GAL3ST2 from mammary gland epithelial cells affects differentiation of 3T3-L1 preadipocytes

Introduction. In the mammary gland, the involution that occurs when lactation ends is an important period for cancer development. We have previously demonstrated stromal–epithelium interactions evaluating conditioned medium of adipose tissue on breast epithelial metalloproteases activity (Creydt et al., Clin Transl Oncol 15:124–131, 2013). Here, we evaluated the effects of conditioned medium of breast epithelial mammary cells on stromal cells. Materials and methods. Conditioned medium from normal murine mammary gland cell line (NMuMG) and conditioned medium proteins were obtained. Then, they were evaluated on modulation of adipocyte differentiation, using 3T3-L1 cell line. Results. We described, for the first time, that breast epithelial mammary cells could produce the enzyme galactose 3-O-sulfotransferase 2 (GAL3ST2). Importantly, GAL3ST2 is present in NMMuMG and two human breast cancer cell lines, and it is more strongly expressed in more metastatic tumors. When 3T3-L1 preadipocyte differentiation was triggered in the presence of conditioned medium from NMuMG or GAL3ST2, triglyceride accumulation was decreased by 40 % and C/EBPβ expression by 80 % in adipocytes. In addition, the expression of FABP4 (aP2), another marker of adipocyte differentiation, was inhibited by 40 % in GAL3ST2-treated cells. Conclusions. Taken together, these results suggest that GAL3ST2 would interfere with normal differentiation of 3T3-L1 preadipocytes; raising the possibility that it may affect normal differentiation of stromal preadipocytes and be a link to tumor metastatic capacity (AU)

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Effects of uremic toxin p-cresol on proliferation, apoptosis, differentiation, and glucose uptake in 3T3-L1 cells.

Malnutrition is a common feature seen in chronic dialysis patients, and the survival rate of obese patients receiving such treatment is higher than that of lean patients. Irrespective of obesity or diabetes, dialysis patients commonly have insulin resistance, and the leading cause of death is cardiovascular (CV) disease. It has been reported that the concentration of p-cresol, a uremic toxin, is highly associated with CV events. As uremic toxin levels are high in dialysis patients, they may be involved in the pathogenesis of insulin resistance and CV disease in this population. However, little is known so far. Thus, we focused on this uremic toxin to examine its effects on adipocytes and their precursors. 3T3-L1 cells, a mouse preadipocyte cell line, were cultured until 90% confluency. The cells were then differentiated with 500 µM 3-isobutyl-methylxanthine, 250 nM dexamethasone, and 10 µg/mL insulin. Cell proliferation was evaluated by cell counting and bromodeoxyuridine (Brd-U) incorporation assay. Glucose uptake was estimated using radiolabeled 2-deoxyglucose. The range of concentrations of p-cresol used in the experiments was from 2 to 200 µM. The investigation of cell proliferation by cell counting revealed that, compared with control, 3T3-L1 cells treated with 100 and 200 µM p-cresol were significantly decreased in number at day 3 and day 7 of culture. The Brd-U incorporation assay also demonstrated similar inhibitory effects on cell proliferation, suggesting that p-cresol affected the normal cell cycle. Oil Red O staining at day 7 showed that the number of mature adipocytes was decreased by treatment with 200 µM p-cresol. Consistent with that finding, the number of apoptotic cells at day 7 was increased by treatment with 100 and 200 µM p-cresol. Peroxisome proliferator-activated receptor γ (PPARγ) mRNA expression increased time-dependently during the differentiation process of 3T3-L1 cells. p-Cresol dose-dependently decreased differentiation-induced mRNA expression of PPARγ. Uptake of 3H-labeled 2-deoxyglucose was markedly decreased by 200 µM p-cresol in the presence or in the absence of insulin, mainly because of the decreased number of mature adipocytes. High concentrations of p-cresol disturbed the cell cycle, induced apoptosis, inhibited the differentiation of preadipocytes into mature adipocytes, and decreased glucose uptake at baseline and after insulin stimulation. These findings indicate that accumulated p-cresol may induce reduction in adipose tissue, insulin resistance, and malnutrition, eventually leading to poor outcomes in chronic dialysis patients.

The SGBS cell strain as a model for the in vitro study of obesity and cancer

INTRODUCTION: The murine adipocyte cell line 3T3-L1 is well characterised and used widely, while the human pre-adipocyte cell strain, Simpson-Golabi-Behmel Syndrome (SGBS), requires validation for use in human studies. Obesity is currently estimated to account for up to 41 % of the worldwide cancer burden. A human in vitro model system is required to elucidate the molecular mechanisms for this poorly understood association. This work investigates the relevance of the SGBS cell strain for obesity and cancer research in humans. MATERIALS AND METHODS: Pre-adipocyte 3T3-L1 and SGBS were differentiated according to standard protocols. Morphology was assessed by Oil Red O staining. Adipocyte-specific gene expression was measured by qPCR and biochemical function was assessed by glycerol-3-phosphate dehydrogenase (GPDH) enzyme activity. Differential gene expression in oesophageal adenocarcinoma cell line OE33 following co-culture with SGBS or primary omental human adipocytes was investigated using Human Cancer Profiler qPCR arrays. RESULTS: During the process of differentiation, SGBS expressed higher levels of adipocyte-specific transcripts and fully differentiated SGBS expressed more similar morphology, transcript levels and biochemical function to primary omental adipocytes, relative to 3T3-L1. Co-culture with SGBS or primary omental adipocytes induced differential expression of genes involved in adhesion (ITGB3), angiogenesis (IGF1, TEK, TNF, VEGFA), apoptosis (GZMA, TERT) and invasion and metastasis (MMP9, TIMP3) in OE33 tumour cells. CONCLUSIONS: Comparable adipocyte-specific gene expression, biochemical function and a shared induced gene signature in co-cultured OE33 cells indicate that SGBS is a relevant in vitro model for obesity and cancer research in humans (AU)

Chlorophyll a, an active anti-proliferative compound of Ludwigia octovalvis, activates the CD95 (APO-1/CD95) system and AMPK pathway in 3T3-L1 cells.

Ludwigia octovalvis is an aquatic plant widely distributed in Taiwan. It is traditionally used as a diuretic and is consumed as health drink. In this study, we evaluated the anti-proliferative activity of extracts and active constituent (chlorophyll a; CHL-a) of L. octovalvis in 3T3-L1 adipocytes; its mode of action on apoptosis was also investigated. Results showed that, among the different extracts and fractions, the ethylacetate layer (EAL) possessed the most potent anti-proliferative activity. Activity guided fractionation of the EAL obtained the bioactive constituent CHL-a (IC50: 24.10+/-0.83 nM). At concentrations 5-30 nM, CHL-a exhibited a dose-dependent accumulation of the Sub-G1 peak and caused cell cycle arrest at the G0/G1 phase. At 30 nM, it significantly reduced the cell viability, induced the appearance of DNA fragments, and enhanced the activation of caspase-3. Western blot data revealed that CHL-a decreased the level of Bcl-2, and increased the expression of CD95 (APO-1/CD95) and Bax. Furthermore, CHL-a up-regulated the AMPK and p-AMPK levels, and down-regulated the expression of PPAR-gamma. These results conclude that CHL-a possesses potent anti-proliferative activity, and its apoptotic effects on 3T3-L1 adipocytes are mediated through the activation of CD95 (APO-1/CD95) system and the AMPK signaling pathway.

Inhibitory effect of (-)-epigallocatechin-3-gallate on lipid accumulation of 3T3-L1 cells.

OBJECTIVE: The objective of this study was to investigate the molecular mechanisms underlying the attenuating effect of (-)-epigallocatechin-3-gallate (EGCG) on proliferation and lipid accumulation of 3T3-L1 cells, with a focus on the duration of EGCG treatment. RESEARCH METHODS AND PROCEDURES: Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium assay and diamidino-2-phenylindole staining. The anti-adipogenic effect of EGCG on 3T3-L1 cells was analyzed by glycerol-3-phosphate dehydrogenase activity and Oil red O staining. Western blot analysis was used to detect adenosine monophosphate-activated protein kinase (AMPK) activation and phosphorylation of its substrate, acetyl-CoA carboxylase (ACC), and expression of insulin (INS) receptor, INS receptor substrate-1 (IRS-1), and adipocyte marker proteins. RESULTS: Exposure to EGCG during the early period of adipogenesis (7 days) was sufficient to prevent lipid accumulation. During this period, EGCG greatly decreased expression of the adipocyte marker proteins peroxisome proliferator-activated receptor gamma2 (PPARgamma2) and liver X receptor (LXR)-alpha. Furthermore, EGCG significantly induced generation of reactive oxygen species (ROS), which led to AMPK activation, and these effects were eliminated by N-acetylcysteine (NAC) treatment. Also, EGCG increased the tyrosine phosphorylation of INS receptor and INS-1 with increasing incubation time. In contrast, EGCG treatment did not alter glycerol release in the presence or absence of 2',5'-dideoxyadenosine (DDA), indicating that EGCG had no effect on lipolysis. DISCUSSION: Our data demonstrate that EGCG decreased cell viability and inhibited differentiation of 3T3-L1 cells in a manner dependent on the duration of treatment. Also, we showed that inhibition of adipocyte differentiation by EGCG was associated with decreased glycerol-3-phosphate dehydrogenase (GPDH) activity accompanied by a strong inhibition of PPARgamma2-induced transcriptional activity. Furthermore, the inhibition of adipocyte differentiation by EGCG involved generation of ROS and activation of AMPK.

Effect of 4-nonylphenol on cell proliferation and adipocyte formation in cultures of fully differentiated 3T3-L1 cells.

The effect of 4-nonylphenol (NP) on cell proliferation and adipocyte formation was examined in cultures of fully differentiated 3T3-L1 cells. Following the hormonal induction of differentiation into adipocytes, 3T3-L1 cells were treated for 8 days with or without NP. NP at 5 and 10 microg/ml increased the DNA content by 32% and 68%, respectively, compared with that of the untreated cultures, in which NP was absent during the treatment period. There were many more bromodeoxyuridine (BrdU)-positive cells in the NP-treated cultures, in which NP was present at a concentration of 10 microg/ml during the treatment period, compared to the untreated cultures. These results indicate that NP had the ability to stimulate the proliferation of fully differentiated 3T3-L1 cells. NP at 5 and 10 microg/ml decreased the triacylglycerol (TG) content by 26% and 58%, respectively, and decreased the lipoprotein lipase (LPL) activity by 51% and 71%, respectively. The lipid droplets in individual cells of the NP-treated cultures were smaller than those of the untreated cultures. The mRNA levels of LPL and adipocyte-specific fatty acid binding protein (aP2) were considerably lower in the NP-treated cultures than in the untreated cultures. Thus, NP also had the ability to inhibit adipocyte formation in cultures of fully differentiated 3T3-L1 cells. A study using an antiestrogen ICI 182,780 showed that the NP-stimulated cell proliferation was mediated partly by the estrogen receptor, while the NP-induced inhibition of adipocyte formation was mediated by a mechanism other than the estrogen receptor.