Obesity, an intractable metabolic disease, currently has no medical treatment without side effects, so studies have been actively carried out to find natural compounds that have anti-obesity activity with minimum side effects. In this study, the anti-obesity effects of water extracts of seven Capsicum annuum L. varieties being Putgochu (Pca), Oyee gochu (Oca), Kwari putgochu (Kca), Green pepper (Gca), Yellow paprika (Yca), Red paprika (Rca) and Cheongyang gochu (Cca), were examined through the evaluation of lipoprotein lipase (LPL) mRNA expression level in 3T3-L1 cells (mouse pre-adipocytes). After capsaicin elimination by chloroform defatting, freeze-dried powder of Cca was treated to 3T3-L1 cells and anti-obesity effects were examined by determining the LPL mRNA level using the RT-PCR method. Of the primary fractions, only proven fractions underwent secondary and tertiary refractionating to determine anti-obesity effects. From seven different Capsicum annuum L., there was a significant decrease of the LPL mRNA expression level of 50.9% in Cca treatment compared to the control group. A significant decrease of the LPL mRNA expression level was shown in primary fractions (Fr) 5 (36.2% decrease) and 6 (30.5% decrease) of the Cca water extracts. Due to the impurities checked by UPLC chromatography, Fr 5 and 6 were refractionated to determine the LPL mRNA expression level. Treatment of Fr 6-6 (35.8% decrease) and Fr 5-6 (35.3% decrease) showed a significant decrease in the LPL mRNA expression level. When analyzed using UPLC, major compounds of Fr 6-6 and Fr 5-6 were very similar. Subsequently, we refractionated Fr 6-6 and Fr 5-6 to isolate the major peak for structure elucidation. Treatment of Fr 5-6-1 (26.6% decrease) and Fr 6-6-1 (29.7% decrease) showed a significant decrease in the LPL mRNA expression level. Consequently, the fractions may have a possibility to ameliorate obesity through the decrease of the LPL mRNA expression level.
3T3-L1 Cells ; Capsaicin ; Capsicum ; Chloroform ; Chromatography ; Lipoprotein Lipase ; Lipoproteins ; Metabolic Diseases ; Obesity ; RNA, Messenger ; Water

Differentiation of preadipocyte, also named adipogenesis, leads to the phenotype of mature adipocyte that is filled with many lipid droplets. Excessive lipid accumulation in adipocytes leads to the development of obesity. In this study, we investigated the effect of 11 different natural compounds on lipid accumulation during the differentiation of 3T3-L1 preadipocytes into 3T3-L1 adipocytes. Strikingly, among the natural compounds, cryptotanshinone at 10 µM most strongly reduced triglyceride (TG) contents in 3T3-L1 cells after 8 days of the differentiation. Furthermore, cryptotanshinone at 10 µM significantly suppressed lipid accumulation in 3T3-L1 cells after 8 days of the differentiation. Cryptotanshinone at 1 to 10 µM tested did not affect the survival of 3T3-L1 cells after 8 days of the differentiation. On mechanistic levels, cryptotanshinone time-differentially decreased the expression levels of CCAAT/enhancer-binding protein-α (C/EBP-α), peroxisome proliferator-activated receptor-γ (PPAR-γ), fatty acid synthase (FAS), and perilipin A but also the phosphorylation levels of signal transducer and activator of transcription-3 (STAT-3) during the 3T3-L1 cell differentiation. Taken together, these findings demonstrate that cryptotanshinone inhibits lipid accumulation in differentiating 3T3-L1 cells, which appears to be mediated through the reduced expression and/or phosphorylation levels of C/EBP-α, PPAR-γ, FAS, Perilipin A, and STAT-3.
3T3-L1 Cells ; Adipocytes ; Adipogenesis ; Lipid Droplets ; Obesity ; Peroxisomes ; Phenotype ; Phosphorylation ; Transducers ; Triglycerides

It is noted that chalcone derivatives have characteristic diverse pharmacological properties, and that precise evidence has been growing that they could regulate a tumor necrosis factor-α (TNF-α) induced insulin resistance. The purpose of the present investigation is to elucidate the effects of the identified chalcone derivatives on adipogenesis, and to find the underlying mechanism of action in that case. Consequently, we first investigated whether the chalcone derivatives could affect the identified PPARγ-induced transcriptional activity on the proliferator-activated receptor response elements (PPRE) at target promoters, and find that trans-chalcone most significantly increased the PPARγ-induced transcriptional activity. Additionally, we confirmed that there were up-regulatory effects of trans-chalcone during the adipogenesis and lipid accumulation, and on the mRNA of adipogenic factors in 3T3-L1 cells. Next, we examined the effect of trans-chalcone on the inhibition induced by TNF-α on adipogenesis. To that end, we noted that the treatment with trans-chalcone attenuated the effect of TNF-α mediated secretion of various adipokines that are involved in insulin sensitivity. For this reason, we noted that this study clearly demonstrates that trans-chalcone enhanced adipogenesis, in part, by its potent effect on PPARγ activation and by its reverse effect on TNF-α.
3T3-L1 Cells ; Adipogenesis ; Adipokines ; Chalcone ; Insulin Resistance ; Necrosis ; Response Elements ; RNA, Messenger

OBJECTIVE: To establish a simple, low-cost and time-saving method for primary culture of mature white adipocytes from mice. METHODS: Mature white adipocytes were isolated from the epididymis and perirenal area of mice for primary culture using a modified mature adipocyte culture method or the ceiling culture method. The morphology of the cultured mature adipocytes was observed using Oil Red O staining, and the cell viability was assessed with CCK8 method. The expression of PPARγ protein in the cells was detected with Western blotting, and the mRNA expressions of CD36, FAS, CPT1A and FABP4 were detected using RT-qPCR. RESULTS: Oil Red O staining showed a good and uniform morphology of the adipocytes in primary culture using the modified culture method, while the cells cultured using the ceiling culture method exhibited obvious morphological changes. CCK8 assay showed no significant difference in cell viability between freshly isolated mature white adipocytes and the cells obtained with the modified culture method. Western blotting showed that the freshly isolated adipocytes and the cells cultured for 72 h did not differ significantly in the expression levels of PPARγ protein (P=0.759), which was significantly lowered in response to treatment with GW9662 (P < 0.001). GW9662 treatment of the cells upregulated mRNA expressions of CD36 (P < 0.001) and CPT1A (P=0.003) and down-regulated those of FAS (P=0.001) and FABP4 (P < 0.001). CONCLUSION We established a convenient and time-saving method for primary culture mature white adipocytes from mice to facilitate further functional studies of mature adipocytes.
Male ; Mice ; Animals ; Adipocytes, White/metabolism* ; PPAR gamma/metabolism* ; RNA, Messenger ; Cell Differentiation ; 3T3-L1 Cells

3T3-L1 preadipocyte were differentiated to adipocytes, and then treated with 0, 10, 20, and 40 microg/mL of peanut sprout ethanol extract (PSEE). The main component of PSEE is resveratrol which contained 5.55 mg/mL of resveratrol. The MTT assay, Oil-Red O staining, glycerol-3-phosphate dehydrogenase (GPDH) activity, and the triglyceride concentration were determined in 3T3-L1 cells. MMP-2 and MMP-9 activities as well as mRNA expressions of C/EBP beta and C/EBP alpha were also investigated. As the concentration of PSEE in adipocytes increased, the cell proliferation was decreased in a dose-dependent manner from 4 days of incubation (P < 0.05). The GDPH activity (P < 0.05) and the triglyceride concentration (P < 0.05) were decreased as the PSEE treatment concentration increased. The mRNA expression of C/EBPbeta in 3T3-L1 cells was significantly low in groups of PSEE-treated, compared with control group (P < 0.05). The MMP-9 (P < 0.05) and MMP-2 (P < 0.05) activities were decreased in a dose-dependent manner as the PSEE concentration increased from 20 microg/mL. In conclusion, it was found that PSEE has an effect on restricting proliferation and differentiation of adipocytes.
3T3-L1 Cells ; Adipocytes ; Animals ; Cell Proliferation ; Ethanol ; Fibroblasts ; Glycerolphosphate Dehydrogenase ; Matrix Metalloproteinases ; Mice ; RNA, Messenger ; Stilbenes

BACKGROUND: The target of the treatment of metabolic syndrome and diabetes is an improvement of insulin resistance. D-chiro-inositol (DCI) plays a role in a phospholipid mediating intracellular insulin action. In the previous studies, the urine level of DCI were decreased in the diabetic animal with insulin resistance. Some clinical studies showed that DCI improved a glucose level and HbA1c. Therefore we studied the relationship between DCI and glucose metabolism, especially insulin resistance. METHODS: To investigate the mechanism of DCI affecting the glucose metabolism, we examined the effects of DCI on 2-deoxyglucose uptake, gene expression of adipocytokines and AMPK pathway by using RT-PCR and western blot in 3T3-L1 cells. RESULTS: Insulin-stimulated 2-deoxyglucose uptake increased in DCI-treated cells by about 1.2-fold (relative to the control) and was inhibited by phosphoinositide 3-kinase (PI3 Kinase) inhibitors (Wortmanin, LY294002) and AMPK inhibitor (STO-609). In Western blot analysis, it didn't show the difference of phosphorylation of Akt and AMPK between DCI-treated group and control in 3T3-L1 cells. However, DCI decreased the gene expression of resistin in 3T3-L1 cells. CONCLUSION: DCI may involve other pathway of insulin signaling, but not PI3 Kinase and AMPK signaling pathways and it may be useful in managing metabolic syndrome by improving insulin resistance through increasing glucose uptake and decreasing resistin relevant to insulin resistance.
3T3-L1 Cells ; Adipokines ; Animals ; Blotting, Western ; Deoxyglucose ; Gene Expression ; Glucose ; Insulin ; Insulin Resistance ; Negotiating ; Phosphorylation ; Phosphotransferases ; Resistin

Ten novel compounds were designed and synthesized on the basis of compound 1, their insulin-sensitizing activities were evaluated in 3T3-L1 cells. Results showed that compound 10 exhibited strong differentiation-stimulating activity on 3T3-L1 cells model, which indicated that compound 10 may possess well insulin-sensitizing activity.
3T3-L1 Cells ; Animals ; Benzopyrans ; chemical synthesis ; pharmacology ; Drug Design ; Hypoglycemic Agents ; chemical synthesis ; pharmacology ; Insulin ; pharmacology ; Mice

Objective To explore the effect of the action time of inducers on the differentiation of 3T3-L1 cells to adipocytes. Methods According to the "Cocktail" method,3T3-L1 cells were divided into three groups according to the action time of inducers,with the action time being 2,3 or 4 days,respectively. Cell morphology was observed using inverted microscope and adipose content were detected by Oil red "O" staining and detection of triglyceride. The cell viability was identified by trypan blue staining method. Results The proportion of samples (n=12) with differentiation rate above 80% in group A was 66% (12/18),while the differentiation rate of all the samples (n=18)in group B and group C were above 80%. For the Oil red "O",the OD value at 510 nm in group C was 2.59±0.17,which was significantly higher than that in group A (2.12±0.47;F=6.62,P=0.0001)and group B (2.20±0.17;F=5.15,P=0.0001),while no significant difference was found between group A and group B (F=1.14,P=0.74). As for the triglyceride,the value in group C was (1351.04±119.01)ng/ml,which was significantly higher than that in group A[ (1077.88±272.75)ng/ml;F=6.73,P=0.001] and group B [(1089.38±115.39)ng/ml;F=5.78,P=0.001],while no difference was found between group A and group B (F=0.27,P=0.64). The cell viability in group A,B,and C was (98.3±1.2)%,(98.5±1.8)%,and (98.9±2.1)%,respectively,showing no significant difference (F=0.18,P=0.83). Conclusions The modified procedure for the differentiation of 3T3-L1 cells to adipocytes can increase the differentiation rate and thus may be applied for establishing adipocyte models. The recommended action time is three days.
3T3-L1 Cells ; Adipocytes ; cytology ; drug effects ; Animals ; Cell Culture Techniques ; Cell Differentiation ; drug effects ; Mice ; Time Factors

AIMTo investigate the effect of interleukin-8 (IL-8) on the differentiation and clonal expansion of 3T3-L1 preadipocyte during the differentiation period.

METHODSThe morphological changes of 3T3-L1 cells during differentiation after the treatment of IL-8 was observed by Oil-Red O staining. Glycerol-3-phosphate dehydrogenase (GPDH) activity was measured by a spectrophotometric method. MTT method and 3H-TdR incorporation were applied to examine the changes of cell proliferation and DNA synthesis in clonal expansion of 3T3-L1 cells. Cell cycle analysis was taken by flow cytometry.

RESULTSIL-8 could inhibit the differentiation and GDPH activity in a dose dependent manner. IL-8 decreased the cell proliferation and DNA synthesis in clonal expansion after induction. Also, the proportion of cells in G1 phase was increased and that of cells in S and G2 phase was declined after the treatment of IL-8.

CONCLUSIONIL-8 inhibits the differentiation of 3T3-L1 preadipocytes by decreasing the clonal expansion of the cells.

3T3-L1 Cells ; Adipocytes ; cytology ; metabolism ; Animals ; Cell Cycle ; Cell Differentiation ; Cell Proliferation ; drug effects ; Interleukin-8 ; pharmacology ; Mice

OBJECTIVETo investigate the effects of rapamycin on cholesterol homeostasis and secretory function of 3T3-L1 cells.

METHODSThe in vitro cultured 3T3-L1 cells (preadipocytes) were divided into control group, rapamycin 50 nmol/L group, rapamycin 100 nmol/L group, and rapamycin 200 nmol/L group. Intracellular cholesterol level was measured by oil red O staining and high performance liquid chromatography. The secretion levels of leptin and adiponectin were assayed by enzyme-linked immunosorbent assay. The mRNA and protein expressions of peroxisome proliferator-activated receptor (PPARgamma) were assayed by quantitative real-time polymerase chain reaction and Western blot.

RESULTSOil red O staining showed rapamycin down-regulated 3T3-L1 cells differentiation and lipid accumulation. Quantitative measurement of cholesterol with high performance liquid chromatography showed that the concentrations of free cholesterol in rapamycin treatment groups had a significant reduction. The concentrations of free cholesterol in the control group, rapamycin 50 nmol/L group, rapamycin 100 nmol/L group, and rapamycin 200 nmol/L group were (12.89 +/- 0.16), (9.84 +/- 0.45), (9.39 +/- 0.46), and (8.61 +/- 0.34) mg/ml, respectively (P < 0.05), and the concentrations of total cholesterol were (12.91 +/- 0.50), (9.94 +/- 0.96), (10.45 +/- 2.51), and (9.53 +/- 0.63) mg/ml, respectively. The leptin concentrations in the control group, rapamycin 50 nmol/L group, rapamycin 100 nmol/L group, and rapamycin 200 nmol/L group were (19.02 +/- 0.52), (16.98 +/- 0.11), (15.62 +/- 0.01), and (13.84 +/- 0.66) ng/ml, respectively. The mRNA expressions of PPARgamma in the rapamycin 50 nmol/L group, rapamycin 100 nmol/L group, and rapamycin 200 nmol/L group were significantly lower than that in control group (P < 0.05). The protein expressions of PPARgamma in the rapamycin 50 nmol/L group, rapamycin 100 nmol/L group, and rapamycin 200 nmol/L group were 80%, 74%, and 61% of that in control group (P < 0.05). After the cells were treated with rapamycin 100 nmol/L, PPARgamma blocking agent GW9662 10 micromol/L, and PPARgamma agonist troglitazone 10 micromol/L, respectively, for 96 hours, the mRNA expression of PPARgamma was (0.60 +/- 0.14), (0.67 +/- 0.03), and (1.30 +/- 0.14) of that in control group (P < 0.05). The protein expression showed a similar trend with mRNA expression (P < 0.05). After the cells were treated with rapamycin 100 nmol/L, PPARgamma blocking agent GW9662 10 micromol/L, and PPARgamma agonist troglitazone 10 micromol/L, respectively, for 96 hours, the expression of leptin in the control group, rapamycin 50 nmol/L group, rapamycin 100 nmol/L group, and rapamycin 200 nmol/L group was (19.02 +/- 0.52), (15.62 +/- 0.10), and (14.45 +/- 1.01) and (18.07 +/- 0.66) ng/ml, respectively (P < 0.05 compared with the control group).

CONCLUSIONSBy downregulating the expression of PPARgamma, rapamycin can decrease cholesterol accumulation in 3T3-L1 cells and inhibit its leptin-secreting capability. This finding may provide a possible explanation for rapamycin-induced hyperlipidemia in clinical practice.

3T3-L1 Cells ; Adipocytes ; drug effects ; metabolism ; Animals ; Cholesterol ; metabolism ; Leptin ; metabolism ; Mice ; PPAR gamma ; genetics ; metabolism ; Sirolimus ; pharmacology