Reorganization of the DNA replication landscape during adipogenesis is closely linked with adipogenic gene expression.

The temporal order of DNA replication along the chromosomes is thought to reflect the transcriptional competence of the genome. During differentiation of mouse 3T3-L1 cells into adipocytes, cells undergo one or two rounds of cell division called mitotic clonal expansion (MCE). MCE is an essential step for adipogenesis; however, little is known about the regulation of DNA replication during this period. Here, we performed genome-wide mapping of replication timing (RT) in mouse 3T3-L1 cells before and during MCE, and identified a number of chromosomal regions shifting toward either earlier or later replication through two rounds of replication. These RT changes were confirmed in individual cells by single-cell DNA-replication sequencing. Coordinate changes between a shift toward earlier replication and transcriptional activation of adipogenesis-associated genes were observed. RT changes occurred before the full expression of these genes, indicating that RT reorganization might contribute to the mature adipocyte phenotype. To support this, cells undergoing two rounds of DNA replication during MCE had a higher potential to differentiate into lipid droplet-accumulating adipocytes, compared with cells undergoing a single round of DNA replication and non-replicating cells.

Tipepidine activates AMPK and improves adipose tissue fibrosis and glucose intolerance in high-fat diet-induced obese mice.

Tipepidine (3-[di-2-thienylmethylene]-1-methylpiperidine) (TP) is a non-narcotic antitussive used in Japan. Recently, the potential application of TP in the treatment of neuropsychiatric disorders, such as depression and attention deficit hyperactivity disorder, has been suggested; however, its functions in energy metabolism are unknown. Here, we demonstrate that TP exhibits a metabolism-improving action. The administration of TP reduced high-fat diet-induced body weight gain in mice and lipid accumulation in the liver and increased the weight of epididymal white adipose tissue (eWAT) in diet-induced obese (DIO) mice. Furthermore, TP inhibited obesity-induced fibrosis in the eWAT. We also found that TP induced AMP-activated protein kinase (AMPK) activation in the eWAT of DIO mice and 3T3-L1 cells. TP-induced AMPK activation was abrogated by the transfection of liver kinase B1 siRNA in 3T3-L1 cells. The metabolic effects of TP were almost equivalent to those of metformin, an AMPK activator that is used as a first-line antidiabetic drug. In summary, TP is a potent AMPK activator, suggesting its novel role as an antidiabetic drug owing to its antifibrotic effect on adipose tissues.

IL-33 regulates adipogenesis via Wnt/ß-catenin/PPAR-γ signaling pathway in preadipocytes.

Interleukin-33 (IL-33), an emerging cytokine within the IL-1 family, assumes a pivotal function in the control of obesity. However, the specific mechanism of its regulation of obesity formation remains unclear. In this study, we found that the expression level of IL-33 increased in visceral adipose tissue in mice fed with a high-fat diet (HFD) compared with that in mice fed with a normal diet (ND). In vitro, we also found the expression level of IL-33 was upregulated during the adipogenesis of 3T3-L1 cells. Functional test results showed that knockdown of IL-33 in 3T3-L1 cells differentiation could promote the accumulation of lipid droplets, the content of triglyceride and the expression of adipogenic-related genes (i.e. PPAR-γ, C/EBPα, FABP4, LPL, Adipoq and CD36). In contrast, overexpression of IL-33 inhibits adipogenic differentiation. Meanwhile, the above tests were repeated after over-differentiation of 3T3-L1 cells induced by oleic acid, and the results showed that IL-33 played a more significant role in the regulation of adipogenesis. To explore the mechanism, transcriptome sequencing was performed and results showed that IL-33 regulated the PPAR signaling pathway in 3T3-L1 cells. Further, Western blot and confocal microscopy showed that the inhibition of IL-33 could promote PPAR-γ expression by inhibiting the Wnt/ß-catenin signal in 3T3-L1 cells. This study demonstrated that IL-33 was an important regulator of preadipocyte differentiation and inhibited adipogenesis by regulating the Wnt/ß-catenin/PPAR-γ signaling pathway, which provided a new insight for further research on IL-33 as a new intervention target for metabolic disorders.

ATAD3 is a limiting factor in mitochondrial biogenesis and adipogenesis of white adipocyte-like 3T3-L1 cells.

ATAD3 is a vital ATPase of the inner mitochondrial membrane of pluri-cellular eukaryotes, with largely unknown functions but early required for organism development as necessary for mitochondrial biogenesis. ATAD3 knock-down in C. elegans inhibits at first the development of adipocyte-like intestinal tissue so we used mouse adipocyte model 3T3-L1 cells to analyze ATAD3 functions during adipogenesis and lipogenesis in a mammalian model. ATAD3 function was studied by stable and transient modulation of ATAD3 expression in adipogenesis- induced 3T3-L1 cells using Knock-Down and overexpression strategies, exploring different steps of adipocyte differentiation and lipogenesis. We show that (i) an increase in ATAD3 is preceding differentiation-induced mitochondrial biogenesis; (ii) downregulation of ATAD3 inhibits adipogenesis, lipogenesis, and impedes overexpression of many mitochondrial proteins; (iii) ATAD3 re-expression rescues the phenotype of ATAD3 KD, and (iv) differentiation and lipogenesis are accelerated by ATAD3 overexpression, but inhibited by expression of a dominant-negative mutant. We further show that the ATAD3 KD phenotype is not due to altered insulin signal but involves a limitation of mitochondrial biogenesis linked to Drp1. These results demonstrate that ATAD3 is limiting for in vitro mitochondrial biogenesis and adipogenesis/lipogenesis and therefore that ATAD3 mutation/over- or under-expression could be involved in adipogenic and lipogenic pathologies.

Bacoside-A repressed the differentiation and lipid accumulation of 3T3-L1 preadipocytes by modulating the expression of adipogenic genes.

Obesity is one of the more complicated diseases, it can induce numerous life-threatening diseases mainly diabetes mellitus, cardiovascular disease, hypertension, and certain cancers. In this study, we assessed the efficacy of bacoside-A (a dammarane-type triterpenoid saponin derived from the plant Bacopa monniera Linn.) on the adipogenesis of 3T3-L1 preadipocytes. Results of this study illustrated that bacoside-A decreased the differentiation of 3T3-L1 cell, as evidenced by diminution of lipid droplets, which contains triglycerides and other lipids. During the differentiation process, transcription factors, which are mainly participating in adipogenesis such us CCAAT/enhancer-binding protein α (C/EBPα) and C/EBPß, peroxisome proliferator-activated receptor-γ (PPARγ), and sterol regulatory element-binding protein-1c (SREBP-1c), expressions were significantly suppressed by bacoside-A. In addition, bacoside-A showed a potent reduction in genes precise to adipocytes such as lipoprotein lipase (LPL), fatty acid synthase (FAS), adipocyte fatty acid-binding protein (FABP4), and leptin expressions. Further, bacoside-A stimulated the phosphorylation of acetyl CoA carboxylase (ACC) and AMP-activated protein kinase (AMPK). These results demonstrated that bacoside-A has anti-adipogenic effects by regulating the transcription factors involved in adipocyte differentiation. Therefore, bacoside-A might be considered as a potent therapeutic agent for alleviating obesity and hyperlipidemia.

Effect of four hydroxy fatty acids on lipid accumulation in the 3T3-L1 cells: A comparative study.

Notwithstanding the several investigations of the hydroxy fatty acids (hFAs)' physiological functions, studies focusing on their anti-obesity effects are limited. This study investigated the anti-obesity effects of four hFAs, 10-hydroxy stearic acid (10-hSA), 12-hydroxy stearic acid (12-hSA), 9,12-hydroxy stearic acid (9,12-dhSA), and 12-hydroxy oleic acid (12-hOA), on the 3T3-L1 cells. All hFAs suppressed lipid accumulation, with 10-hSA and 12-hOA exhibiting the strongest suppression, followed by 12-hSA and 9, 12-hSA. This trend was similar to that observed for the glycerol-3-phosphate dehydrogenase (GPDH) activity degree. Contrastingly, only 9,12-dhSA suppressed cell viability. The mRNA levels of HK1 and Aldoa were markedly suppressed by 10-hSA and 12-hSA compared to the control. Additionally, mRNA expression of Gyk was considerably suppressed by 12-hSA. Thus, all hFAs suppressed lipid accumulation by suppressing GPDH activity, although their molecular mechanisms were different. These findings will aid the application of hFAs in the food and medical industries.

Effects of Low-Intensity Pulsed Ultrasound on the Regulation of Free Fatty Acid Release in 3T3-L1 Cells.

OBJECTIVES: To investigate the effects of low-intensity pulsed ultrasound (LIPUS) on the proliferation, differentiation, and tumor necrosis factor-α (TNF-α)-induced lipolysis of 3T3-L1 cells, and to explore the feasibility of regulating the release of free fatty acids (FFA) to prevent lipotoxicity. METHODS: Different intensities (30, 60, 90, and 120 mW/cm2) of LIPUS were applied to 3T3-L1 preadipocytes for different durations (5, 10, 15, 20, 25, and 30 minutes). Appropriate parameters for subsequent experiments were selected by assessing cell viability. The effect of LIPUS on the proliferation and differentiation of 3T3-L1 cells was evaluated by microscope observation, flow cytometry, and lipid content determination. After treated with LIPUS and TNF-α (50 ng/mL), the degree of lipolysis was assessed by measuring the extracellular FFA content. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the mRNA expression of relevant genes. RESULTS: Different parameters of LIPUS significantly enhance the viability of 3T3-L1 cells (P < .05), with 20 minutes and 30 mW/cm2 as the most suitable settings. After LIPUS treatment, 3T3-L1 cell proliferation accelerated, apoptosis rate and G1 phase cell proportion decreased, the content of lipid droplets and TG was increased in differentiated cells, while FFA release decreased (P < .05). The expression of PCNA, PPARγ, C/EBPα, Perilipin A mRNA increased, and the expression of TNF-α, ATGL, HSL mRNA decreased (P < .05). CONCLUSIONS: LIPUS could promote the proliferation and differentiation of 3T3-L1 cells and inhibit TNF-α-induced lipolysis, indicating its potential as a therapy for mitigating lipotoxicity caused by decompensated adipocytes.

Assessment of the disruption effects of tetrabromobisphenol A and its analogues on lipid metabolism using multiple in vitro models.

Tetrabromobisphenol A (TBBPA), a widely-used brominated flame retardant, has been revealed to exert endocrine disrupting effects and induce adipogenesis. Given the high structural similarities of TBBPA analogues and their increasing exposure risks, their effects on lipid metabolism are necessary to be explored. Herein, 9 representative TBBPA analogues were screened for their interference on 3T3-L1 preadipocyte adipogenesis, differentiation of C3H10T1/2 mesenchymal stem cells (MSCs) to brown adipocytes, and lipid accumulation of HepG2 cells. TBBPA bis(2-hydroxyethyl ether) (TBBPA-BHEE), TBBPA mono(2-hydroxyethyl ether) (TBBPA-MHEE), TBBPA bis(glycidyl ether) (TBBPA-BGE), and TBBPA mono(glycidyl ether) (TBBPA-MGE) were found to induce adipogenesis in 3T3-L1 preadipocytes to different extends, as evidenced by the upregulated intracellular lipid generation and expressions of adipogenesis-related biomarkers. TBBPA-BHEE exhibited a stronger obesogenic effect than did TBBPA. In contrast, the test chemicals had a weak impact on the differentiation process of C3H10T1/2 MSCs to brown adipocytes. As for hepatic lipid formation test, only TBBPA mono(allyl ether) (TBBPA-MAE) was found to significantly promote triglyceride (TG) accumulation in HepG2 cells, and the effective exposure concentration of the chemical under oleic acid (OA) co-exposure was lower than that without OA co-exposure. Collectively, TBBPA analogues may perturb lipid metabolism in multiple tissues, which varies with the test tissues. The findings highlight the potential health risks of this kind of emerging chemicals in inducing obesity, non-alcoholic fatty liver disease (NAFLD) and other lipid metabolism disorders, especially under the conditions in conjunction with high-fat diets.

Pennogenin 3-O-ß-Chacotrioside Attenuates Hypertrophied Lipid Accumulation by Enhancing Mitochondrial Oxidative Capacity.

Excessive lipid accumulation in adipocytes is a primary contributor to the development of metabolic disorders, including obesity. The consumption of bioactive compounds derived from natural sources has been recognized as being safe and effective in preventing and alleviating obesity. Therefore, we aimed to explore the antilipidemic effects of pennogenin 3-O-ß-chacotrioside (P3C), a steroid glycoside, on hypertrophied 3T3-L1 adipocytes. Oil Red O and Nile red staining demonstrated a P3C-induced reduction in lipid droplet accumulation. Additionally, the increased expression of adipogenic and lipogenic factors, including PPARγ and C/EBPα, during the differentiation process was significantly decreased by P3C treatment at both the protein and mRNA levels. Furthermore, P3C treatment upregulated the expression of fatty acid oxidation-related genes such as PGC1α and CPT1a. Moreover, mitochondrial respiration and ATP generation increased following P3C treatment, as determined using the Seahorse XF analyzer. P3C treatment also increased the protein expression of mitochondrial oxidative phosphorylation in hypertrophied adipocytes. Our findings suggest that P3C could serve as a natural lipid-lowering agent, reducing lipogenesis and enhancing mitochondrial oxidative capacity. Therefore, P3C may be a promising candidate as a therapeutic agent for obesity-related diseases.

Cinnamyl Alcohol Attenuates Adipogenesis in 3T3-L1 Cells by Arresting the Cell Cycle.

Cinnamyl alcohol (CA) is an aromatic compound found in several plant-based resources and has been shown to exert anti-inflammatory and anti-microbial activities. However, the anti-adipogenic mechanism of CA has not been sufficiently studied. The present study aimed to investigate the effect and mechanism of CA on the regulation of adipogenesis. As evidenced by Oil Red O staining, Western blotting, and real-time PCR (RT-PCR) analyses, CA treatment (6.25-25 µM) for 8 d significantly inhibited lipid accumulation in a concentration-dependent manner and downregulated adipogenesis-related markers (peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer-binding protein α (C/EBPα), fatty acid binding protein 4 (FABP4), adiponectin, fatty acid synthase (FAS)) in 3-isobutyl-1-methylxanthine, dexamethasone, and insulin(MDI)-treated 3T3-L1 adipocytes. In particular, among the various differentiation stages, the early stage of adipogenesis was critical for the inhibitory effect of CA. Cell cycle analysis using flow cytometry and Western blotting showed that CA effectively inhibited MDI-induced initiation of mitotic clonal expansion (MCE) by arresting the cell cycle in the G0/G1 phase and downregulating the expression of C/EBPß, C/EBPδ, and cell cycle markers (cyclin D1, cyclin-dependent kinase 6 (CDK6), cyclin E1, CDK2, and cyclin B1). Moreover, AMP-activated protein kinase α (AMPKα), acetyl-CoA carboxylase (ACC), and extracellular signal-regulated kinase 1/2 (ERK1/2), markers of upstream signaling pathways, were phosphorylated during MCE by CA. In conclusion, CA can act as an anti-adipogenic agent by inhibiting the AMPKα and ERK1/2 signaling pathways and the cell cycle and may also act as a potential therapeutic agent for obesity.

Anti-Obesity Effects of GABA in C57BL/6J Mice with High-Fat Diet-Induced Obesity and 3T3-L1 Adipocytes.

Obesity is the excessive accumulation of body fat resulting from impairment in energy balance mechanisms. In this study, we aimed to investigate the mechanism whereby GABA (γ-aminobutyric acid) prevents high-fat diet-induced obesity, and whether it induces lipolysis and browning in white adipose tissue (WAT), using high-fat diet (HFD)-fed obese mice and 3T3-L1 adipocytes. We demonstrated that GABA substantially inhibits the body mass gain of mice by suppressing adipogenesis and lipogenesis. Consistent with this result, histological analysis of WAT demonstrated that GABA decreases adipocyte size. Moreover, we show that GABA administration decreases fasting blood glucose and improves serum lipid profiles and hepatic lipogenesis in HFD-fed obese mice. Furthermore, Western blot and immunofluorescence analyses showed that GABA activates protein kinase A (PKA) signaling pathways that increase lipolysis and promote uncoupling protein 1 (UCP1)-mediated WAT browning. Overall, these results suggest that GABA exerts an anti-obesity effect via the regulation of lipid metabolism.

Hypocholesterolemic potential of Bacillus amyloliquefaciens KAVK1 modulates lipid accumulation on 3T3-L1 adipose cells and high fat diet-induced obese rat model.

The significance of microorganisms occurring in foods is predominantly targeted due to their application for identifying a novel range of the bacterial spectrum. Diverse microbial species are capable of exhibiting potential pharmacological activities like antimicrobial and anticancer. Microbial strains capable of reducing obesity-related syndromes have also been reported. In the present study, the hypocholesterolemic efficacy of Bacillus amyloliquefaciens isolated from dairy products was scrutinised by in vitro (3T3-L1 adipose cells) and in vivo (high-fat diet-induced obese Wistar albino rats) methods. Potential cholesterol-lowering isolates were screened using a plate assay method and optimised by physical parameters. Molecular identification of the topmost five cholesterol-lowering isolates was acquired by amplification of the 16 S rRNA gene region. Bacillus amyloliquefaciens strain KAVK1, followed by strains KAVK2, KAVK3, KAVK4, and KAVK5 were molecularly determined. Further, cholesterol-lowering strains degraded the spectral patterns determined by the side chain of a cholesterol molecule. The anti-lipase activity was demonstrated using the porcine pancreatic lipase inhibitory method and compared with the reference compound Atorvastatin. Lyophilised strain KAVK1 revealed maximum pancreatic lipase inhibition. Strain KAVK1 attenuated lipid accumulation in 3T3-L1 adipose cell line predicted by Oil Red O staining method. Significant reduction of body weight and change in lipid profile was recognised after the supplement of KAVK1 to obese rats. Histopathological changes in organs were predominantly marked. The result of this study implies that the cholesterol-lowering B. amyloliquefaciens KAVK1 strain was used to treat hypercholesterolemia.

In Vitro Screening and Lipid-Lowering Effect of Prickly Pear (Opuntia Ficus-Indica L. Mill.) Fruit Extracts in 3T3-L1 Pre-Adipocytes and Mature Adipocytes.

Opuntia ficus-indica fruits have been widely used due to their nutritional composition and beneficial effects on health, particularly against chronic diseases such as diabetes, obesity, cardiovascular diseases and cancer, among others. In recent years, prickly pear peel and pulp extracts have been characterised, and a high number of bioactive compounds have been identified. This study aimed to analyse the triglyceride-lowering effect of prickly pear peel and pulp extracts obtained from fruits of three varieties (Pelota, Sanguinos, and Colorada) in 3T3-L1 maturing and mature adipocytes. At a concentration of 50 µg/mL, peel extracts from Colorada reduced triglyceride accumulation in pre-adipocytes and mature adipocytes. Additionally, at 25 µg/mL, Pelota peel extract decreased triglyceride content in mature adipocytes. Moreover, maturing pre-adipocytes treated with 50 and 25 µg/mL of Sanguinos pulp extract showed a reduction of triglyceride accumulation. In addition, the lipid-lowering effect of the main individual betalain and phenolic compounds standards were assayed. Piscidic acid and isorhamnetin glycoside (IG2), found in Colorada peel extract, were identified as the bioactive compounds that could contribute more notably to the triglyceride-lowering effect of the extract. Thus, the betalain and phenolic-rich extracts from Opuntia ficus indica fruits may serve as an effective tool in obesity management.

New role for the anandamide metabolite prostaglandin F2α ethanolamide: Rolling preadipocyte proliferation.

White adipose tissue regulation is key to metabolic health, yet still perplexing. The chief endocannabinoid anandamide metabolite, prostaglandin F2α ethanolamide (PGF2αEA), inhibits adipogenesis, that is, the formation of mature adipocytes. We observed that adipocyte progenitor cells-preadipocytes-following treatment with PGF2αEA yielded larger pellet sizes. Thus, we hypothesized that PGF2αEA might augment preadipocyte proliferation. Cell viability MTT and crystal violet assays, cell counting, and 5-bromo-2'-deoxyuridine incorporation in cell proliferation ELISA analyses confirmed our prediction. Additionally, we discovered that PGF2αEA promotes cell cycle progression through suppression of the expression of cell cycle inhibitors, p21 and p27, as shown by flow cytometry and qPCR. Enticingly, concentrations of this compound that showed no visible effect on cell proliferation or basal transcriptional activity of peroxisome proliferator-activated receptor gamma could, in contrast, reverse the anti-proliferative and peroxisome proliferator-activated receptor gamma-transcription activating effects of rosiglitazone (Rosi). MTT and luciferase reporter examinations supported this finding. The PGF2αEA pharmaceutical analog, bimatoprost, was also investigated and showed very similar effects. Importantly, we suggest the implication of the mitogen-activated protein kinase pathway in these effects, as they were blocked by the selective mitogen-activated protein kinase kinase inhibitor, PD98059. We propose that PGF2αEA is a pivotal regulator of white adipose tissue plasticity, acting as a regulator of the preadipocyte pool in adipose tissue.

Dopamine receptor D4 (DRD4) negatively regulates UCP1- and ATP-dependent thermogenesis in 3T3-L1 adipocytes and C2C12 muscle cells.

The activation of beige fat and muscle tissues is an interesting and encouraging target for therapeutic intervention in obesity owing to their remarkable lipolytic activity and energy-consuming futile cycles. This study examined the effect of dopamine receptor D4 (DRD4) on lipid metabolisms as well as UCP1- and ATP-dependent thermogenesis in Drd4-silenced 3T3-L1 adipocytes and C2C12 muscle cells. Silencing of Drd4, followed by quantitative real-time PCR, immunoblot analysis, immunofluorescence, and staining methods, were applied to evaluate the effects of DRD4 on diverse target genes and proteins of both cells. The findings showed that DRD4 was expressed in the adipose and muscle tissues of normal and obese mice. Furthermore, the knockdown of Drd4 upregulated the expression of brown adipocyte-specific genes and proteins while downregulating lipogenesis and the adipogenesis marker proteins. Drd4 silencing also upregulated the expression of key signaling molecules involved in ATP-dependent thermogenesis in both cells. This was further elucidated by mechanistic studies showing that a Drd4 knockdown mediates UCP1-dependent thermogenesis via the cAMP/PKA/p38MAPK pathway in 3T3-L1 adipocytes and UCP1-independent thermogenesis via the cAMP/SLN/SERCA2a pathway in C2C12 muscle cells. In addition, siDrd4 also mediates myogenesis via the cAMP/PKA/ERK1/2/Cyclin D3 pathway in C2C12 muscle cells. Silencing of Drd4 promotes ß3-AR-dependent browning in 3T3-L1 adipocytes and α1-AR/SERCA-based thermogenesis through an ATP-consuming futile process in C2C12 muscle cells. Understanding the novel functions of DRD4 on adipose and muscle tissues in terms of its ability to enhance energy expenditure and regulate whole-body energy metabolism will aid in developing novel obesity intervention techniques.

AIP4 regulates adipocyte differentiation by targeting C/EBPα for ubiquitin-mediated proteasomal degradation.

Adipogenesis, that is, the formation of terminally differentiated adipocytes is intricately regulated by transcription factors where CCAAT/enhancer binding protein alpha (C/EBPα) plays a key role. In the current study, we demonstrate that E3 ubiquitin ligase AIP4 negatively regulates C/EBPα protein stability leading to reduced adipogenesis. While AIP4 overexpression in 3T3-L1 cells preadipocytes inhibited lipid accumulation when treated with differentiation inducing media (MDI), AIP4 depletion was sufficient to partially promote lipid accumulation even in the absence of MDI. Mechanistically, overexpression of AIP4 inhibited protein levels of both ectopically expressed as well as endogenous C/EBPα while catalytically inactive AIP4 failed. On the contrary, AIP4 depletion profoundly enhanced endogenous C/EBPα protein levels. The observation that AIP4 levels decrease with concomitant increase in C/EBPα levels during adipocyte differentiation further indicated that AIP4 negatively regulates C/EBPα levels. We further show that AIP4 physically interacts with C/EBPα and ubiquitinates it leading to its proteasomal degradation. AIP4 promoted K48-linked ubiquitination of C/EBPα while catalytically inactive AIP4-C830A failed. Taken together, our data demonstrate that AIP4 inhibits adipogenesis by targeting C/EBPα for ubiquitin-mediated proteasome degradation.

The inhibitory effect of Gremlin-2 on adipogenesis suppresses breast cancer cell growth and metastasis.

BACKGROUND: Gremlin-1 (GREM1) and Gremlin-2 (GREM2) are bone morphogenetic protein antagonists that play important roles in organogenesis, tissue differentiation, and tissue homeostasis. Although GREM1 has been reported to be involved in promoting various cancers, little has been reported about effects of GREM2 on cancer. Recently, it has been reported that GREM2 can inhibit adipogenesis in adipose-derived stromal/stem cells. However, as an inhibitor of adipogenesis, the role of GREM2 in cancer progression is not well understood yet. METHODS: Pre-adipocyte 3T3-L1 cells overexpressing mock or Grem2 were established using a lentiviral transduction system and differentiated into adipocytes-mock and adipocytes-Grem2, respectively. To investigate the effect of adipocyte-Grem2 on breast cancer cells, we analyzed the proliferative and invasion abilities of spheroids using a 3D co-culture system of breast cancer cells and adipocytes or conditioned medium (CM) of adipocytes. An orthotopic breast cancer mouse model was used to examine the role of adipocytes-Grem2 in breast cancer progression. RESULTS: Grem2 overexpression suppressed adipogenesis of 3T3-L1 cells. Proliferative and invasion abilities of spheroids formed by co-culturing MTV/TM-011 breast cancer cells and adipocytes-Grem2 were significantly reduced compared to those of spheroids formed by co-culturing MTV/TM-011 cells and adipocytes-mock. Compared to adipocytes-mock, adipocytes-Grem2 showed decreased mRNA expression of several adipokines, notably IL-6. The concentration of IL-6 in the CM of these cells was also decreased. Proliferative and invasive abilities of breast cancer cells reduced by adipocytes-Grem2 were restored by IL-6 treatment. Expression levels of vimentin, slug, and twist1 in breast cancer cells were decreased by treatment with CM of adipocytes-Grem2 but increased by IL-6 treatment. In orthotopic breast cancer mouse model, mice injected with both MTV/TM-011 cells and adipocytes-Grem2 showed smaller primary tumors and lower lung metastasis than controls. However, IL-6 administration increased both the size of primary tumor and the number of metastatic lung lesions, which were reduced by adipocytes-Grem2. CONCLUSIONS: Our study suggests that GREM2 overexpression in adipocytes can inhibit adipogenesis, reduce the expression and secretion of several adipokines, including IL-6, and ultimately inhibit breast cancer progression.

Gene regulation of RMR-related DNAJC6 on adipogenesis and mitochondria function in 3T3-L1 preadipocytes.

In the pilot GWAS of children obesity, DNAJC6 gene was found as a regulator for resting metabolic rate (RMR) and obesity in children aged 8-9 years. To investigate whether DNAJC6 gene regulated obesity and energy metabolism, the physiological mechanisms during adipogenesis of 3T3-L1 preadipocytes were confirmed after DNAJC6 gene was overexpressed or inhibited. Overexpressing DNAJC6 gene maintained a 3T3-L1 preadipocyte status during cell differentiation (MTT, ORO, DAPI/BODIPY). It suppressed adipogenesis and adipokine production (leptin, adiponectin), insulin signaling with IRS-GLUT4 system (RT-PCR, Western blotting), and mitochondrial function (Mito Stress Test). DNAJC6 overexpressed cells inhibited mTOR expression, but maintained LC3 expression at a high level, indicating that autophagy occurred and energy was obtained. However, when DNAJC6 gene was inhibited, fat synthesis factor was highly expressed during differentiation (PPARr, C/EBPa, aP2, etc) and the intracellular stress level increased accordingly, which affected the reduction of reserve respiratory capacity during mitochondrial respiration. Our study confirmed gene regulation of DNAJC6, overexpression or inhibition, affects adipogenesis with energy metabolism and mitochondrial functions. This basic data can be used for clinic obesity studies to control an energy imbalance.

13C-Metabolic flux analysis of 3T3-L1 adipocytes illuminates its core metabolism under hypoxia.

Hypoxia has been identified as a major factor in the pathogenesis of adipose tissue inflammation, which is a hallmark of obesity and obesity-linked type 2 diabetes mellitus. In this study, we have investigated the impact of hypoxia (1% oxygen) on the physiology and metabolism of 3T3-L1 adipocytes, a widely used cell culture model of adipose. Specifically, we applied parallel labeling experiments, isotopomer spectral analysis, and 13C-metabolic flux analysis to quantify the impact of hypoxia on adipogenesis, de novo lipogenesis and metabolic flux reprogramming in adipocytes. We found that 3T3-L1 cells can successfully differentiate into lipid-accumulating adipocytes under hypoxia, although the production of lipids was reduced by about 40%. Quantitative flux analysis demonstrated that short-term (1 day) and long-term (7 days) exposure to hypoxia resulted in similar reprogramming of cellular metabolism. Overall, we found that hypoxia: 1) reduced redox and energy generation by more than 2-fold and altered the patterns of metabolic pathway contributions to production and consumption of energy and redox cofactors; 2) redirected glucose metabolism from pentose phosphate pathway and citric acid cycle to lactate production; 3) rewired glutamine metabolism, from net glutamine production to net glutamine catabolism; 4) suppressed branched chain amino acid consumption; and 5) reduced biosynthesis of odd-chain fatty acids and mono-unsaturated fatty acids, while synthesis of saturated even-chain fatty acids was not affected. Together, these results highlight the profound impact of extracellular microenvironment on adipocyte metabolic activity and function.

Dual mechanism of silver nanoparticle-mediated upregulation of adipogenesis in mouse fibroblasts (3T3-L1) in vitro.

Silver nanoparticles (AgNPs) are widespread in the environment due to the increase in their application e.g. in medicine as part of hard-to-heal wound dressings. Many studies have revealed easy diffusion of AgNPs into deep skin layers through damaged epidermis and contact with e.g. fibroblasts. Therefore, the aim of this study was to evaluate the impact of small-size AgNPs (10 nm) in ppm concentrations on the adipogenesis process in mouse embryo fibroblasts (3T3-L1). The results showed a decrease in the metabolic activity, followed by an increase in the reactive oxygen species (ROS) level in a dose- and time-dependent manner (0-20 ppm). The increased caspase-3 activity was observed only at the highest concentration (20 ppm) of AgNPs. Further analysis showed the ability of the tested NPs to increase the lipid accumulation in adipocytes, similar to ROSI [peroxisome proliferator-activated receptor gamma (PPARγ) agonist], measured by Oil-Red-O staining. Moreover, the analyses evidenced the ability of AgNPs to increase the lipoxygenase activity and malondialdehyde levels, which is probably based on ROS-dependent enhancement of lipid hydroperoxidation. Lastly, a significant increase in the PPARγ, Adiponectin, Resistin, Vegf, and Serpine mRNA expression was shown 6 h after the induction of the differentiation process. Based on the obtained results, it can be concluded that small-size AgNPs increase adipogenesis via ROS- and PPARγ-based mechanisms with potential engagement of crosstalk with the aryl hydrocarbon receptor, which is important due to the widespread application of AgNPs in medicine. However, more studies are needed to elucidate the full mechanism of these NPs in the tested cell model in depth.