Qin-hui Tuo

CHANGES IN microRNA (miR) profile and effects of miR-320 in insulin-resistant 3T3-L1 adipocytes.

1 MicroRNAs (miRNAs) play essential roles in many biological processes. It is known that aberrant miRNA expression contributes to some pathological conditions. However, it is not known whether miRNAs play any role in the development of insulin resistance in adipocytes, a key pathophysiological link between obesity and diabetes. 2 To investigate the function of miRNAs in the development of insulin resistance, using miRNA microarray analysis we compared miRNA expression profiles between normal insulin‐sensitive 3T3‐L1 adipocytes and 3T3‐L1 adipocytes rendered insulin resistant following treatment with high glucose (25 mmol/L) and high insulin (1 µmol/L). Furthermore, adipocytes were transfected with specific antisense oligonucleotides against miRNA‐320 (anti‐miR‐320 oligo) and the effects on the development of insulin resistance were evaluated. 3 We identified 50 upregulated and 29 downregulated miRNAs in insulin‐resistant (IR) adipocytes, including a 50‐fold increase in miRNA‐320 (miR‐320) expression. Using bioinformatic techniques, the p85 subunit of phosphatidylinositol 3‐kinase (PI3‐K) was found to be a potential target of miR‐320. In experiments with anti‐miR‐320 oligo, insulin sensitivity was increased in IR adipocytes, as evidenced by increases in p85 expression, phosphorylation of Akt and the protein expression of the glucose transporter GLUT‐4, as well as insulin‐stimulated glucose uptake. These beneficial effects of anti‐miR‐320 oligo were observed only in IR adipocytes and not in normal adipocytes. 4 In conclusion, the miRNA profile changes in IR adipocytes compared with normal 3T3‐L1 adipocytes. Anti‐miR‐320 oligo was found to regulate insulin resistance in adipocytes by improving insulin–PI3‐K signalling pathways. The findings provide information regarding a potentially new therapeutic strategy to control insulin resistance.

MicroRNA-375 promotes 3T3-L1 adipocyte differentiation through modulation of extracellular signal-regulated kinase signalling

1. Adipocyte hypertrophy and hyperplasia are important processes in the development of obesity. To understand obesity and its associated diseases, it is important to elucidate the molecular mechanisms governing adipogenesis. MicroRNA‐375 has been shown to inhibit differentiation of neurites, and participate in the regulation of insulin secretion and blood homeostasis. However, it is unknown whether miR‐375 plays a role in adipocyte differentiation.

Caveolae and caveolin-1 mediate endocytosis and transcytosis of oxidized low density lipoprotein in endothelial cells

AbstractAim:To explore the mechanisms involved in ox-LDL transcytosis across endothelial cells and the role of caveolae in this process.Methods:An in vitro model was established to investigate the passage of oxidized low density lipoprotein (ox-LDL) through a tight monolayer of human umbilical vein endothelial cells (HUVEC) cultured on a collagen-coated filter. Passage of DiI-labeled ox-LDL through the monolayer was measured using a fluorescence spectrophotometer. The uptake and efflux of ox-LDL by HUVEC were determined using fluorescence microscopy and HPLC.Results:Caveolae inhibitors – carrageenan (250 μg/mL), filipin (5 μg/mL), and nocodazole (33 μmol/L)–decreased the transport of ox-LDL across the monolayer by 48.9%, 72.4%, and 79.8% as compared to the control group. In addition, they effectively decreased ox-LDL uptake and inhibited the efflux of ox-LDL. Caveolin-1 and LOX-1 were up-regulated by ox-LDL in a time-dependent manner and decreased gradually after depletion of ox-LDL (P<0.05). After treatment HUVEC with ox-LDL and silencing caveolin-1, NF-κB translocation to the nucleus was blocked and LOX-1 expression decreased (P<0.05).Conclusion:Caveolae can be a carrier for ox-LDL and may be involved in the uptake and transcytosis of ox-LDL by HUVEC.

Effects and underlying mechanisms of curcumin on the proliferation of vascular smooth muscle cells induced by Chol:MβCD.

Proliferation of vascular smooth muscle cells (VSMCs) contributes to the development of various cardiovascular diseases. Curcumin, extracted from Curcumae longae, has been shown a variety of beneficial effects on human health, including anti-atherosclerosis by mechanisms poorly understood. In the present study, we attempted to investigate whether curcumin has any effect on VSMCs proliferation and the potential mechanisms involved. Our data showed curcumin concentration-dependently abrogated the proliferation of primary rat VSMCs induced by Chol:MbetaCD. To explore the underlying cellular and molecular mechanisms, we found that curcumin was capable of restoring caveolin-1 expression which was reduced by Chol:MbetaCD treatment. Moreover, curcumin abrogated the increment of phospho-ERK1/2 and nuclear accumulation of ERK1/2 in primary rat VSMCs induced by Chol:MbetaCD, which led to a suppression of AP-1 promoter activity stimulated by Chol:MbetaCD. In addition, curcumin was able to reverse cell cycle progression induced by Chol:MbetaCD, which was further supported by its down-regulation of cyclinD1 and E2F promoter activities in the presence of Chol:MbetaCD. Taking together, our data suggest curcumin inhibits Chol:MbetaCD-induced VSMCs proliferation via restoring caveolin-1 expression that leads to the suppression of over-activated ERK signaling and causes cell cycle arrest at G1/S phase. These novel findings support the beneficial potential of curcumin in cardiovascular disease.

Onychin inhibits proliferation of vascular smooth muscle cells by regulating cell cycle.

AbstractAim:To investigate the effects of onychin on the proliferation of cultured rat artery vascular smooth muscle cells (VSMCs) in the presence of 10% new-born calf serum (NCS).Methods:Rat VSMCs were incubated with onychin 1–50 μmol/L or genistein 10 μmol/L in the presence of 10% NCS for 24 h. The proliferation of VSMCs was measured by cell counting and MTS/PMS colorimetric assays. Cell cycle progression was evaluated by flow cytometry. Retinoblastoma (Rb) phosphorylation, and expression of cyclin D1 and cyclin E were measured by Western blot assays. The tyrosine phosphorylation of ERK1/2 was examined by immunoprecipitation techniques using anti-phospho-tyrosine antibodies.Results:The proliferation of VSMCs was accelerated significantly in the presence of 10% NCS. Onychin reduced the metabolic rate of MTS and the cell number of VSMCs in the presence of 10% NCS in a dose-dependent manner. Flow cytometry analysis revealed that the G1-phase fraction ratio in the onychin group was higher than that in the 10% NCS group (85.2% vs 70.0%, P < 0.01), while the S-phase fraction ratio in the onychin group was lower than that in 10% NCS group (4.3% vs 16.4%, P < 0.01). Western blot analysis showed that onychin inhibited Rb phosphorylation and reduced the expression of cyclin D1 and cyclin E. The effects of onychin on proliferation, the cell cycle and the expression of cyclins in VSMCs were similar to those of genistein, an inhibitor of tyrosine kinase. Furthermore immunoprecipitation studies showed that both onychin and genistein markedly inhibited the tyrosine phosphorylation of ERK1/2 induced by 10% NCS.Conclusion:Onychin inhibits the proliferation of VSMCs through G1 phase cell cycle arrest by decreasing the tyrosine phosphorylation of ERK1/2, and the expression of cyclin D1 and cyclin E, and sequentially inhibiting Rb phosphorylation.

Angiopoietin-1 protects myocardial endothelial cell function blunted by angiopoietin-2 and high glucose condition

Aim:To evaluate the effects of angiopoietin-1 (Ang-1) on myocardial endothelial cell function under high glucose (HG) condition.Methods:Mouse heart myocardial endothelial cells (MHMECs) were cultured and incubated under HG (25 mmol/L) or normal glucose (NG, 5 mmol/L) conditions for 72 h. MTT was used to determine cellular viability, and TUNEL assay and caspase-3 enzyme linked immunosorbent assays were used to assay endothelial apoptosis induced by serum starvation. Immunoprecipitation and Western blot analysis were used to analyze protein phosphorylation and expression. Endothelial tube formation was used as an in vitro assay for angiogenesis.Results:Exposure of MHMECs to HG resulted in dramatic decreases in phosphorylation of the Tie-2 receptor and its downstream signaling partners, Akt/eNOS, compared to that under NG conditions. Ang-1 (250 ng/mL) increased Tie-2 activation, inhibited cell apoptosis, and promoted angiogenesis. Ang-1-mediated protection of endothelial function was blunted by Ang-2 (25 ng/mL).Conclusion:Ang-1 activates the Tie-2 pathway and restores hyperglycemia-induced myocardial microvascular endothelial dysfunction. This suggests a protective role of Ang-1 in the ischemic myocardium, particularly in hearts affected by hyperglycemia or diabetes.

MiRNA-375 promotes 3T3-L1 adipocyte differentiation via modulation of ERK signaling

1. Adipocyte hypertrophy and hyperplasia are important processes in the development of obesity. To understand obesity and its associated diseases, it is important to elucidate the molecular mechanisms governing adipogenesis. MicroRNA‐375 has been shown to inhibit differentiation of neurites, and participate in the regulation of insulin secretion and blood homeostasis. However, it is unknown whether miR‐375 plays a role in adipocyte differentiation.

Curcumin enhances vascular contractility via induction of myocardin in mouse smooth muscle cells

A variety of cardiovascular diseases is accompanied by the loss of vascular contractility. This study sought to investigate the effects of curcumin, a natural polyphenolic compound present in turmeric, on mouse vascular contractility and the underlying mechanisms. After mice were administered curcumin (100 mg·kg-1·d-1, ig) for 6 weeks, the contractile responses of the thoracic aorta to KCl and phenylephrine were significantly enhanced compared with the control group. Furthermore, the contractility of vascular smooth muscle (SM) was significantly enhanced after incubation in curcumin (25 μmol/L) for 4 days, which was accompanied by upregulated expression of SM marker contractile proteins SM22α and SM α-actin. In cultured vascular smooth muscle cells (VSMCs), curcumin (10, 25, 50 μmol/L) significantly increased the expression of myocardin, a “master regulator” of SM gene expression. Curcumin treatment also significantly increased the levels of caveolin-1 in VSMCs. We found that as a result of the upregulation of caveolin-1, curcumin blocked the activation of notch1 and thereby abolished Notch1-inhibited myocardin expression. Knockdown of caveolin-1 or activation of Notch1 signaling with Jagged1 (2 μg/mL) diminished these effects of curcumin in VSMCs. These findings suggest that curcumin induces the expression of myocardin in mouse smooth muscle cells via a variety of mechanisms, including caveolin-1-mediated inhibition of notch1 activation and Notch1-mediated repression of myocardin expression. This may represent a novel pathway, through which curcumin protects blood vessels via the beneficial regulation of SM contractility.

MicroRNA-375 promotes 3T3-L1 adipocyte differentiation through modulation of extracellular signal-regulated kinase signalling: MicroRNA-375 and adipocyte differentiation

1. Adipocyte hypertrophy and hyperplasia are important processes in the development of obesity. To understand obesity and its associated diseases, it is important to elucidate the molecular mechanisms governing adipogenesis. MicroRNA‐375 has been shown to inhibit differentiation of neurites, and participate in the regulation of insulin secretion and blood homeostasis. However, it is unknown whether miR‐375 plays a role in adipocyte differentiation.