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 insulinsensitive 3T3-L1 adipocytes and 3T3-L1 adipocytes rendered insulin resistant following treatment with high glucose (25mmol/L) and high insulin (1 mol/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.

SUMMARY 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. MiR-375 has been demonstrated 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.To investigate the role of miR-375 in adipocyte differentiation, we compared miR-375 expression level between 3T3-L1 pre-adipocytes and adipocytes using miRNA microarray and quantitative real-time RT-PCR (qRT-PCR) analysis. Furthermore, we evaluated the effects of overexpression or inhibition of miR-375 on 3T3-L1 adipocyte differentiation.In this study, we found that miR-375 expression was increased after induction of adipogenic differentiation. Overexpression of miR-375 enhanced 3T3-L1 adipocyte differentiation: as evidenced by its ability to increase mRNA levels of both CCAAT/enhancer binding proteinα (C/EBPα) and peroxisome proliferator-activated receptor gamma (PPARγ2) and induction of adipocyte fatty acid-binding protein (aP2) and triglyceride (TG) accumulation. Furthermore, we found overexpression of miR-375 suppressed phosphorylation levels of extracellular signal-regulated kinases 1/2 (ERK1/2). In contrast, Anti-miR-375 increased ERK1/2 phosphorylation levels and inhibited mRNA expression of C/EBPα, PPARγ2 and aP2 in 3T3-L1 adipocyte, accompanied by decreased adipocyte differentiation.Taken together, these data suggest that miR-375 promotes 3T3-L1 adipocyte differentiation, possibly via modulating ERK - PPARγ2 - aP2 pathway.

Recent studies reveal a crucial role of pericyte loss in sepsis-associated microvascular dysfunction. Sirtuin 3 (SIRT3) mediates histone protein post-translational modification related to aging and ischemic disease. This study investigated the involvement of SIRT3 in LPS-induced pericyte loss and microvascular dysfunction. Mice were exposed to LPS, expression of Sirt3, HIF-2α, Notch3 and angiopoietins/Tie-2, pericyte/endothelial (EC) coverage and vascular permeability were assessed. Mice treated with LPS significantly reduced the expression of SIRT3, HIF-2α and Notch3 in the lung. Furthermore, exposure to LPS increased Ang-2 while inhibited Ang-1/Tie-2 expression with a reduced pericyte/EC coverage. Intriguingly, knockout of Sirt3 upregulated Ang-2, but downregulated Tie-2 and HIF-2α/Notch3 expression which resulted in a dramatic reduction of pericyte/EC coverage and exacerbation of LPS-induced vascular leakage. Conversely, overexpression of Sirt3 reduced Ang-2 expression and increased Ang-1/Tie-2 and HIF-2α/Notch3 expression in the LPS treated mice. Overexpression of Sirt3 further prevented LPS-induced pericyte loss and vascular leakage. This was accompanied by a significant reduction of the mortality rate. Specific knockout of prolyl hydroxylase-2 (PHD2) increased HIF-2α/Notch3 expression, improved pericyte/EC coverage and reduced the mortality rate in the LPS-treated mice. Our study demonstrates the importance of SIRT3 in preserving vascular integrity by targeting pericytes in the setting of LPS-induced sepsis.

Aim: 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.Keywords: caveolae; caveolin-1; oxidized low density lipoprotein; atherosclerosis; transcytosis; human umbilical vein endothelial cells Acta Pharmacologica Sinica (2010Sinica ( ) 31: 1336Sinica ( -1342 doi: 10.1038/aps.2010 published online 13 Sep 2010 Original Article * To whom correspondence should be addressed. E-mail dfliao66@yahoo.com.cn (Duan-fang LIAO); tchaoke@yahoo.com.cn (Chao-ke TANG) Received 2010-03-09 Accepted 2010-06-03 the vessel lumen and the arterial wall, and the control may be disturbed in arteriosclerotic blood vessels. Understanding the mechanisms of lipoprotein translocation, especially from arterial wall to vessel lumen, will be beneficial to the therapy of cardiovascular disorders. Caveolae are distinctive, flaskshaped invaginations of the plasma membrane generated in the Golgi complex. These organelles have a characteristic lipid composition and are associated with a 22 kDa protein called caveolin-1. Zhang reported that two ox-LDL receptors, CD36 and the related receptor SR-B1, localize to caveolae [4,5] . Several human studies have indicated transcytosis of macromolecules by means of caveolae in endothelial cells [6][7][8][9][10][11] . We hypothesized that ox-LDL may internalize and transcytose in this way. Bruneau described a model to study the passage of macromolecules through a monolayer of INT-407 cells [12] . We used this model to investigate the characteristics and mechanism of ox-LDL passage. LOX-1 is the major receptor for ox-LDL in endothelial cells [13] . [14] . Caveolin-1 is the main structural protein of caveolae, and it plays a crucial role in the formation of these invaginations of the plasma membrane. Caveolin-1 is a cholesterolbinding protein. Hu and Wu demonstrated the correlation between caveolin-1 and cellular choles...

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