3T3-L1 cell line with Gulp1 knockdown (shGulp1) and a 3T3-L1 control group (U6) were established. Changes in shGulp1 cells due to GULP1 deficiency were examined and compared to those in U6 cells using microarray analysis. Glucose uptake was monitored via insulin stimulation in shGulp1 and U6 cells using a 2-NBDG glucose uptake assay, and the insulin signaling pathway was investigated by western blot analysis. Adipogenesis was significantly delayed, lipid metabolism was altered, and several adipogenesis-related genes were downregulated in shGulp1 cells compared to those in U6 cells. Microarray analysis revealed significant inhibition of peroxisome proliferator-activated receptor signaling in shGulp1 cells compared with U6 cells. The production and secretion of adiponectin as well as the expression of adiponectin receptor were decreased in shGulp1 cells. In particular, compared with U6 cells, glucose uptake via insulin stimulation was significantly decreased in shGulp1 cells through the disturbance of ERK1/2 phosphorylation. This is the first study to identify the role of GULP1 in adipogenesis and insulin-stimulated glucose uptake by adipocytes, thereby providing new insights into the differentiation and functions of adipocytes and the metabolism of lipids and glucose, which can help better understand metabolic diseases.     

Calcineurin-independent inhibition of 3T3-L1 adipogenesis by Pasteurella multocida toxin: suppression of Notch1, stabilization of beta-catenin and pre-adipocyte factor 1

Pasteurella multocida toxin (PMT) is a potent mitogen and a specific activator of Gq-dependent signalling pathways. PMT impairs osteoblast differentiation and causes bone loss and fat reduction in vivo. We examined the effect of PMT on cell signalling pathways involved in 3T3-L1 adipocyte differentiation. We demonstrate that PMT treatment before or together with differentiation induction factors inhibits adipogenesis and prevents upregulation of important adipocyte markers - peroxisome-proliferator-activated receptor gamma (PPARgamma) and CAATT enhancer-binding protein alpha (C/EBPalpha). Moreover, PMT completely downregulates PPARgamma and C/EBPalpha expression in mature adipocytes. Differentiation of pre-adipocytes into adipocytes requires the suppression of pre-adipocyte factor 1 (Pref1) and Wnt signalling, along with the degradation of beta-catenin. PMT prevents downregulation of Pref1 and beta-catenin under differentiation-inducing conditions. In addition, PMT treatment downregulates expression of Notch1, a protein responsible for cell fate decision and implicated in regulation of adipogenesis in 3T3-L1 cells. PMT action on adipogenesis was not reversed by cyclosporin A, an inhibitor of Galphaq-PLC-calcium-dependent calcineurin activation. Our results reveal new pathways involved in PMT action on cellular physiology and differentiation. Our study further demonstrates that the effect of PMT on Pref1/PPARgamma/C/EBPalpha expression and adipogenesis does not occur just through activation of the Galphaq-calcium-calcineurin pathway, but involves Wnt/beta-catenin and Notch1 signalling pathways, two signalling pathways strongly linked to cancer predisposition, neurological and immunological dysfunctions, and fat and bone development.     

Co-treatment with dexamethasone and octanoate induces adipogenesis in 3T3-L1 cells

We report here that octanoate, a medium chain fatty acid, induces adipocyte differentiation in 3T3-L1 cells by co-treatment with dexamethasone, although octanoate has been known not to stimulate 3T3-L1 adipogenesis. A low concentration of exogenous glucose prevented 3T3-L1 adipogenesis induced by 1-methyl 3-isobutylxanthine, dexamethasone, and insulin (MDI) treatment (a common protocol for adipocyte differentiation). In contrast, co-treatment with dexamethasone and octanoate (D-OCT) induced adipogenesis under the same conditions. These findings imply that octanoate, rather than glucose, is the source of accumulated lipids in D-OCT-induced adipogenesis. D-OCT increased expression of the differentiation markers peroxisome proliferator-activated receptor (PPAR)gamma2 and caveolin-1. A specific inhibitor of p38 mitogen-activated protein (MAP) kinase inhibited D-OCT-induced adipogenesis. These results suggest that the p38 MAP kinase pathway followed by up-regulation of PPARgamma2 may be involved in 3T3-L1 adipocyte differentiation induced by D-OCT, as well as by MDI.     

Esculetin induces apoptosis and inhibits adipogenesis in 3T3-L1 cells

Objective: To determine the effects of esculetin, a plant phenolic compound with apoptotic activity in cancer cells, on 3T3‐L1 adipocyte apoptosis and adipogenesis. Research Methods and Procedures: 3T3‐L1 pre‐confluent preadipocytes and lipid‐filled adipocytes were incubated with esculetin (0 to 800 μM) for up to 48 hours. Viability was determined using the Cell Titer 96 Aqueous One Solution cell proliferation assay; apoptosis was quantified by measurement of single‐stranded DNA. Post‐confluent preadipocytes were incubated with esculetin for up to 6 days during maturation. Adipogenesis was quantified by measuring lipid content using Nile Red dye; cells were also stained with Oil Red O for visual confirmation of effects on lipid accumulation. Results: In mature adipocytes, esculetin caused a time‐ and dose‐related increase in adipocyte apoptosis and a decrease in viability. Apoptosis was increased after only 6 hours by 400 and 800 μM esculetin (p < 0.05), and after 48 hours, as little as 50 μM esculetin increased apoptosis (p < 0.05). In preadipocytes, apoptosis was detectable only after 48 hours (p < 0.05) with 200 μM esculetin and higher concentrations. However, results of the cell viability assay indicated a reduction in preadipocyte number in a time‐ and dose‐related manner, beginning as early as 6 hours with 400 and 800 μM esculetin (p < 0.05). Esculetin also inhibited adipogenesis of 3T3‐L1 preadipocytes. Esculetin‐mediated inhibition of adipocyte differentiation occurred during the early, intermediate, and late stages of the differentiation process. In addition, esculetin induced apoptosis during the late stage of differentiation. Discussion: These findings suggest that esculetin can alter fat cell number by direct effects on cell viability, adipogenesis, and apoptosis in 3T3‐L1 cells.