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 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.     

Differential roles of Gβ/Gαq-PI3K-PKC signaling pathway in the regulation of 3T3-L1 pre-adipocyte differentiation

AIM:To investigate the role of individual protein isoforms in the regulation of 3T3-L1 adipocyte development through detecting temporal patterns of Gβ, Gα/q, phosphorylated phosphoinositide 3-kinase IA and IB isoforms and activated protein kinase C α and ζ subtypes involved in Gβ/Gαq-PI3K-PKC signaling pathway during 3T3-L1 preadipocyte differentiation. METHODS:The cells were induced by 1-methyl-3-isobutylxanthine, dexamethasone and insulin in vitro, and harvested at indicated time points (0 d, 6 h,12 h, 1 d, 3 d, 6 d, 9 d), then the total proteins of these cells were extracted. The expressions of Gβ, Gα/q, p101, phosphorylated p85, p55, p110γ, PKCα and ζ were assayed by Western blotting. RESULTS:(1) Gαq/11 and Gβ increased after induction of differentiation, reaching maxima respectively at 3 d and 1 d when cellular level of Gβ was 1.97±0.16-fold higher and expression of Gαq/11 was 2.34±0.22-fold higher than that in just-confluent (0 d) cells. Subsequently the expression declined. (2) Compared to 0 d, phosphorylated p110γ, p55 and p85 elevated slightly at 12 h, and decreased significantly by the end of the treatment period (9 d) which coincided with maximal differentiation. While the expression of p101 elevated slightly at 12 h, no statistical significance through differentiation was found. (3) Phosphorylated PKCα increased significantly, peaking at 3 d of differentiation. Expression of phosphorylated PKCζ decreased during differentiation and its level 45.52% lower in adipocytes than that in preadipocyte was observed. CONCLUSION:The protein levels elevating at the early stage of differentiation correlate with the time point at which clonal expansion of cells is observed. Phosphorylated p55 and PKCζ decrease in adipocytes than that in preadipocyte, indicating an inhibitory influence upon the late differentiation process.     

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.     

Acylation stimulating protein induces preadipocyte differentiation

AIM: To study the role of acylation stimulating protein (ASP) in the differentiation of 3T3-F442A preadipocytes. METHODS: Differentiation of 3T3-F442A preadipocytes was induced by ASP. The morphological changes were observed by Oil-Red O staining and the differentiation rate was compared. TG synthesis and TG mass in these cells were also assayed. DNA synthesis was measured by ［3H］-TdR incorporation. A typical differentiation inducer, insulin, was used as a positive control to compare these results. RESULTS: (1) 3T3-F442A preadipocytes were induced to differentiate by ASP alone. Fat droplets were clearly visible in the cytoplasm of 3T3-F442A cells. The differentiation rate was high (90%), but no significant difference was observed, compared with that in insulin group (95%). (2) In ASP group, TG synthesis and TG mass were significantly increased, both of them were higher than that in control group (P<0.05), but there was no significant difference, compared with insulin group (P>0.05). CONCLUSION: As a new adipocytes hormone, ASP plays an important biological role in the differentiation of preadipocytes.     

Mechanisms of PGF2α on the glucose-induced insulin secretion in NIT-1β cells

AIM:To investigate the cellular mechanisms by which PGF₂α promotes glucose-stimulated insulin secretion in NIT-1 beta cells. METHODS:Using the radioimmunoassay (RIA), the amount of the PGF₂α augmentation of glucose stimulated insulin secession was determined in different conditions, and the confocal laser scanning methods by Fluo-3AM as a fluorescent probe were used to analyze the changes of intracellular calcium in NIT-1β cells. RESULTS:At the lower glucose (0, 5.5 mmol/L), PGF₂α (5 μmol/L) failed to potentiate insulin secretion (P>0.05). However, in the presence of 16.5 mmol/L glucose, PGF₂α increased significantly in insulin secretion (P<0.05). Neither the AC inhibitor ddA nor the GC inhibitor Ly-83583 altered PGF₂α-potentiated insulin secretion in the presence of 16.5 mmol/L (P<0.05 or P<0.01). Otherwise, the PLC inhibitor U-73122 and the PKC blocker calphostin C both counteracted the insulinotropic of PGF₂α (P<0.01 or P<0.05). Moreover, exposure of the NIT-1β cells to 5 μmol/L PGF₂α induced a rapid increase of intracellular calcium (P<0.01). The inhibitor, ddA or Ly-83583 had no impact on PGF₂α-induced elevation of the intracellular calcium (P<0.01). Pretreatment of the cells with U-73122 completely prevented the calcium response induced by PGF₂α (P<0.01). CONCLUSION:Efects of PGF₂α was independent of cAMP or cGMP, potentiated glucose (16.5 mmol/L)-induced insulin secretion in NIT-1β cells through stimulation of phospholipase C, which subsequently mediated the elevation of intracellular calcium and activation of protein kinase C.     

Lipotoxicity of free fatty acid mixture and effect of free fatty acid mixture on lipid metabolism-related genes in L-02 cells

AIM：To investigate the lipotoxicity of free fatty acid (FFA) mixture and the effect of the FFA mixture on lipid metabolism-related genes in L-02 cells. METHODS：A normal human hepatocytes-derived cell line L-02 was treated with 0.5, 1 and 2 mmol/L FFA mixture (oleate and palmitate, 2∶1) for 24 h. The cellular total lipid accumulation was determined after Nile red staining by confocal laser scanning microscopy and flow cytometry. Intracellular triglyceride (TG) content was measured using an enzymatic kit. The viability of L-02 cells was determined by MTT assay and the apoptosis-inducing effect of FFA mixture was evaluated by annexin V/PI staining. The levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the culture medium were detected by ALT and AST kits. The mRNA levels of lipid metabolism-related genes, adipose differentiation-related protein (ADRP) and sterol regulatory element-binding protein 1 (SREBP-1), were examined by quantitative real-time PCR. RESULTS：All the different concentrations of FFA mixture increased intracellular lipid accumulation and TG content in a dose-dependent manner. FFA mixture at concentration of 1 mmol/L increased intracellular TG by 2.6 folds, which matched with the change in non-alcoholic fatty liver disease patients. Treatment for 24 h with 0.5 mmol/L and 1 mmol/L FFA mixture did not trigger apparent cell death and apoptosis, while treatment with 2 mmol/L FFA mixture resulted in a marked decrease in cell viability and induced early and late stages of apoptosis in L-02 cells. The levels of ALT and AST in the culture supernatant had no significant difference between control group and FFA treatment group. Treatment with 1 mmol/L FFA mixture up-regulated the expression of ADRP and SREBP-1 by 2.660 and 2.758 folds, respectively. CONCLUSION：FFA mixture induces the hepatic steatosis and 2 mmol/L FFA mixture causes mild cells damage in L-02 cells. The up-regulation of ADRP and SREBP-1 may be involved in FFA-induced hepatic steatosis.     

The mechanism of lipopolysaccharide-activated rat alveolar macrophages producing tumor necrosis factor alpha

AIM: To investigate the expression of hypoxia inducible factor-1alpha (HIF-1α) and the role of HIF-1α in tumor necrosis factor alpha (TNF-α) production in rat alveolar macrophages activated by lipopolysaccharide (LPS). METHODS: HIF-1α function was inhibited by using the method of HIF-1α decoy. Western blotting and semiquantitative RT-PCR were applied to determine the expression of HIF-1α protein and mRNA, respectively. The production of TNF-α was determined with ELISA. RESULTS: The content of HIF-1α protein in LPS group (1.95±0.57) and HIF-1α decoy group (1.89±0.59) were 4.8 times and 4.6 times higher than that in control group (0.41±0.14), respectively. The expression of HIF-1α mRNA showed no difference among three groups (F=3.14，P>0.05). The production of TNF-α in LPS group was higher than that in control group (61 ng/L vs 156 ng/L, q=5.12, P<0.05) and HIF-1α decoy group (90 ng/L vs 156 ng/L, q=4.63, P<0.05), respectively. However, the content of TNF-α in HIF-1α decoy group was still higher than that in control group (61 ng/L vs 94 ng/L, q=4.47, P<0.05). CONCLUSION: The enhanced stability of HIF-1α protein results in the marked upregulation of its protein and HIF-1α is contributed to the production of TNF-α in LPS-stimulating rat alveolar macrophages. It is indicated that HIF-1α plays important role in the pathogenesis of chronic inflammation involved in diseases such as COPD.     

Role of GPR40 mediates the effects of FFAs on lipoapoptosis in mouse β-cell line NIT-1

AIM: To evaluate the role of G protein-coupled receptor 40 (GPR40) mediates the effects of free fatty acids (FFAs) on lipoapoptosis in mouse β-cell line NIT-1 and the mechanisms involved in this process.METHODS: NIT-1 cells were supplemented with palmitate (500 μmol/L) or oleate (500 μmol/L) for 48 h, then apoptosis of the cells was determined by the methods of Hoechst 33342, TUNEL and flow cytometry (Annexin V/PI). The small interfering RNA technique was used to inhibit the expression of GPR40 in NIT-1 cell. The mock, control siRNA and GPR40 siRNA transfected cells were either supplemented with palmitate (500 μmol/L) or co-incubated with palmitate and oleate (500 μmol/L for each) for 48 h. The percentages of apoptotic cells were quantified. The expression of p-c-Jun, Bcl-2 and Bax were detected by Western blotting.RESULTS: Palmitate induced β cell lipoapoptosis, whereas oleate inhibited NIT-1 cells from palmitate-induced lipoapoptosis. No significant difference of the percentages of apoptotic cells was indicated among the mock, control siRNA and GPR40 siRNA transfected cells treated with palmitate (P>0.05). However, after co-incubated with palmitate and oleate (500 μmol/L for each) for 48 h, the percentage of apoptotic cells in GPR40 siRNA transfected cells was greater than that in mock (P<0.05), while the expression of p-c-Jun was decreased. The expressions of Bcl-2 and Bax were not affected.CONCLUSION: Palmitate induced β cell lipoapoptosis might not be mediated through GPR40, whereas oleate inhibits NIT-1 cells from palmitate-induced lipoapoptosis, which is mediated at least in part through GPR40, the change of c-Jun expression may play an role in this process, suggesting that GPR40 might be implicated in the control of β cell mass plasticity and GPR40 probably provides a link between obesity and type 2 diabetes.     

Mechanisms of hypoxia-induced tumor necrosis factor-α production in rat alveolar macrophages

AIM: To study the effect of hypoxia inducible factor-1 alpha (HIF-1α) on tumor necrosis factor alpha (TNF-α) production in rat alveolar macrophages cultured under hypoxic condition. METHODS: Using HIF-1α decoy inhibiting its function, Immunohistochemistry, Western blot, semiquantitative RT-PCR and ELISA were used to determine the expression of HIF-1α protein and mRNA and the production of TNF-α in rat alveolar macrophages cultured under hypoxic condition (3% O2, 5% CO2, 92% N2), respectively. RESULTS: Expression of HIF-1α was positive in cultured macrophage nucleoli in hypoxia group and HIF-1α decoy group but it was negative in nomoxic control group. The content of HIF-1α protein in hypoxia group and HIF-1α decoy group were significantly higher than that in nomoxic control group (P<0.05). The content of HIF-1α mRNA in hypoxia group and HIF-1α decoy group were markedly higher than that in nomoxic control group (P<0.05), respectively. The content of TNF-α in hypoxia group (115±17 ng/L) was higher than that in control group ［（69±13） ng/L, P<0.05］ and HIF-1α decoy group ［（81±15） ng/L, P<0.05］. CONCLUSION: Hypoxia can increase significantly expression and activity of HIF-1α， which can promote the production of TNF-α in rat alveolar macrophages. It suggests that HIF-1α plays an important role in the pathogenesis of chronic inflammation-related diseases that can give rise to lung hypoxia such as COPD.     

Inflammatory cytokines up-regulate FAT/CD36 expression in renal cells loaded by fatty acids

AIM: To investigate the effects of inflammatory cytokines on the expression of fatty acid transporter (FAT/CD36) in renal cells loaded by fatty acids. METHODS: Human mesangial cells (HMCs) and renal tubular epithelial HK-2 cells were treated with palmitate at concentrations of 0 mmol/L, 0.02 mmol/L, 0.04 mmol/L, 0.08 mmol/L, 0.16 mmol/L and 0.32 mmol/L for 24 h. The expression of FAT/CD36 at mRNA and protein levels was detected by real-time PCR and Western blotting,respectively. The renal cells were treated with palmitate at concentration of 0.04 mmol/L combined with TNF-α (25 μg/L) or IL-6 (20 μg/L) for 24 h. The effect of inflammatory cytokines on the mRNA and protein levels of FAT/CD36 in the renal cells was also investigated. Oil red O staining was used to determine the intracellular lipid droplet formation. The intracellular triglyceride (TG) and free fatty acid (FFA) were measured by enzymic assay and ELISA, respectively. RESULTS: Palmitate loading dose-dependently increased the expression of FAT/CD36 at mRNA and protein levels in both HMCs and HK-2 cells. The inflammatory cytokines further increased the expression of FAT/CD36 at mRNA and protein levels in both cells loaded by palmitate. Oil red O staining, TG detection and FFA assay showed that the inflammatory cytokines increased intracellular lipid levels in both HMCs and HK-2 cells. CONCLUSION: Inflammatory cytokines up-regulate the expression of FAT/CD36 in renal cells loaded by fatty acids and exacerbate the intracellular lipid accumulation.     

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.     

PPAR-α involves in cardiomyocyte hypertrophy induced by high glucose and insulin

AIM: To study the role of peroxisome proliferator-activated receptor-α (PPAR-α) signal transduction pathway in cardiac hypertrophy induced by high glucose and insulin (HGI). METHODS: The cultured neonatal rat cardiomyocytes were used to observe the effect of fenofibrate (FF), a selective PPAR-α agonist, on cardiomyocyte hypertrophy induced by HGI (glucose at concentration of 25.5 mmol/L and insulin at 0.1 μmol/L). The cardiomyocyte hypertrophic responses were assayed by measuring the cell surface area, protein content, and mRNA expression of atrial natriuretic factor (ANF). The expressions of mRNA and protein were assayed by real -time PCR and Western blotting. RESULTS: In cultured cardiomyocytes, HGI induced profound change of hypertrophic morphology, the significant increase in cell surface area, protein content and ANF mRNA expression compared to those in vehicle control (P<0.01), but the expressions of PPAR-α mRNA and protein decreased significantly (P<0.05). At the same time, the expression of cyclooxygenase 2 (COX-2), one of the PPAR-α downstream effectors was obviously elevated (P<0.05). However, FF (0.1, 0.3 and 1 μmol/L) inhibited the cardiomyocyte hypertrophy induced by HGI in a concentration-dependent manner (P<0.01). FF at concentration of 0.3 μmol/L increased the expressions of PPAR-α in both mRNA and protein levels (P<0.05) and inhibited the expressions of COX-2 (P<0.05), which were abolished by MK 886 (0.3 μmol/L), a selective PPAR-α antagonist (P<0.05). CONCLUSION: PPAR-α signal transduction pathway and its downstream effector COX-2 might involve in the cardiomyocyte hypertrophy induced by HGI.     

Sinomenine inhibits TNF-α-induced VCAM-1 expression on human umbilical vein endothelial cells

AIM: To investigate whether sinomenine(SN) can decrease TNF-α-induced VCAM-1 expression in human umbilical vein endothelial cells (HUVECs). METHODS: HUVECs were isolated from freshly collected umbilical cords.Positive control samples were stimulated with TNF-α, omitting SN. Negative control samples were treated in the same way, omitting TNF-α and SN. Experiment samples were co-cultured with TNF-α and SN at different concentration (0.25, 0.5, and 1.0 mol/L),or TNF-α and dexamethasone(Dex) at concentration of 1.0×10^-6mol/L.Cells were harvested after cultivation with the drugs for 12 hours. VCAM-1 mRNA expression was detected by real-time quantitative PCR, and VCAM-1 expression was detected by flow cytometry (FCM).RESULTS: VCAM-1 mRNA and VCAM-1 were induced by TNF-α. Compared with the positives, the relative VCAM-1 mRNA expression decreased to varying degrees in the experiment groups (P<0.05), and SN at concentration of 0.5 mol/L and 1.0 mol/L inhibited expression of VCAM-1 (P<0.05). SN at concentration of 1.0 mol/L decreased VCAM-1 expression by 28.8%(P<0.05), and SN at concentration of 0.5 mol/L reduced VCAM-1 expression by 21.68%(P<0.05). But SN at concentration of 0.25 mol/L and Dex at concentration of 1.0×10^-6mol/L didnt depress expression of VCAM-1. CONCLUSION: SN may inhibit TNF-α-induced VCAM-1 expression in HUVECs in vitro.     

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.     

Change of Gq protein-phosphatidyl inositol signaling in brain of rats with acute respiratory distress syndrome

AIM: To study the change of Gq protein-phosphatidyl inositol signaling in the brain of rats with acute respiratory distress syndrome (ARDS). METHODS: Forty male Wistar rats were randomly divided into oleic acid groups (OA groups of 30 min, 60 min, 90 min and 120 min exposure) and control group. The ARDS model in rats was reproduced by intravenous injection of oleic acid (0.2 mL/kg in 2 min). The mean arterial blood pressure (MABP), blood gas indexes, malondialdehyde (MDA), lactate dehydrogenase (LDH) and creatine kinase (CK) in plasma and brain tissues were measured. Gαq/11 protein and phospholipase C in brain tissues were detected by Western blotting. RESULTS: Compared to control group, MABP and PaO2 in all OA groups obviously decreased (P<0.05). MDA concentration and LDH activity in plasma and brain tissues of OA rats at time points of 90 min and 120 min were significantly higher than those in the controls (P<0.05). The CK activity in plasma of all OA groups increased (P<0.05). The CK activity in the brain tissues peaked at 60 min after OA exposure (P<0.05) and then decreased at 90 min and 120 min after treated with OA (P<0.05). Compared to control group, the concentration of Gαq/11 protein in the brain tissues of OA 60 min, 90 min and 120 min groups, and PLC concentration in the brain tissues of all OA groups obviously increased (P<0.05). A negative correlation between the change of Gαq/11 protein in the brain tissues and PaO2 (r=-0.579, P<0.05), a positive correlation between the change of Gαq/11 protein and MDA in the brain tissues (r=0.538, P<0.05), and a positive correlation between the change of Gαq/11 protein and LDH in the brain tissues (r=0.624, P<0.05) were observed. CONCLUSION: Up-regulation of Gq protein-phosphatidyl inositol signaling in the brain may play a role in the brain injury during ARDS.     

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.     

Effects of pentoxifylline on ventricular remodeling and cardiac function of dilated cardiomyopathy rats

AIM: To explore the effects of pentoxifylline (PTX) on ventricular remodeling and cardiac function in dilated cardiomyopathy (DCM) rats.METHODS: Lewis rats were randomly allocated to a myocin-induced dilated cardiomyopathy (DCM) group receiving saline (n=10), a DCM group receiving PTX (PTX group; 25 mg·kg^-1·d^-1, ip, for 30 days, n=10) or healthy control group (n=10). The levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and IL-10 in the blood plasma were analyzed by ELISA. The extent of fibrosis was estimated using Massons staining and immunohistochemistry analyses. Cardiac structure and function were measured by echocardiography.RESULTS: PTX decreased plasma levels of TNF-α and IL-6, and increased IL-10 level in DCM animals compared with DCM group ［TNF-α: (7.21±0.24) μg/L vs (19.30±1.31) μg/L, P<0.01; IL-6: (119.60±36.58) ng/L vs (189.50±13.25) ng/L, P<0.05; IL-10: (41.26±3.27) μg/L vs (32.45±4.32) μg/L, P<0.05］. Collagen volume fraction (CVF), perivascular collagen area (PVCA) and collagen Ⅰ/Ⅲ ratio were lower in PTX group than those in DCM group ［CVF: (16.45±3.01)% vs (23.33±4.43)%, P<0.05; PVCA: 4.58±2.10 vs 13.74±4.29, P<0.05; Ⅰ/Ⅲ ratio: 2.84±0.67 vs 4.22±0.54, P<0.01］. Left ventricular end-diastolic dimension reduced ［(6.11±0.51) mm vs (6.46±0.28) mm, P<0.05］ and left ventricular ejection fraction elevated ［(77.29±5.20)% vs (62.73±10.11)%, P<0.01］ by PTX compared with DCM.CONCLUSION: PTX modulates plasma levels of inflammatory cytokines, delays the ventricle remodeling and improves the heart function in DCM rats.