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ObjectiveTo investigate the effect of rutin on the browning of 3T3-L1 preadipocytes and the mechanism. MethodCell counting kit-8 （CCK-8） assay was used to detect the effect of different concentration of rutin （3.125， 6.25， 12.5， 25， 50， 100， 200 μmol·L-1） on 3T3-L1 cell activity， and Western blot to examine the effect of rutin （12.5， 25， 50 μmol·L-1） on the expression of thermogenesis-associated proteins uncoupling protein 1 （UCP1）， PR domain containing 16 （PRDM16） and peroxisome proliferator-activated receptor γ coactivator-1α （PGC-1α） in adipocytes. After the optimal concentration of rutin was determined， the effect of rutin on lipid droplet formation in adipocytes was observed based on oil red O staining， and the expression of nuclear respiratory factor 1 （NRF1）， nuclear respiratory factor 2 （NRF2） and mitochondrial transcription factor A （TFAM）， which were the landmark proteins of mitochondrial biosynthesis， was detected by Western blot. ResultCompared with the blank group， 200 μmol·L-1 rutin inhibited 3T3-L1 cell activity （P<0.01）. Compared with the blank group， at the concentration of 12.5， 25， 50 μmol·L-1 rutin significantly promoted the expression of thermogenesis-associated proteins （UCP1， PRDM16， and PGC-1α） （P<0.01）， which was determined as the optimal concentration. Compared with the blank group， 50 μmol·L-1 rutin significantly increased the immunofluorescence intensity of mitochondrial UCP1 protein in 3T3-L1 cells （P<0.01） and the expression of the markers of mitochondrial biosynthesis （NRF1， NRF2， and TFAM） （P<0.01）. In addition， 50 μmol·L-1 rutin significantly inhibited lipid droplet formation of 3T3-L1 adipocytes （P<0.01）. ConclusionRutin inhibited lipid droplet deposition in 3T3-L1 adipocytes and increased the expression of thermogenesis-related proteins （UCP1， PRDM16， and PGC-1α） and markers of mitochondrial biosynthesis （NRF1， NRF2， and TFAM）， thereby inducing the browning of 3T3-L1 adipocytes. This lays a basis for the development of drugs that safely regulate the browning of white cells.
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Aim To explore whether alisol B 23-acetate possesses the therapeutic potential for treatment of type 2 diabetes mellitus ( T2DM ). Methods T2DM mouse model was established by combined administration of streptozotocin and nicotinamide. After three weeks of oral administration of rosiglitazone or alisol B 23-acetate, the blood glucose of type 2 diabetic mice was measured. Oral glucose tolerance test(OGTT) was carried out the next day. Rosiglitazone was chosen as positive drug. 2-[N-(7-nitrobenz-2-oxa-l, 3-diazol-4-yl) amino]-2-deoxy-D-glucose (2-NBDG) uptake assay in adipocytes was adopted to test whether alisol B 23-acetate had effect on glucose uptake in cells. 3T3-Ll pre-adipocytes differentiation model was performed to evaluate whether alisol B 23-acetate promoted adipo-genesis. Results Mice exhibited significantly higher blood glucose concentration after intraperitoneal injection of streptozotocin and nicotinamide for three weeks, as examined by blood glucose concentration on day 21 and OGTT on day 22, compared with normal mice in blank control group. After orally administrating alisol B 23-acetate at dose of 5 mg • kg-1 , 10 mg • kg-1 ,20 mg • kg-1 daily for three weeks, respectively, or orally administered rosiglitazone at dose of 10 mg • kg-1 daily for three weeks, blood glucose greatly decreased in type 2 diabetic mice. Moreover, insulin resistance was also improved to a certain degree during OGTT. Furthermore, alisol B 23-acetate not only increased insulin-induced glucose uptake in adipocytes at the concentration of 30 mmol • L-1 glucose, but also accelerated 3T3-L1 pre-adipocytes differentiation process at concentration of 1 μmol • L-1 and 10 μmol • L-1 . Conclusions Alisol B 23-acetate reduces blood glucose of type 2 diabetic mice, promotes pre-adipocyte differentiation and increases glucose uptake in adipocytes; however, the mechanism of action needs further exploration.
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Aim To investigate the hypoglycemic effect of alismoxide. Methods Type 2 diabetes mellitus (DM) mouse model induced by combined administration of streptozotocin and nicotinamide was adopted. Three weeks later, blood glucose of blank control group and type 2 diabetic mouse model group was measured on day 21 , and oral glucose tolerance test(OGTT) was carried out on day 2 2 , respectively. After type 2 diabetic mouse model was successfully established, rosiglitazorie was chosen as positive drug. Oral administration of rosiglitazone at dose of 10 mgk g-1 daily was performed for three weeks in positive group. Oral administration of alismoxide at dose of 5 , 10 and 20 mg kg"1 daily for three weeks was carried out in alismoxide different dose group, respectively. Furthermore, influence of alismoxide on differentiation was investigated in 3T3-L1 pre-adipocytes, and Oil red 0 staining was adopted. Results Not only blood glucose was decreased by alismoxide in type 2 DM mice, but also hypoglycemic trend was exhibited during OGTT. Furthermore, at concentration of 0. 5 and 1 fimol L " 1 , alismoxide promoted 3T3-L1 pre-adipocyte differentiation. Conclusions It suggests that alismoxide might possess hypoglycemic property and accelerate pre-adipocyte differentiation; however, the mechanism involved needs further study.
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Aim To explore the effect of water extract of Radix Isatidis(WERI)on proliferation and differentiation of 3T3-L1 preadipocytes.Methods 3T3-L1 preadipocytes were cultured to explore the effect of WERI on their proliferation measured by MTT and flow cytometry.Oil red O staining was applied to investigate the effect of different concentrations of WERI on the differentiation of 3T3-L1 preadipocytes.
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AIM:To discuss the effect of Shenmai injection on insulin resistance ( IR) in 3T3-L1 cells and its mechanisms.METHODS:3T3-L1 preadipocytes were induced by chemical reagents to differentiate into fully differentiated adipocytes.Oil red O staining was used to detect the differentiation level of the adipocytes .The insulin-resistant 3T3-L1 cell model was demonstrated using insulin , which was confirmed by glucose concentration in cell supernatant .The IR cell model was given 10 μmol/L rosiglitazone , 25 and 50 g/L Shenmai injection and normal saline for comparison .MTT assay was used to assess the cell activity of 3T3-L1 cells which was treated with drugs for 8, 16, 24 and 36 h.Glucose oxidase method was used to detect the glucose concentration in the cell supernatant at 8, 16 and 24 h.The protein levels of glucose transporter-4 (GLUT4), phosphatidylinositol 3-kinase (PI3K), AKT and p-AKT were determined by Western blot .RE-SULTS:3T3-L1 adipocytes were successfully induced as shown by the positive oil red O staining .The IR cell model was demonstrated , and glucose concentration in the cell supernatant after treatment with Shenmai injection showed that Shenmai injection reduced the IR in 3T3-L1 cell model.The protein levels of GLUT4, PI3K and p-AKT increased compared to con-trol group.CONCLUSION:Shenmai injection reduces the IR in 3T3-L1 cell model, which functions by increasing the protein levels of GLUT4, PI3K and p-AKT.
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The 3T3-L1 preadipocytes were used as carriers in the investigation of total extract, n-butanol extract, CB-1 and CB-2 of Coreopsis tinctoria Nutt. on cell proliferation and differentiation. Three groups at different doses were set for each of the four extract regions of C. tinctoria Nutt., respectively. MTT assay was used to detect 3T3-L1cell proliferation by four extract regions of C. tinctoria Nutt. Oil Red O staining was used to analyze the formation and accumulation of cytoplasmic lipid during cell differentiation. The results showed that compared with the control group, there were significant inhibition on cell proliferation when thetotal extract of C. tinctoriaNutt. at 100 μg·mL-1, n-butanol extract at 0.5, 5, and 50 μg·mL-1, CB-1 and CB-2 at 50 μg·mL-1 (P< 0.01). N-butanol extract showed certain dose-dependent manner (r = -0.903). Oil Red O staining showed that compared with the control group, thetotal extract of C. tinctoria Nutt. at 1, 10, 100 μg·mL-1 can obviously inhibit cell differentiation, reduce the formation of cytoplasmic lipid (P< 0.01). N-butanol extract can inhibit cell differentiation in a dose-dependent manner (r= -0.779). CB-1 and CB-2 obviously inhibited cell differentiation at the concentration of 50 μg·mL-1 (P < 0.01). It was concluded that thetotal extract, n-butanol extract, CB-1 and CB-2 of C. tinctoria Nutt. can inhibit the proliferation and differentiation of 3T3-L1 preadipocytes and reduce the formation of cytoplasmic lipid.
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Objective To investigate the synergistic effect of 11 β-hydroxysteriod dehydrogenase (11 β-HSD1) and S100A16 on the differentiation of3T3-L1 preadipocytes and its mechanism.Methods Lentiviral vectors PLJM1-11β-HSD1 and PLJM1-S100A16-GFP were respectively constructed and co-transfected into 3T3-L1 preadipocytes.The cell strains expressing 11 β-HSD1/S100A16 were screened with 2.5 μg/ml puromycin for two weeks.Western blot was employed to verify the lentiviral carrier transfection effects.The expressions of marker genes related to the adipocyte differentiation were detected by mean of realtime PCR.Oil red O staining was used to observe the lipid droplet accumulation and the content of triglyceride was measured after differentiation of preadipocytes.The effect of 11β-HSD1 and S100A16 on PPARγ promoter activity was detected by luciferase reporter gene.Results Compared with the empty vector group,the expressions of 11β-HSD1 and S100A16 protein in the lentivirus cotransfected 3T3-L1 cell strain were significantly higher.After 3T3-L1 cell strain co-expressing 1 1β-HSD1 and S100A16 was induced to differentiate for 8 days,the lipid droplets accumulation and triglyceride content were siginificantly increased,along with increased expressions of adipocyte differentiation marker genes such as PPARγ,CCAAT/enhancer binding protein α,lipoprotein lipase,fatty acid synthase,and adipocyte fatty acid-binding protein,in comparison with 11 β-HSD1 or S100A16 overexpression.The result of reporter gene indicated that 11 β-HSD1/ S100A16 enhanced PPARγ promoter activity.Conclusions 11β-HSD1 and S100A16 may jointly promote the differentiation of 3T3-L1 preadipocytes through a synergistic effect on PPARγexpression and play a critical role in the development of obesity.
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It has been reported that CLA decreases fat deposition in vivo and in vitro experiments. Among CLA isomers, c9t11 and t10c12 have been shown to exert active biological activities. For example, t10c12 reduces body weight and increases lean body mass, whereas, c9t11 has little effect on body fattness. However, the underlying molecular mechanism for the anti-obesity action of CLA isomers are not well understood. The purpose of this study was to examine the effects of t10c12 and c9t11 on lipid accumulation, cell proliferation, cell death and the expression levels of Ucp genes which are proposed as targets for anti-obesity in 3T3-L1 preadipocytes. Isomers of CLA at 50 micrometer were added into preadipocyte differentiation medium for 3, 6 and 9days. Control cells received only the vehicle in the differentiation medium. Cytochemical analyses for lipid accumulation, cell proliferation and apotosis were carried out to compare lipidogenesis and cellular activity. RT-PCR analysis of GAPDH, Ucp 2, 3 and 4 were also performed to find any modulatory effects of CLA isomers on the metabolic genes. Lipid accumulation indicated by Oil Red-O staining was inhibited in CLA isomers as compared to the control. T10c12 isomer showed less lipidogenesis than c9t11 did. A decrease occurred in CLA isomers as shown by BrdU incorporation. Apotosis has occured at higher level in t10c12 when compared to that of t9c11. Ucp 2, 3 and 4 genes were also upregulated in CLA isomers. T10c12 showed higher level of Ucp gene expressions than the c9t11 did. The biological activities of CLA isomers were also found to be different during differentiation of 3T3-L1 preadipocytes, suggesting that different isomers may be active in certain stage of lipidogenesis. The results indicate that both c9t11 and t10c12 CLA isomers decrease lipidogenesis, inhibit cell proliferation, increase cell death and upregulate in Ucp gene expressions during 3T3-L1 preadipocyte differentiation. T10c12 isomer was more effective than c9t11 in overall anti-obesity activity.
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Body Weight , Bromodeoxyuridine , Cell Death , Cell Proliferation , Gene Expression , Linoleic AcidABSTRACT
Objective To investigate the protein level of protein tyrosine phosphatase 1B (PTP1 B) during the differentiation of 3T3-L1 preadipocytes and the effects of tumor necrosis factor-?(TNF-?) and rosiglitazone on the expression of PTP1B during the differentiation,and to explore the relationship between PTP1B and adipecyte differentiation.Methods 3T3-LI preadipecytes were cultured in vitro and differentiated by three groups of inducers: basic inducers only (3-isobutyl-1-methylxanthine+dexamethasone+insulin,group C),20?g/L TNF-?+basic inducers (group CT) and 10~(-5) mol/L rosiglitazone+basic inducers (group CR).Protein level of PTP-1B in adipocytes during differentiation was detected by Western blot.Results Each group showed the relatively high level of PTP1B in 3T3-L1 preadipecytes,going down with the differentiation of adipecytes,and reaching the lowest level in fully-matured adipecytes.Comparing the late period of differentiation in these three groups,CT group was sluggishly differentiated with more PTP1B protein,and CR group showed active differentiation with the lowest level of PTP1B.Conclusion PTP1B decreases with the differentiation of adipoeytes.The effects of TNF-?and rosiglitazone on insulin sensitivity perhaps partly via their influences on PTP1B level in adipecytes.
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Objective To investigate the effect and underling mechanism of genistein(GEN)on adipogenesis in 3T3-L1 preadipocytes.Method 3T3-L1 preadipocytes were induced to differentiate in the presence of vehicle(MDI),GEN or SB203580.On D 6 after induction of differentiation,lipid accumulation was assessed using the dye Oil Red O,medium was collected and assayed for the content of NEFA and TG,and fatty acid synthase(FAS) expression was analyzed by Western blotting.After pretreatment with GEN for 30 min,3T3-L1 preadipocytes were stimulated with insulin for 5 or 10 min.The phosphorylation of p38 MAPK(p38) was analyzed by Western blotting.Results GEN inhibited lipid accumulation and decreased NEFA and TG content of 3T3-L1 on D 6 after the induction of differentiation.Pretreatment with GEN inhibited phosphorylation of p38 when stimulated with insulin.Furthermore,GEN inhibited the expression of FAS.SB203580,a p38 inhibitor,mimicked the FAS inhibition effect of genistein,which suggested that the inhibitory effect of GEN on FAS was partially via the p38 pathway.Conclusion Geinstein attenuates the differentiation of 3T3-L1 via the inhibition of p38 and FAS.