Dictyostelium Differentiation-Inducing Factor 1 Promotes Glucose Uptake via Direct Inhibition of Mitochondrial Malate Dehydrogenase in Mouse 3T3-L1 Cells.

Differentiation-inducing factor 1 (DIF-1), found in Dictyostelium discoideum, has antiproliferative and glucose-uptake-promoting activities in mammalian cells. DIF-1 is a potential lead for the development of antitumor and/or antiobesity/antidiabetes drugs, but the mechanisms underlying its actions have not been fully elucidated. In this study, we searched for target molecules of DIF-1 that mediate the actions of DIF-1 in mammalian cells by identifying DIF-1-binding proteins in human cervical cancer HeLa cells and mouse 3T3-L1 fibroblast cells using affinity chromatography and liquid chromatography-tandem mass spectrometry and found mitochondrial malate dehydrogenase (MDH2) to be a DIF-1-binding protein in both cell lines. Since DIF-1 has been shown to directly inhibit MDH2 activity, we compared the effects of DIF-1 and the MDH2 inhibitor LW6 on the growth of HeLa and 3T3-L1 cells and on glucose uptake in confluent 3T3-L1 cells in vitro. In both HeLa and 3T3-L1 cells, DIF-1 at 10-40 µM dose-dependently suppressed growth, whereas LW6 at 20 µM, but not at 2-10 µM, significantly suppressed growth in these cells. In confluent 3T3-L1 cells, DIF-1 at 10-40 µM significantly promoted glucose uptake, with the strongest effect at 20 µM DIF-1, whereas LW6 at 2-20 µM significantly promoted glucose uptake, with the strongest effect at 10 µM LW6. Western blot analyses showed that LW6 (10 µM) and DIF-1 (20 µM) phosphorylated and, thus, activated AMP kinase in 3T3-L1 cells. Our results suggest that MDH2 inhibition can suppress cell growth and promote glucose uptake in the cells, but appears to promote glucose uptake more strongly than it suppresses cell growth. Thus, DIF-1 may promote glucose uptake, at least in part, via direct inhibition of MDH2 and a subsequent activation of AMP kinase in 3T3-L1 cells.

Somatostatin receptor ligands suppressed proliferation and lipogenesis in 3T3-L1 preadipocytes.

Somatostatin and its analogues, known as somatostatin receptor ligands (SRLs), have been reported to attenuate weight gain in some clinical settings. However, their direct effects on preadipocytes are barely investigated. Therefore, this study aimed to evaluate the influence of SRLs on preadipocytes and to further explore the potential mechanisms. Cell Counting Kit-8 assay, Oil Red O staining, triglyceride contents measurements, quantitative polymerase chain reaction (qPCR) and western blot were used to investigate the effects of SRLs on preadipocytes. We found that three SRLs (octreotide, TT232 and pasireotide) inhibited cell viability after 8-48 h but not 4 h. Further western blot results showed that they significantly suppressed activation of PI3K/Akt pathway. Besides, lipid accumulation was also significantly inhibited by these SRLs. Moreover, mRNA levels of some critical adipogenic markers, including Pparg, Cebpa, Fasn, Fabp4, Acaca and Lpl, were downregulated by the treatments of all these SRLs. Consistently, the protein expression of peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer binding protein α (C/EBPα) and fatty acid synthase (FAS) was also suppressed by SRLs. SRLs inhibit the proliferation and lipogenesis in preadipocytes. Their inhibitory effects on cell proliferation may be mediated by the downregulated PI3K/Akt pathway, and the suppressive actions on lipogenesis may be related to the decreased PPARγ and C/EBPα expression.

Oncostatin M Induces Lipolysis and Suppresses Insulin Response in 3T3-L1 Adipocytes.

Oncostatin M (OSM) is an immune cell-derived cytokine that is upregulated in adipose tissue in obesity. Upon binding its receptor (OSMR), OSM induces the phosphorylation of the p66 subunit of Src homology 2 domain-containing transforming protein 1 (SHC1), called p66Shc, and activates the extracellular signal-related kinase (ERK) pathway. Mice with adipocyte-specific OSMR deletion (OsmrFKO) are insulin resistant and exhibit adipose tissue inflammation, suggesting that intact adipocyte OSM-OSMR signaling is necessary for maintaining adipose tissue health. How OSM affects specific adipocyte functions is still unclear. Here, we examined the effects of OSM on adipocyte lipolysis. We treated 3T3-L1 adipocytes with OSM, insulin, and/or inhibitors of SHC1 and ERK and measured glycerol release. We also measured phosphorylation of p66Shc, ERK, and insulin receptor substrate-1 (IRS1) and the expression of lipolysis-associated genes in OSM-exposed 3T3-L1 adipocytes and primary adipocytes from control and OsmrFKO mice. We found that OSM induces adipocyte lipolysis via a p66Shc-ERK pathway and inhibits the suppression of lipolysis by insulin. Further, OSM induces phosphorylation of inhibitory IRS1 residues. We conclude that OSM is a stimulator of lipolysis and inhibits adipocyte insulin response. Future studies will determine how these roles of OSM affect adipose tissue function in health and disease.

Effects of IL-33 on 3T3-L1 cells and obese mice models induced by a high-fat diet.

Obesity is a syndrome that attributes to many factors such as genetics, diet, lifestyle and environment, which includes an imbalance of immune regulation. IL-33, as a new member of the IL-1 family, is classically associated with type 2 immune responses. Here, IL-33 was investigated for its ability to optimize lipid aggregation and ameliorate the inflammatory response in obesity. In vitro experimental results showed that, compared with the induction group, the treatment with 30 ng/mL IL-33 displayed a reduction in the number of lipid droplets. The expression levels of AceCS1 and PPARγ also decreased in the 30 ng/mL IL-33 group compared to the induction group. For confirmation in vivo, three groups of C57BL/6 mice were treated for 14 weeks: mice in control were fed with a normal diet; mice in the HFD and IL-33 groups were fed with a high-fat diet (HFD) and with sterile PBS or recombinant IL-33, respectively. Liver, muscle, spleen and four types of adipose tissue, as well as serum, were collected for further testing. Our data demonstrated that after 4-week treatment with recombinant IL-33, metabolic parameters in mice were improved significantly (visceral fat weight, glucose and insulin tolerance, liver steatosis, expression of lipid synthesis index and inflammatory response). Moreover, IL-33 treatment regulated the original distribution of IL-33 among different tissues. Hence, IL-33 modulated lipid metabolism and inflammatory response in obesity, which would be a novel therapeutic target for obesity and related metabolic diseases.

Trigonelline induces browning in 3T3-L1 white adipocytes.

Trigonelline, a major alkaloid component of fenugreek, has been demonstrated to have several biological activities, including antidiabetic and anticancer effects. This study aimed to examine the possible application of trigonelline as an anti-obesity compound based on an investigation of its enhancement of lipid catabolism and induction of browning in white adipocytes. Trigonelline induces browning of 3T3-L1 white adipocytes by enhancing the expressions of brown-fat signature proteins and genes as well as beige-specific genes, including Cd137, Cited1, Tbx1, and Tmem26. Trigonelline also improves lipid metabolism in white adipocytes by decreasing adipogenesis and lipogenesis as well as promotes lipolysis and fatty acid oxidation. Moreover, trigonelline increases the expression of Cox4, Nrf1, and Tfam genes that are responsible for mitochondrial biogenesis. Mechanistic studies revealed that the browning effect of trigonelline in 3T3-L1 white adipocytes is mediated by activating ß3-AR and inhibiting PDE4, thereby stimulating the p38 MAPK/ATF-2 signaling pathway. Considering its high bioavailability in humans and the results of this study, trigonelline may have potential as an anti-obesity compound.

4ß-Hydroxywithanolide E and withanolide E from Physalis peruviana L. inhibit adipocyte differentiation of 3T3-L1 cells through modulation of mitotic clonal expansion.

Obesity is a risk factor for many diseases, including type 2 diabetes and cardiovascular disease, and is related to the rising morbidity and mortality. Discovery of agents targeting adipogenesis, especially from natural sources, is important for the treatment of obesity. Here, we aimed to identify anti-adipogenic substances in methanol extracts of Physalis peruviana and to investigate their effect, along with underlying mechanisms. Activity-guided fractionation of the extract revealed 4ß-hydroxywithanolide E (HWE) and withanolide E (WE) as the adipogenesis inhibitors. Both compounds suppressed mRNA expression of central adipogenic transcription factors, peroxisome proliferator-activated receptor γ, and CCAAT/enhancer-binding protein α in the early stage of adipocyte differentiation. The inhibitory action of these two withanolides on adipogenesis was largely limited to this stage. The proliferation of preadipocytes was markedly suppressed by treatment with HWE and WE for 24 and 48 h in the differentiation medium, and cell-cycle arrest in the G0/G1 phase was observed. Therefore, our results suggested that withanolides from P. peruviana to be novel anti-adipogenic compounds that modulate mitotic clonal expansion.

Interleukin-38 inhibits adipogenesis and inflammatory cytokine production in 3T3-L1 preadipocytes.

Interleukin-38 (IL-38) is a novel member of the IL-1 cytokine family with anti-inflammatory activity. However, its effect on adipogenesis and inflammatory cytokines secretion of adipocytes in vitro has not been reported. To address whether IL-38 inhibits adipogenesis and inflammation in vitro, adipose precursor 3T3-L1 cells were cultured with or without IL-38. The morphology and size of lipid droplets in 3T3-L1 cells were measured by Oil red O staining. The mRNA expression levels of GATA-binding protein-3 (GATA-3), glucose transporter type 4 (GLUT4), peroxisome proliferator-associated receptor γ2, IL-1ß, IL-6, and monocyte chemoattractant protein-1 (MCP-1) in 3T3-L1 cells were detected by real-time PCR, The contents of IL-6, IL-1ß, and MCP-1 in 3T3-L1 cell medium supernatants were determined by enzyme-linked immunosorbent assay. IL-38 significantly decreased the number of lipid droplets in 3T3-L1 cells. IL-38 also increased GATA-3 and GLUT4 mRNA expression and inhibited IL-1ß, IL-6, and MCP-1 secretion by 3T3-L1 cells. It is concluded that IL-38 can inhibit the differentiation of human adipocytes and inflammatory cytokine production by 3T3-L1 cells.

MicroRNA 182 is a Novel Negative Regulator of Adipogenesis by Targeting CCAAT/Enhancer-Binding Protein α.

OBJECTIVE: Recent studies have shown that microRNAs (miRNAs/miRs) play key roles in adipogenesis. This study aimed to investigate the role and underlying mechanism of miR-182 in adipogenesis. METHODS: This study used the 3T3-L1 cell line and human visceral adipose tissue (VAT)-derived adipocytes to determine the role of miR-182 in adipogenesis. Adipose tissues from mice with high-fat diet-induced obesity, ob/ob mice, or human individuals with obesity were used to determine the association of miR-182 levels with obesity. A luciferase reporter assay was used to determine the target of miR-182. RESULTS: The expression level of miR-182 was greatly downregulated during white adipogenesis and markedly lower in the VAT of mice and humans with obesity. Ectopic expression of miR-182 in 3T3-L1 cells and human adipocytes suppressed the formation of lipid droplets and the expression of adipogenic genes. The luciferase reporter assay showed that miR-182 targeted the 3'-untranslated sequence of CCAAT/enhancer-binding protein α (C/EBPα) directly. In addition, glucocorticoids negatively regulated miR-182 expression, which, in turn, suppressed the glucocorticoid-induced expression of C/EBPα. CONCLUSIONS: Taken together, our studies identified miR-182 as a novel negative regulator of adipogenesis and a potential therapeutic target for obesity.

Anti-obesity Effect of Fermented Persimmon Extracts via Activation of AMP-Activated Protein Kinase.

There is significant cultivation of persimmon (Diospyros kaki) in East Asia, a plant whose fruit has abundant nutrients, including vitamins, polyphenols, and dietary fiber. Persimmon dietary supplements can benefit health by amelioration of diabetes, cardiovascular disease, and obesity. There are also persimmon-based beverages produced via fermentation, such as wines and vinegars, and increasing consumption of these products in East Asia. Although there is great interest in functional foods, the health effects of fermented persimmon extract (FPE) are completely unknown. We examined the effects of FPE on the metabolic parameters of mice fed a high-fat diet (HFD). Our results indicated that FPE supplementation led to an approx. 15% reduction of body weight, reduced abdominal and liver fat, and reduced serum levels of triglycerides, total cholesterol, and glucose. FPE also blocked the differentiation of murine 3T3-L1 pre-adipocyte cells into mature adipocytes. We suggest that gallic acid is a major bioactive component of FPE, and that AMP-activated protein kinase mediates the beneficial effects of FPE and gallic acid.

Spatiotemporal contact between peroxisomes and lipid droplets regulates fasting-induced lipolysis via PEX5.

Lipid droplets (LDs) are key subcellular organelles for regulating lipid metabolism. Although several subcellular organelles participate in lipid metabolism, it remains elusive whether physical contacts between subcellular organelles and LDs might be involved in lipolysis upon nutritional deprivation. Here, we demonstrate that peroxisomes and peroxisomal protein PEX5 mediate fasting-induced lipolysis by stimulating adipose triglyceride lipase (ATGL) translocation onto LDs. During fasting, physical contacts between peroxisomes and LDs are increased by KIFC3-dependent movement of peroxisomes toward LDs, which facilitates spatial translocations of ATGL onto LDs. In addition, PEX5 could escort ATGL to contact points between peroxisomes and LDs in the presence of fasting cues. Moreover, in adipocyte-specific PEX5-knockout mice, the recruitment of ATGL onto LDs was defective and fasting-induced lipolysis is attenuated. Collectively, these data suggest that physical contacts between peroxisomes and LDs are required for spatiotemporal translocation of ATGL, which is escorted by PEX5 upon fasting, to maintain energy homeostasis.

Effects of the Selective EP2 Receptor Agonist Omidenepag on Adipocyte Differentiation in 3T3-L1 Cells.

Purpose: We aimed at comparing the effects of omidenepag (OMD) with those of prostaglandin F (FP) receptor agonists (FP agonists) on adipogenesis in mouse 3T3-L1 cells. Methods: To evaluate the agonistic activities of OMD against the mouse EP2 (mEP2) receptor, we determined cAMP contents in mEP2 receptor-expressing CHO cells by using radioimmunoassays. Overall, 3T3-L1 cells were cultured in differentiation medium for 10 days and adipocyte differentiation was assessed according to Oil Red O-stained cell areas. Changes in expression levels of the adipogenic transcription factors Pparg, Cebpa, and Cebpb were determined by using real-time polymerase chain reaction (PCR). OMD at 0.1, 1, 10, and 40 µmol/L, latanoprost free acid (LAT-A) at 0.1 µmol/L, or prostaglandin F2α (PGF2α), at 0.1 µmol/L were added to cell culture media during adipogenesis. Oil Red O-stained areas and expression patterns of transcription factor targets of OMD or FP agonists were compared with those of untreated controls. Results: The 50% effective concentration (EC50) of OMD against the mEP2 receptor was 3.9 nmol/L. Accumulations of Oil Red O-stained lipid droplets were observed inside control cells on day 10. LAT-A and PGF2α significantly inhibited the accumulation of lipid droplets; however, OMD had no effect on this process even at concentrations up to 40 µmol/L. LAT-A and PGF2α significantly suppressed Pparg, Cebpa, and Cebpb gene expression levels during adipocyte differentiation. Conversely, OMD had no obvious effects on the expression levels of these genes. Conclusions: A selective EP2 receptor agonist, OMD, did not affect the adipocyte differentiation in 3T3-L1 cells, whereas FP agonists significantly inhibited this process.

Functional expression of TRPM7 as a Ca2+ influx pathway in adipocytes.

In adipocytes, intracellular Ca2+ and Mg2+ modulates physiological functions, such as insulin action and the secretion of adipokines. TRPM7 is a Ca2+ /Mg2+ -permeable non-selective cation channel. TRPM7 mRNA is highly expressed in adipose tissue, however, its functional expression in adipocytes remains to be elucidated. In this study, we demonstrated for the first time that TRPM7 was functionally expressed in both freshly isolated white adipocytes and in 3T3-L1 adipocytes differentiated from a 3T3-L1 pre-adipocyte cell line by whole-cell patch-clamp recordings. Consistent with known properties of TRPM7 current, the current in adipocytes was activated by the elimination of extracellular divalent cations and the reduction of intracellular free Mg2+ concentrations, and was inhibited by the TRPM7 inhibitors, 2-aminoethyl diphenylborinate (2-APB), hydrogen peroxide (H2 O2 ), N-methyl maleimide (NMM), NS8593, and 2-amino-2-[2-(4-octylphenyl)ethyl]-1,3-propanediol (FTY720). Treatment with small-interfering (si) RNA targeting TRPM7 resulted in a reduction in the current to 23 ± 7% of nontargeting siRNA-treated adipocytes. Moreover a TRPM7 activator, naltriben, increased the TRPM7-like current and [Ca2+ ]i in 3T3-L1 adipocytes but not in TRPM7-knockdown adipocytes. These findings indicate that TRPM7 is functionally expressed, and plays a role as a Ca2+ influx pathway in adipocytes.

Fibroblast growth factor 21 secretion enhances glucose uptake in mono(2-ethylhexyl)phthalate-treated adipocytes.

Previous studies revealed that cellular accumulation of mono(2-ethylhexyl)phthalate (MEHP) disturbed energy metabolism in adipocytes, where glucose uptake was significantly increased. The present study aimed to determine the mechanisms underlying the increased glucose uptake. MEHP-treated 3T3-L1 adipocytes exhibited a significantly increased glucose uptake activity. Immunoblot analysis suggested that the insulin-induced signals were not responsible for the increased glucose uptake. qPCR analysis revealed that both Glut1 and Glut4 genes were highly expressed during adipogenesis; Glut1 mRNA levels in MEHP-treated adipocytes were significantly increased. Moreover, MEHP-treated adipocytes exhibited significantly increased levels of fibroblast growth factor 21 (FGF21) in both mRNA and secreted protein. FGF21 is a peptide hormone with pleiotropic effects on regulation of insulin sensitivity and glucose/lipid homeostasis. We found that MEHP, FGF21, and lactate in culture medium together enhanced Fgf21 gene expression in MEHP-treated adipocytes. FGF21 signaling requires fibroblast growth factor receptor (FGFR) and ßKlotho. Fgfr family and ßKlotho genes were actively expressed during adipogenesis; mRNA levels of Fgfr3 and Fgfr4 genes in MEHP-treated adipocytes were significantly increased. Roles of FGF21/FGFR and phosphoinositide 3-kinase (PI3K)/AKT signal axes in regulation of glucose uptake were determined. We demonstrated that FGF21/FGFR signals played the major roles in up-regulation of the basal glucose uptake in MEHP-treated adipocytes. The in vitro evidence suggests that cellular FGF21 secretion enhances the basal glucose uptake in MEHP-treated adipocytes.

Comparison of the Effects of Long-chain Monounsaturated Fatty Acid Positional Isomers on Lipid Metabolism in 3T3-L1 Cells.

Long chain monounsaturated fatty acids (LC-MUFAs) have shown beneficial health effects in previous studies. They occur as mixtures of positional isomers (PIs) in food. The functionalities of LC-MUFA PIs have not been studied extensively. Common LC-MUFA PIs, namely cis-octadecenoic acid (c-18:1), cis-eicosenoic acid (c-20:1), and cis-docosenoic acid (c-22:1), were screened based on their effects on lipid accumulation. We selected nine fatty acids (FAs) to assess their effects on cellular lipid metabolism using 3T3-L1 preadipocytes. Lipid accumulation was found to be higher in cells treated with LC-MUFAs than in the non-treated cells. When comparing the influence of chain length of LC-MUFAs, TG levels tended to be higher in cells treated with c-22:1 group than that of the c18:1 and c-20:1 groups. Among the c-22:1 group, c9-22:1 treatment showed higher lipid accumulation, and was accompanied with elevated expression of transcription factors related to adipogenesis and lipogenesis, such as PPARγ and C/EBPα, and SREBP-1, respectively. In contrast, the effects of c-20:1 FAs were less pronounced than those of c-18:1 and c-22:1. Levels of accumulated lipid in cells treated with c15-20:1 were the same as in non-treated control. PPARγ, C/EBPα, and SREBP-1 were expressed at lower levels with c15-20:1 FA. Furthermore, mRNA levels of SCD-1 and FAS were lowered more by c15- and c11-20:1 than by other MUFAs. These results revealed that differences in the effects of LC-MUFAs on lipid metabolism depend on their chain lengths and on the position of the double bond.

Cerium Oxide Nanoparticles Regulate Insulin Sensitivity and Oxidative Markers in 3T3-L1 Adipocytes and C2C12 Myotubes.

Insulin resistance is associated with oxidative stress, mitochondrial dysfunction, and a chronic low-grade inflammatory status. In this sense, cerium oxide nanoparticles (CeO2 NPs) are promising nanomaterials with antioxidant and anti-inflammatory properties. Thus, we aimed to evaluate the effect of CeO2 NPs in mouse 3T3-L1 adipocytes, RAW 264.7 macrophages, and C2C12 myotubes under control or proinflammatory conditions. Macrophages were treated with LPS, and both adipocytes and myotubes with conditioned medium (25% LPS-activated macrophages medium) to promote inflammation. CeO2 NPs showed a mean size of ≤25.3 nm (96.7%) and a zeta potential of 30.57 ± 0.58 mV, suitable for cell internalization. CeO2 NPs reduced extracellular reactive oxygen species (ROS) in adipocytes with inflammation while increased in myotubes with control medium. The CeO2 NPs increased mitochondrial content was observed in adipocytes under proinflammatory conditions. Furthermore, the expression of Adipoq and Il10 increased in adipocytes treated with CeO2 NPs. In myotubes, both Il1b and Adipoq were downregulated while Irs1 was upregulated. Overall, our results suggest that CeO2 NPs could potentially have an insulin-sensitizing effect specifically on adipose tissue and skeletal muscle. However, further research is needed to confirm these findings.

Mixture of Two Lactobacillus plantarum Strains Modulates the Gut Microbiota Structure and Regulatory T Cell Response in Diet-Induced Obese Mice.

SCOPE: The gut microbiota has been linked to diet-induced obesity, and microorganisms that influence obesity have important health implications. In this study, the anti-obesity effects of two Lactobacillus plantarum strains (DSR M2 and DSR 920) isolated from kimchi are investigated. METHODS AND RESULTS: Mice are fed a normal or high-fat diet with or without DSR M2 and DSR 920 (DSR, 1 × 109 CFU d-1 ) for 12 weeks. DSR improves the obesity state, as evidenced by the i) suppressed obesity-related markers, e.g., gains in body weight and fat mass, ii) reduced serum and liver triglyceride levels, iii) upregulated ß-oxidation and downregulated lipogenesis-related genes in the liver, iv) reduced serum leptin levels, v) altered microbial communities, vi) increased regulatory T cell immunity, and vii) suppressed inflammatory response. In addition, correlation analysis shows that Akkermansia muciniphila and the genus Anaerostipes, which are increased in the DSR group, are negatively correlated with obesity-related markers, but Mucispirillum schaedleri, which is increased in the high-fat-diet (HFD) group, is positively correlated with serum leptin level. CONCLUSION: Lactobacillus plantarum DSR M2 and DSR 920 are candidate probiotics for the prevention and amelioration of obesity.

Antiobesity and Cholesterol-Lowering Effects of Dendropanax morbifera Water Extracts in Mouse 3T3-L1 Cells.

Obesity is the most common metabolic disease in developed countries and has become a global epidemic in recent years. Obesity is associated with various metabolic abnormalities, including glucose intolerance, insulin resistance, type 2 diabetes, dyslipidemia, and hypertension. Leaves from the plant Dendropanax morbiferus are beneficial to health as they contain high levels of vitamin C and tannin. There have been seminal studies on the anticancer, antimicrobial, antidiabetes, and antihyperglycemic effects of treatments with D. morbiferus trees. Herein, we investigated the toxicity of D. morbiferus water (DLW) extracts in vitro, and demonstrated no toxicity at 5-500 µg/mL in 24-72-h experiments with 3T3-L1 cells. The DLW increased cell viability at 48 h and inhibited adipogenesis in 3T3-L1 cells by reducing intracellular triglyceride levels and glucose uptake. In addition, mRNA and protein expression levels of adipogenesis-related genes were lowered by DLW, suggesting antiobesity effects in mouse 3T3-L1 cells. Because few studies have demonstrated cholesterol-lowering effects of D. morbiferus, we investigated the activities of adipogenic transcriptional factors following treatments of 3T3-L1 cells with D. morbiferus and observed increased CEBPα, CEBPß, PPARγ, and SREBP1 activities in the cells, indicating that DLW extracts inhibit adipogenesis.

Effects of Aldosterone on Chemerin Expression And Secretion in 3T3-L1 Adipocytes.

Aldosterone plays a pivotal role in the pathogenesis of metabolic syndrome and cardiovascular disease; however, the underlying mechanisms have not been clarified. Chemerin has been characterized as an adipokine with crucial roles in obesity-associated disorders and cardiovascular homeostasis. The aim of the present study was to investigate the direct effects of aldosterone on chemerin expression and secretion in 3T3-L1 adipocytes and to identify the potential signalling pathways involved. Chemerin mRNA levels were measured using real-time PCR, whereas the levels of secreted chemerin in the culture media were determined using ELISA. Treatment with aldosterone induced time- and dose-dependent increases of chemerin gene expression and protein secretion, and effect that was mediated through the mineralocorticoid receptor. Signalling studies suggested that the NF-κB pathway is involved in aldosterone-induced chemerin expression. Taken together, our data demonstrate a direct interaction between aldosterone and chemerin in adipocytes, which may be an underlying mechanism linking aldosterone-associated metabolic abnormalities and cardiovascular disease.

Heme oxygenase-1 mediates anti-adipogenesis effect of raspberry ketone in 3T3-L1 cells.

BACKGROUND: Obesity is caused by excessive accumulation of body fat and is closely related to complex metabolic diseases. Raspberry ketone (RK), a major aromatic compound in red raspberry, was recently reported to possess anti-obesity effects. However, its mechanisms are unclear. AIM: Adipogenesis plays a critical role in obesity and, therefore, this study aimed to investigate the effect and mechanisms of action of RK on adipogenesis in 3T3-L1 preadipocytes. MATERIALS AND METHODS: 3T3-L1 preadipocytes were differentiated in medium containing insulin, dexamethasone, and 1-methyl-3-isobutylxanthine. Adipocyte lipid contents were determined using oil-red O staining while adipogenic transcription factor and lipogenic protein expressions were determined using western blotting. RESULTS: RK (300-400µM) strongly inhibited lipid accumulation during 3T3-L1 preadipocyte differentiation into adipocytes. RK reduced the CCAAT/enhancer-binding protein-α (C/EBP-α), peroxisome proliferation-activated receptor-γ (PPAR-γ), fatty acid synthase (FAS), and fatty acid-binding protein 4 (FABP4) expressions and increased heme oxygenase-1 (HO-1), Wnt10b, and ß-catenin expressions in 3T3-L1 adipocytes. Additionally, RK inhibited lipid accumulation, and adipogenic transcription factor and lipogenic protein expressions were all decreased by inhibiting HO-1 or ß-catenin using tin protoporphyrin (SnPP) or ß-catenin short-interfering RNA (siRNA), respectively. Furthermore, Wnt10b and ß-catenin expressions were negatively regulation by SnPP. CONCLUSION: RK may exert anti-adipogenic effects through modulation of the HO-1/Wnt/beta-catenin signaling pathway.

Chemical Compounds in Natural Medicines That Affect Macropharges and Adipocyte Cells.

Macrophages play major roles in inflammation, immunity and host defense mechanisms. Once activated they produce and release cytokines, oxygen and nitrogen species, and eicosanoids. The best characterized stimuli to induce the transcription of genes encoding pro-inflammatory proteins in macrophages in vitro is bacterial lipopolysaccharide (LPS). LPS could be used alone or in combination with recombinant mouse interferon-γ (IFN-γ). Such stimulation results in cytokine release and the synthesis of enzymes such as cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS). The nitric oxide (NO) radical is known to play a central role in inflammatory and immune reactions for self-protection. However, the excessive production of NO may lead to tissue damage. In inflammatory diseases such as rheumatoid arthritis, excessive NO production by activated macrophages has been observed. Adipose tissue is composed of various cell types such as mature adipocytes, preadipocytes, fibroblasts, endothelial cells, vascular cells, and macrophages. Recent studies indicate that obesity is associated with low-grade chronic inflammation of adipose tissues, and that such inflammation is one of the potential mechanisms leading to the insulin resistance. It has been demonstrated that obese adipose tissue is characterized by the increased infiltration of macrophages. Therefore, we attempted to identify natural anti-inflammatory compounds that not only inhibit the secretion of NO from RAW 264.7 cells, but also inhibit triglyceride accumulation in 3T3-L1 adipocytes. This review describes the NO prpduction inhibitory activity or the TG accumulation inhibitory activity of the compounds obtained from 18 plants and a fungi that have been used as traditional medicines.