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.     

Fructose promotes differentiation of 3T3-L1 preadipocytes by activating Akt signaling pathway

AIM: To study the effect of fructose on the differentiation of 3T3-L1 preadipocytes and the specific mechanism. METHODS: 3T3-L1 preadipocytes were cultured in vitro, induced to differentiate by cocktail method and treated with fructose at 1 g/L. The intracellular lipid content was identified and quantified by oil red O staining. The mRNA expression of perilipin-2 (Plin2), CCAAT/enhancer binding protein (C/EBP) α and C/EBPβ was detected by RT-qPCR. The protein expression of peroxisome proliferator-activated receptor γ (PPARγ) and adipocyte protein 2 (aP2) was determined by Western blot. RESULTS: The volume of differentiated adipocytes and the accumulation of cytoplasmic lipid droplets in the 3T3-L1 cells with fructose intervention were increased compared with control group (P<0.05). Compared with control group, the expression levels of the marker proteins PPARγ and aP2 were up-regulated (P<0.01). The mRNA expression levels of Plin2, C/EBPα and C/EBPβ were up-regulated (P<0.05). In addition, the phosphorylation level of the key molecule Akt in the Akt signaling pathway was significantly increased (P<0.01) after the addition of fructose. After the addition of Akt blocker, the expression levels of PPARγ and aP2 were decreased. CONCLUSION: Fructose promotes the adipose differentiation of 3T3-L1 cells possibly by activating the Akt signaling pathway.     

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.     

Role of cyclin D1 in PKC regulating the proliferation of airway smooth muscle cells in asthmatic rat

AIM: To observe the expression of cyclin D1 following PKC activation and the correlation between PKC-α and cyclin D1 in asthmatic rat airway smooth muscle cells (ASMCs), and to discuss the effects of cyclin D1 on the proliferation of airway smooth muscle cells regulated by PKC in asthmatic rat. METHODS: Twelve SD rats were randomly divided into control group (group N) and asthmatic 2-week group (group A), and then subdivided into 4 groups based on the drug interventions: (1) control group; (2) PMA; (3) PMA+5 μmol/L Ro-31-8220 group; (4) 5 μmol/L Ro-31-8220 group. The proliferations of ASMCss were examined with cell cycle analysis, MTT colorimetric assay and proliferating cell nuclear antigen (PCNA) immunocytochemical staining. The expressions of PKC-α and cyclin D1 were analyzed by RT-PCR and Western blotting. The data were subject to correlation analysis. RESULTS: (1) Compared to group A1, there were significant differences in the percentage of S+G2/M phase, absorbance A value of MTT and the rate of positive expression of PCNA protein in group A2 and A4 (P<0.01). The same tendency in group N was observed according with group A. (2) Compared with A1, the ratios of A values of PKC-α mRNA and protein in group A2 and A4 were significantly changed (P<0.01) as well as the ratios of A values of cyclin D1 mRNA and protein. (3) There were positive correlations between PKC-α and cyclin D1 in mRNA (r=0.476, P<0.05) and protein(r=0.899, P<0.01). CONCLUSION: The activation of PKC-α promotes ASMCs proliferation in asthmatic rats, and there are positive correlations between the PKC-α and cyclin D1 in mRNA and protein, indicating that the PKC-α signal pathway may be involved in the process of airway smooth muscle remodeling by regulating the expression of cyclin D1 in asthmatic rats.     

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.     

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.     

Reverse effect of protein kinase C inhibitor Ro-31-8220 on the hypertrophy of cardiomyocytes of neonatal rats induced by high glucose levels

AIM: To study the effect of protein kinase C (PKC) inhibitor Ro-31-8220 on the hypertrophy of cardiomyocytes of neonatal rats induced by high glucose levels, and to investigate the role of PKC and its downstream signal transduction pathway. METHODS: Using cultured neonatal cardiac myocytes as a model, the cells were divided into: (1) control group (glucose 5.5 mmol/L); (2) different high glucose level (10 mmol/L,15 mmol/L, 20 mmol/L, 25.5 mmol/L); (3) high glucose level (25.5 mmol/L) + PKC inhibitor Ro-31-8220 (50 nmol/L); (4) high glucose level (25.5mmol/L) + NF-κB inhibitor (BAY11-7082, 5 mmol/L). The cellular diameters and protein level were measured and the expression of PKC-α, PKC-β2, p-PKC-α, p-PKC-β2, NF-κB and c-Fos were determined by Western blotting. RESULTS: Neonatal cardiomyocytes cultured in high glucose concentration showed increased cellular diameters, protein level and higher expressions of PKC-α, PKC-β2, p-PKC-α, p-PKC-β2, NF-κB and c-Fos, which was consistent with the increased glucose levels and had statistical significance compared to control group (P<0.01). PKC inhibitor Ro-31-8220 reversed these changes induced by high glucose concentration as showed by decreased cellular diameters, protein level and expression of PKC-α, PKC-β2, p-PKC-α, p-PKC-β2, NF-κB and c-Fos, which had statistical significance compared to high glucose groups (P<0.01). CONCLUSION: High glucose levels induce hypertrophy of cardiomyocytes. PKC inhibitor Ro-31-8220 reverses the effect of high glucose on the cardiac myocytes, which may be via PKC/NF-κB/c-Fos pathway.     

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.     

Mechanism of acylation stimulating protein resistance induced by fatty acid in 3T3-L1 adipocytes and preadipocytes

AIM: To evaluate the potential acylation stimulating protein (ASP) resistance in both adipocytes and preadipocytes under the conditions by which insulin resistance is produced by the stimulation of free fatty acids (FFA), and to explore the mechanism of ASP resistance on post-receptor level. METHODS: 3T3-L1 preadipocytes were induced to differentiate. Then the cells were treated with oleate or palmitate at concentration of 0 mmol/L (FFA-free DMEM/F12), 0.125 mmol/L, 0.5 mmol/L or 1.0 mmol/L overnight. Glucose transport was assessed by ［3H］ 2-deoxyglucose uptake to evaluate insulin resistance and ASP resistance. Both non-FFA treated and FFA treated 3T3-L1 cells were cultured with ASP at concentration of 5.0 μmol/L for 4 h, then the cell proteins were extracted, and the expressions of guanine nucleotide binding protein beta (Gβ), guanine nucleotide-binding protein alpha-q/11(Gαq/11), phosphorylated-protein kinase Cα (p-PKCα) and phosphorylated-protein kinase Cζ (p-PKCζ) were measured by Western blotting. RESULTS: Both adipocytes and preadipocytes were responsive to ASP. ASP stimulation increased glucose transport by 198% in adipocytes and by 287% in preadipocytes (P<0.01 vs PBS). FFA at concentration of 0.125 mmol/L did not change ASP-stimulated glucose transport significantly, but high dose of oleate or palmitate effectively reduced the ASP response with a significant reduction by 47% (P<0.05 for oleate) and 34% (P<0.05 for palmitate) at 1 mmol/L FFA in adipocytes. Similarly in preadipocytes, glucose uptake rates were decreased by 43% (P<0.05 for oleate) and 62% (P<0.01 for palmitate) at 1 mmol/L FFA. Effects were comparable to those obtained with insulin. After overnight incubation with oleate or palmitate in adipocytes and preadipocytes, Gβ, Gαq/11, p-PKCα and p-PKCζ were downregulated both in the absence of ASP treatment and in the presence of ASP treatment in adipocytes. At concentration of 1.0 mmol/L, oleate inhibited the expressions of ASP-induced Gβ, Gαq/11, p-PKCα and p-PKCζ in adipocytes by 47％, 44%, 39% (P<0.05, P<0.01) and 20% (P>0.05), respectively. Palmitate also effectively blocked the expressions of ASP (at concentration of 1.0 mmol/L)-induced Gβ, Gαq/11, p-PKCα and p-PKCζ by 50%, 43%, 44% and 43% (P<0.05, P<0.01) in adipocytes. In preadipocytes, oleate only inhibited ASP-induced p-PKCα and p-PKCζ significantly by 39% and 19%, respectively (P<0.05). However, overnight exposure of 3T3-L1 preadipocytes to 1 mmol/L palmitate leaded to 45%, 50%, 52% and 21% (P<0.05, P<0.01) inhibition of ASP-induced expressions of Gβ, Gαq/11, p-PKCα and p-PKCζ, respectively. CONCLUSION: Oleate and palmitate inhibit ASP-mediated stimulation of glucose transport both in adipocytes and preadipocytes. The study provides direct evidence of ASP resistance under the condition of insulin resistance induced by FFA in a cellular model. The mechanism of action involves both changes in expression of C5L2 as well as signaling parameters. Fatty acid-induced ASP resistance may contribute to the physiological abnormalities associated with insulin resistance and obesity phenotype.     

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.     

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.     

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.     

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.     

Effect of GLP-1 receptor agonist on lipolysis in adipose tissue of obese mice and its underlying mechanism

AIM: To investigate the effects of glucagon-like peptide-1(GLP-1) receptor agonist exendin-4 on white adipose tissue (WAT) and the underlying mechanisms. METHODS: Male C57BL/6J mice (8 weeks) were challenged by high-fat diet for 12 weeks, and were randomly divided into saline group and exendin-4 group. The mRNA expression of sirtuin 1(SIRT1), adipose triglyceride lipase (ATGL), TNF-α and adiponectin of WAT was detected by real-time PCR. 3T3-L1 adipocytes or mouse embryonic fibroblasts cells were treated with exendin-4 for 24 h. The protein levels of SIRT1, ATGL and hormone-sensitive lipase (HSL) were determined by Western blot.RESULTS: Exendin-4 significantly decreased epididymal fat weight, fasting blood glucose and serum triglyceride levels (P<0.05), and reduced body weight and serum TNF-α level. The mRNA expression of SIRT1, ATGL and adiponectin in WAT was all significantly up-regulated by exendin-4, which were contrary to the down-regulation of TNF-α mRNA expression (P<0.05). Exendin-4 promoted the protein expression of SIRT1, ATGL, and HSL in 3T3-L1 adipocytes in a dose-dependent manner. Less lipid droplets with up-regulation of lipolytic protein expression were observed when combined with SIRT1 agonist treatment, which were suppressed by SIRT1 inhibitor. Deletion of SIRT1 led to larger adipocytes with more lipid droplets, and the effect of exendin-4 on the lipolysis disappeared when SIRT1 was deficient.CONCLUSION: Exendin-4 promotes lipolysis in WAT of obese mice via activation of SIRT1.     

Effect of pioglitazone on pre-adipocytes differentiation and GILZ expression

AIM: To investigate the roles of pioglitazone on differentiation and expression of GILZ in 3T3-L1 pre-adipocytes. METHODS: The morphological changes during 3T3-L1 cell differentiation were observed. The cells were treated with pioglitazone at concentrations of 1×10^-4～1×10^-2 mmol/L for 48 h, then the relative content of triglyceride were analyzed by oil red O staining at 2nd, 4th and 6th day during adipogenesis. The mRNA expression of peroxisome proliferator-activated receptor γ2 (PPARγ2) and lipoprotein lipase (LPL) was measured by real-time PCR. GILZ protein expression was determined by Western blot after the cells were treated with pioglitazone at concentrations of 1×10^-4 ～1×10^-2 mmol/L for 48 h.RESULTS: Oil-red O staining showed that the relative contents of triglyceride in adipocytes were increased with the increase in the pioglitazone concentration. Compared with the control, the relative contents of triglyceride in group 1×10^-3 mmol/L and group 1×10^-2 mmol/L were significantly increased (P < 0.05). The mRNA expression of PPARγ2 and LPL was also increased with the increase in the pioglitazone concentration. When pioglitazone concentration was more than 1×10^-3 mmol/L, compared with the control, the mRNA expression of PPARγ2 and LPL significantly increased (P < 0.01). The protein expression of GILZ was decreased with the increase in the pioglitazone concentration.CONCLUSION: Pioglitazone down-regulates GILZ expression, and up-regulate PPARγ2 expression and the downstream functional factor such as LPL.     

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.