Shenmai injection improves insulin resistance in 3T3-L1 cells

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. RESULTS: 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 control 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.     

Effect of protein kinase C signal pathway on resistin expression in 3T3-L1 adipocytes

AIM：To investigate the effect of protein kinase C on resistin expression in 3T3-L1 adipocytes.METHODS：The differentiated 3T3-L1 adipocytes were incubated with 50 nmol/L phorbol 12-myristate 13-acetate (PMA) or 5 μmol/L Ro-31-8220 for 24 h.Expression of resistin mRNA was detected by RT-PCR and expression of resistin protein was detected by Western blotting.RESULTS：Compared with control,PMA increased the expression of resistin mRNA and protein in 3T3-L1 adipocytes significantly (P<0.01),while Ro-31-8220 decreased the expression of resistin mRNA and protein in 3T3-L1 adipocytes obviously (P<0.01).CONCLUSION：Protein kinase C signal pathway may regulate resistin expression in 3T3-L1 adipocytes.     

Apelin-13 inhibits nicotine-induced apoptosis of H9c2 cells via PI3K/Akt signaling pathway

AIM:To investigate the effect of apelin-13 on nicotine-induced apoptosis of cardiomyocytes and its potential molecular mechanism. METHODS:Rat H9c2 cells were treated with nicotine (10 μmol/L) to induced apoptosis. Flow cytometry was used to detect apoptotic rate. Western blot was used to determined the expression of related proteins. RESULTS:Compared with control group, nicotine treatment significantly increased the apoptotic rate of the H9c2 cells (P<0.01), and the protein levels of apoptosis-related proteins Bax and cleaved caspase-3, but markedly decreased the protein levels of Bcl-2, p-Akt, p-PI3K and APJ (P<0.05). Compared with nicotine group, apelin-13+nicotine significantly decreased the apoptotic rate of the H9c2 cells (P<0.01) and the the protein levels of Bax and cleaved caspase-3, but markedly increased the protein levels of Bcl-2, p-Akt, p-PI3K and APJ (P<0.05). Compared with apelin-13+nicotine group, apelin-13+nicotine+PI3K/Akt inhibitor LY294002 significantly increased the apoptotic rate of the H9c2 cells (P<0.01) and the protein levels of Bax and cleaved caspase-3, but markedly decreased the protein levels of Bcl-2, p-Akt and p-PI3K (P<0.05). CONCLUSION:Apelin-13 inhibits nicotine-induced apoptosis of H9c2 cells through PI3K/Akt signaling pathway.     

Total triterpenoids from Psidium Guajava leaf improves insulin resistance in 3T3-L1 adipocytes

AIM: To investigate the effects of total triterpenoids from Psidium guajava leaf (TTPGL) on 3T3-L1 adipocyte insulin resistance (IR) and to explore the possible mechanism. METHODS: 3T3-L1 pre-adipocytes were cultured and induced to differentiate into 3T3-L1 adipocytes, then treated with TTPGL (0.3, 1, 3, 10 μg/L) for 48 h. The cells were divided into 0.1% DMSO group, positive drug sodium orthovanadate (Van, 10 μmol/L) group, model group and control group. The effect of TTPGL on the cell activity of pre-adipocytes was detected by MTT assay and its influence on the cellular differentiation was observed by oil red O staining. The IR model of the 3T3-L1 adipocytes was established successfully and then treated with different drugs for 48 h. The glucose consumption in the supernatant of IR adipocyte's culture medium was assayed by glucose oxidase-peroxidase (GOD-POD), free fatty acid (FFA) levels were measured by colorimetric method, and adipocytokines levels were assayed by ELISA. The mRNA expression of protein tyrosine phosphatase-1B (PTP1B) of IR adipocyte was detected by real-time PCR. The protein levels of phosphorylated insulin receptor substrate 1/insulin receptor substrate 1 (p-IRS-1/IRS-1) and phosphorylated protein kinase B/protein kinase B (p-Akt/Akt) were determined by Western blot. RESULTS: Compared with DMSO group, TTPGL treatment significantly promoted the cell activity of 3T3-L1 pre-adipocytes and inhibited its differentiation (P < 0.01). TTPGL (1~10 μg/L) improved glucose consumption of IR adipocytes significantly (P < 0.01), with or without insulin stimulation, and TTPGL (0.3~3 μg/L) restrained FFA production remarkably(P < 0.01). Compared with model group, TTPGL (0.3 and 3 μg/L) significantly increased the secretion of adiponectin in IR adipocytes (P < 0.05), and inhibited the secretion of tumor necrosis factor-α (TNF-α) (P < 0.01). TTPGL (3 μg/L) restrained the secretion of resistin significantly (P < 0.05), and showed no significant effect on secretion of leptin. It also down-regulated the mRNA expression of protein tyrosine phosphates 1B (PTP1B) in IR adipocytes significantly (P < 0.01), and increased the protein levels of p-IRS-1/IRS-1. TTPGL (0.3 and 3 μg/L) up-regulated the protein level of p-Akt/Akt in IR adipocytes significantly (P < 0.05).CONCLUSION: TTPGL reduces IR in 3T3-L1 adipocytes. The mechanism may be that TTPGL significantly down-regulated mRNA expression of PTP1B and increased the protein levels of p-IRS-1/IRS-1 and p-Akt/Akt in IR adipocytes.     

CTRP3 increased insulin sensitivity of insulin resistant 3T3-L1 adipocytes via decreasing expression of inflammatory factors

AIM:To investigate the effects of C1q/TNF related protein 3 (CTRP3) on the insulin sensitivity of insulin resistant 3T3-L1 adipocytes. METHODS:The insulin resistance model of 3T3-L1 adipocytes was induced by palmic acid cultivation. The adipocytes were treated with different concentrations of recombinant CTRP3 protein (10, 50, 250,1 250 μg/L) for 12 h, and for different times (2, 6, 12, 24 h) at the concentration of 250 μg/L. The glucose consumption was detected by the glucose oxidase method. The glucose transport ratio was measured by 2-deoxidation-［3H］-glucose intake method. The contents of TNF-α and IL-6 in the supernatant were detected by ELISA. The mRNA expression of TNF-α, IL-6 and glucose transporter-4 (GLUT-4) was measured by real-time PCR. The protein expression of GLUT-4 was detected by Western blotting. RESULTS:Compared with normal control (NC) group, the glucose consumption and glucose intake ratio of insulin resistance (IR) group was decreased by 50.6% and 57.9%, respectively. Compared with IR group, with the increase in CTRP3 (10, 50, 250,1 250 μg/L) in intervention groups, the glucose consumptions were increased by 22.1%, 42.9%, 76.6% and 80.5%, respectively, and the glucose intake ratios were increased by 39.0%, 68.0%, 108.0% and 111.0%, respectively. With the increased duration (2, 6, 12 and 24 h) of CTRP3 treatment at the concentration of 250 μg/L, the glucose intake ratio was increased by 23.0%, 79.0%, 109.0% and 114.0%, respectively. The contents of TNF-α and IL-6 in the supernatant were decreased by 17.4% and 17.1% respectively as treated with CTRP3 at the concentration of 250 μg/L for 12 h, and the mRNA expression of TNF-α and IL-6 was decreased by 26.0% and 18.9% respectively, while the mRNA and protein expression of GLUT-4 was increased by 61.5% and 55.6% respectively. CONCLUSION: CTRP3 may increase the insulin sensitivity of insulin resistant 3T3-L1 adipocytes by down-regulating the expression of inflammatory factors, improving the insulin signal transduction and increasing the expression of GLUT-4.     

Knockdown of growth hormone receptor prevents growth hormone induced inflammatory cytokine production in 3T3-L1 adipocytes

AIM: To investigate the effect of growth hormone receptor (GHR) knockdown on nuclear factor-κB (NF-κB) activity and inflammatory cytokine production stimulated by growth hormone (GH) in 3T3-L1 adipocytes. METHODS: The specific siRNA for GHR was transfected into 3T3-L1 adipocytes to silence GHR expressions. The effects of GH on NF-κB activation and inflammatory cytokine production in 3T3-L1 adipocytes transfected with siRNA-GHR or siRNA-control were measured by dual-luciferase system analysis, real-time RT-PCR and ELISA. RESULTS: The protein expression of GHR was diminished after transfection with GHR specific siRNA. Dual-luciferase reporter system analysis revealed that GHR knockdown resulted in attenuation of GH-stimulated NF-κB activation in the 3T3-L1 adipocytes. GHR knockdown ameliorated the GH-induced production of inflammatory cytokines TNF-α, IL-1β, IL-6, MCP-1 and MIP-1α in the 3T3-L1 adipocytes. CONCLUSION: Knockdown of GHR might be efficacious to prevent GH-induced inflammatory responses in the 3T3-L1 adipocytes.     

Role of asiatic acid in treatment of insulin resistance in mouse adipocytes

AIM：To investigate the effects of asiatic acid, one of triterpenoids from Psidium guajava leaves, on the proliferation and differentiation of 3T3-L1 preadipocytes, and glucose and lipid metabolism of insulin-resistant adipocytes. METHODS：The proliferation of 3T3-L1 preadipocytes was tested by MTT assay, and the accumulation of lipid droplets in differentiated preadipocytes was measured by oil red O staining. The insulin-resistant cell model was established by exposure of the cells to dexamethasone. The cellular glucose uptake was determined by glucose oxidase-peroxidase assay. The free fat acid (FFA) concentration was detected by colorimetric method. Secreted adiponectin were measured by ELISA. The protein levels of peroxisome proliferator-activated receptor γ (PPARγ) and protein tyrosine phosphatase 1B (PTP1B) in insulin-resistant adipocytes were analyzed by Western blotting. RESULTS：Compared with medium group, asiatic acid increased the proliferation of 3T3-L1 preadipocytes and inhibited their differentiation at a concentration range of 10~100 μmol/L (P<0.05 or P<0.01). At concentrations of 30 μmol/L and 100 μmol/L, asiatic acid enhanced cellular glucose uptake in the insulin-resistant adipocytes both in basic and insulin-stimulation states. Asiatic acid decreased FFA production (P<0.05), and down-regulated the protein expression of PTP1B (P<0.05, or P<0.01). However, no effect on the secretion of adiponectin and the protein expression of PPARγ was observed (P>0.05). CONCLUSION：Asiatic acid enhances glucose uptake and inhibits FFA production in insulin-resistant adipocytes via down-regulating the protein expression of PTP1B, all of which play the roles of increasing insulin signaling sensitivity to improve insulin resistance.     

Effect of glucocorticoid on PI-3K and p38 MAPK signaling pathways in 3T3-L1 adipocytes

AIM: To study the effects of dexamethasone (DEX) on the glucose transport system and the PI-3K/Akt and p38 MAPK insulin signaling pathways in 3T3-L1 adipocytes,and to investigate the possible mechanism in glucocorticoid induced insulin resistance. METHODS: The 3T3-L1 adipocytes were exposed to DEX for 48 h and incubated with 100 nmol/L insulin for additional 30 min. The glucose uptake was measured by detecting the glucose content in cell culture supernatants. Then expression and distribution of Glut4 was measured. The insulin signaling proteins Akt,phospho-Akt,p38MAPK and phospho-p38MAPK were also measured with Western blotting. RESULTS: DEX inhibited insulin stimulated glucose transport capacity in 3T3-L1 adipocytes. DEX did not alter the amount of Glut4 protein in total cell lysates but attenuated the insulin-stimulated Glut4 translocation to the plasma membrane. DEX significantly inhibited insulin stimulated phosphorylation of Akt and p38 MAPK. CONCLUSION: These results suggest that DEX alters insulin stimulated glucose transport capacity in 3T3-L1 adipocytes,which is mediated by attenuating insulin stimulated activation of PI3K-Akt and p38 MAPK pathways,and reducing insulin stimulated Glut4 translocation and transport activity. These may lead to insulin resistance in 3T3-L1 adipocytes.     

Effects of adiponectin siRNA on the glucose transport in 3T3-L1 adipocytes

AIM: To construct the adenovirus vector with adiponectin (Acrp30) siRNA, and to observe its effect on the Acrp30 expression and glucose transport in 3T3-L1 adipocytes. METHODS: Mouse Acrp30 siRNA fragment was designed, synthesized and cloned into the adenovirus vector. 3T3-L1 cells were infected with the two recombinant adenoviruses, respectively. The mRNA expression and protein levels of Acrp30 in these cells were evaluated by RT-PCR and ELISA. Glucose transport was measured by 2-Deoxy-［3H］-D-glucose incorporation method. RESULTS: The recombinant adenoviruses were successfully constructed. They remarkably downregulated the expression of Acrp30 at both mRNA and protein levels in 3T3-L1 cells, and decreased the glucose transport in 3T3-L1 adipocytes (P<0.05). CONCLUSION: The siRNA expression vectors effectively inhibit the expression of Acrp30 in 3T3-L1 adipocytes, and decrease the glucose transport.     

Visfatin gene expression and secretion are regulated by aldosterone in 3T3-L1 preadipocytes and adipocytes

AIM: To explore the effect of aldosterone on visfatin gene expression and secretion in 3T3-L1 preadipocytes or adipocytes. METHODS: Aldosterone at concentration of 10-8 or 10-6 mol/L with or without 10-6 mol/L spironolactone was added to cultured 3T3-L1 preadipocytes or adipocytes for 24 h or 48 h. The mRNA levels of visfatin and mineralocorticoid receptor were measured using real time PCR. The concentration of visfatin in the culture medium was determined by ELISA. RESULTS: In 3T3-L1 preadipocytes treated with aldosterone, the mRNA expression of visfatin reduced and the mRNA expression of mineralocorticoid receptor(MR) increased, but the concentration of visfatin in culture medium was not regulated significantly by aldosterone. In adipocytes with aldosterone treatment, the mRNA expression of visfatin and visfatin concentration in culture medium reduced, and mRNA expression of MR increased. The effect of aldosterone was blocked by spironolactone to some extent. CONCLUSION: Aldosterone inhibits the gene expression and protein secretion of visfatin in 3T3-L1 adipocytes.     

Mechanism of interleukin-6 induced insulin resistance in 3T3-L1 adipocytes

AIM: To investigate the molecular mechanism of interleukin-6 induced insulin resistance in 3T3-L1 adipocytes.METHODS: 3T3-L1 adipocytes were treated with IL-6 at concentration of 20 μg/L within 48 hours. Insulin stimulated glucose uptake was measured by 2-deoxy ［3H］ glucose. Western blotting was used to measure insulin receptor substrate-1(IRS-1), protein kinase B(PKB) expression, tyrosine phosphorylation on IRS-1, and PKB phosphorylation. RESULTS: On basal status, glucose uptake in 3T3-L1 cells, PKB phosphorylation and tyrosine phosphorylation of IRS-1 were all at low level. Insulin stimulation induced a rapid increase in glucose uptake, PKB phosphorylation and IRS-1 tyrosine phosphorylation. IL-6 inhibited insulin-induced glucose uptake and PKB phosphorylation level about 50%. After IL-6 treatment, IRS-1 protein expression and tyrosine phosphorylation of IRS-1 were decreased 35% and 40%, respectively. The inhibitor of mammalian target of rapamycin(mTOR), rapamycin, reversed above effects of IL-6. CONCLUSION: IL-6 induced insulin resistance in 3T3-L1 adipocytes is related to decrease IRS-1 expression and impairs IRS-1 tyrosine phosphorylation. IL-6 induced insulin resistance in adipocytes may be related to the activity of mTOR.     

JNK mediates TNF-α or H2O2-induced insulin resistance in 3T3-L1 adipocytes

AIM: To study the role of c-Jun NH2-terminal kinase (JNK) in the development of insulin resistance induced by tumor necrosis factor-α (TNF-α) or H2O2 in 3T3-L1 adipocytes. METHODS: Differentiated 3T3-L1 adipocytes were pretreated with JNK1 small interfering RNA (siRNA) or JNK inhibitor SP600125, then exposed to 1 nmol/L of TNF-α or micromolar H2O2 generated by adding glucose oxidase (50 U/L) to the medium for 12 h. The cellular glucose uptake was determined by radioactive method. RESULTS: Compared to control adipocytes, 12 h incubation with TNF-α or H2O2 led to 50%-55% reduction (P<0.01) of the insulin-dependent glucose uptake. JNK1 siRNA transfection significantly inhibited JNK1 expression and blocked the TNF-α or H2O2-induced impairments of cellular glucose uptake. Pretreatment with SP600125 (20 μmol/L) resulted in significant increases in insulin-stimulated glucose uptakes in both TNF-α (66%) and H2O2 (62%) treated adipocytes (P<0.01). CONCLUSION: JNK plays a key role in TNF-α or H2O2 induced insulin resistance in 3T3-L1 adipocytes, and inhibition of JNK over-activation may be a new therapeutic target for insulin resistance.     

Regulatory effects of reactive oxygen species on the production of PAI-1 in 3T3-L1 adipocytes and its related possible mechanisms

AIM: To investigate the regulatory effects of reactive oxygen species (ROS) on the production of plasminogen activator inhibitor 1 (PAI-1), and try to determine the signaling cascades involved in it. METHODS: 3T3-L1 cells were cultured and differentiated into mature adipocytes. Cell viability was measured by MTT. The PAI-1 mRNA expression levels were evaluated by quantitative real-time PCR. Quantification of the PAI-1 protein levels secreted into conditioned medium was performed by multiplex immunoassay and sandwich ELISA. The phosphorylation status of protein kinases was determined by Bio-Plex phosphoprotein assays. RESULTS: In 3T3-L1 adipocytes, H2O2 significantly augmented the expression of PAI-1. Also, H2O2 activated several signaling pathways including ERK1/2, JNK, Akt, p70 S6K and JAK/STAT. Verified by protein kinase inhibitors, Akt, JAK/STAT and ERK1/2 may participate in the H2O2-induced increase in PAI-1. CONCLUSION: H2O2 markedly up-regulates the production of PAI-1 in 3T3-L1 adipocytes via some intracellular signaling pathways such as Akt, JAK/STAT and ERK1/2.     

Effects of TNF-α-induced changes of expression and activity of 11-β HSD-1 on the insulin sensitivity in 3T3-L1 adipocytes

AIM: To observe the effects of tumor necrosis factor-α（TNF-α）-induced changes of expression and activity of 11-beta-hydroxysteroid dehydrogenase type1(11-β HSD-1) on the insulin sensitivity in 3T3-L1 adipocytes.METHODS: 3T3-L1 adipocytes were treated with TNF-α and TNF-α combined with aspirin, 2’-hydroxyflavanone or RU486, then mRNA expression and activity of 11-β HSD-1 and insulin-stimulated glucose uptake were examined.RESULTS: TNF-α increased expression and activity of 11-β HSD-1 in 3T3-L1 adipocytes, and decreased insulin-stimulated glucose uptake. Aspirin decreased expression and activity of 11-β HSD-1 induced by TNF-α, and alleviated the inhibiting effect of TNF-α on insulin-stimulated glucose uptake. 11-β HSD-1 specific inhibitor 2’-hydroxyflavanone and cortisol-receptor antagonist RU486 also alleviated the inhibiting effect of TNF-α on insulin-stimulated glucose uptake. CONCLUSION: TNF-α may decrease the insulin sensitivity in 3T3-L1 adipocytes through increasing expression and activity of 11-β HSD-1.     

Effects of Retinervus luffae fructus polysaccharides on differentiation of 3T3-L1 pre-adipocytes

AIM: To observe the influence of polysaccharides extracted from Retinervus luffae fructus (RLF) on the differentiation of 3T3-L1 pre-adipocytes and to investigate its mechanism. METHODS: DEAE-cellulose column was used to isolate and purify RLF. The effect of RLF polysaccharides on 3T3-L1 pre-adipocyte differentiation was determined by oil red O staining. The effect of RLF on the mRNA expression of differentiation-related factors C/EBPβ, PPARγ and C/EBPα was detected by RT-qPCR. RESULTS: Two components of polysaccharides named as RLFⅠand RLFⅡ were acquired by DEAE-cellulose column and identified as polysaccharides by infrared absorption spectrum. RLFⅠsignificantly reduced the differentiation of 3T3-L1 pre-adipocytes into the adipocytes and the content of triglyceride in the cells (P < 0.05). No obvious effect of RLFⅡ was observed. Compared with control group, the mRNA levels of C/EBPβ, PPARγ and C/EBPα in RLFⅠgroup remarkably down-regulated (P < 0.05). CONCLUSION: RLFⅠsignificantly inhibits 3T3-L1 pre-adipocyte differentiation into adipocytes. The mechanism might be related to the down-regulation of differentiation-associated factors C/EBPβ, PPARγ and C/EBPα.     

Effect of deamination reaction mediated by semicarbazide-sensitive amine oxidase on 3T3-L1 adipocytes

AIM: To observe the effect of the metabolites generated from oxidative deamination of methylamine (MA) or benzylamine (BZA) catalyzed by semicarbazide-sensitive amine oxidase (SSAO) on 3T3-L1 adipocytes. METHODS: 3T3-L1 preadipocytes were induced to differentiation. SSAO activity was determined by high performance liquid chromatography (HPLC) at different differentiation time points. MTT assay was applied to detect cell vitality after exposure to different concentrations of MA or BZA. Fluorescence probe DCFH-DA was used to determine the production of reactive oxygen species after incubation of 3T3-L1 adipocytes with MA or BZA. After exposure to 0.5 mmol/L MA or BZA for 4 h, malondialdehyde (MDA), total superoxide dismutase (T-SOD) and glutathione (GSH) in the adipocytes or preadipocytes were measured. RESULTS: SSAO activity increased with the increase in the differentiation days, and reached a maximum at the 8th day. Incubation of the cells with different concentrations of MA or BZA for 4 h did not significantly decreased the cell vitality (P>0.05). After exposure to 0.5 mmol/L MA or BZA, the reactive oxygen species in adipocytes significantly increased, and were about 3 to 4 times as compared with control group (P<0.05). After treatment with 0.5 mmol/L MA or BZA for 4 h, MDA content significantly increased, while the activity of SOD and the expression of GSH decreased in mature adipocytes compared with control group (P<0.05). However, MDA, T-SOD and GSH did not change significantly after treatment with equal molar of MA or BZA in the preadipocytes (P>0.05). CONCLUSION: MA or BZA induces oxidative stress in the mature adipocytes, which might result from the deamination products catalyzed by SSAO.     

Effect of uric acid on the maturation and the biological function of BMDCs

AIM: To investigate the effect of uric acid on the maturation and the biological function of murine bone-marrow derived dendritic cells (BMDCs) in vitro.METHODS: BMDCs were cultured with GM-CSF, IL-4, LPS and uric acid. Cell phenotypes were analyzed by flow cytometry. MTT was used to detect the effect of uric acid on the function of BMDCs in stimulating the proliferation of T cells. IL-12 released by BMDCs was also detected. RESULTS: BMDCs were cultured and identified. Uric acid at concentrations of 200 mg/L and 400 mg/L increased the expression of the molecules (CD11c, CD83, CD86, IA/IE) on BMDCs surface and the IL-12 level in the culture supernatants (P<0.05), promoted the proliferation of T cells at the T: DC rate 5∶1, 10∶1, 20∶1 (P<0.05). However, uric acid at concentration of 70 mg/L had no effect on above molecule expression (P>0.05), no effect on T cell proliferation with BMDCs (P>0.05) was observed. CONCLUSION: Uric acid promotes the differentiation, maturation, the expression of co-stimulatory molecules, the IL-12 production in BMDCs and enhances the ability of BMDCs to stimulate the proliferation of T cell in a specical dose range.