Overexpressing STAMP2 attenuates adipose tissue angiogenesis and insulin resistance in diabetic ApoE−/−/LDLR−/− mouse via a PPARγ/CD36 pathway

The aim of this study was to investigate whether overexpression of STAMP2 improves insulin resistance by regulating angiogenesis in adipose tissues. The characteristics of diabetic mice were measured by serial metabolite and pathology tests. Samples were obtained from epididymal, subcutaneous and brown adipose tissues. Histological and morphological analysis demonstrated that STAMP2 gene overexpression reduced adipocyte size, angiogenesis in epididymal and brown adipose tissues. On aortic ring assay, microvessels sprouting from aortas were significantly inhibited after STAMP2 gene overexpression. The cellular effect of STAMP2 on angiogenesis was explored in human umbilical vein endothelial cells (HUVECs) model. Correlation of STAMP2 and angiogenesis was validated by Ad‐STAMP2 transfection and STAMP2 siRNA inhibition. In vitro, overexpression of STAMP2 significantly inhibited endothelial cell migration, tube formation. The effects of Ad‐STAMP2 transfection on HUVECs were abolished by treatment with PPARγ antagonist GW9662 (2.5 μM), and the roles of STAMP2 siRNA on HUVECs were also reversed by treatment with PPARγ agonist rosiglitazone (RSG) (0.1 mM). RT‐PCR indicated that STAMP2 could regulate levels of adhesion molecules, vascular endothelial growth factor A and CD36. The expression of PPARγ and CD36 was decreased when STAMP2 was inhibited by siRNA, while PPARγ and CD36 were highly expressed after overexpression of STAMP2. Our results suggested that STAMP2 gene overexpression may improve insulin resistance via attenuating angiogenesis in epididymal and brown adipose tissues through the PPARγ/CD36 signalling pathway.     

The Expression of Cyclin D1 during Adipogenesis in Pig Primary Stromal–Vascular Cultures

Objective: Our objective in this study was to measure the expression of cyclin D1 in pig primary stromal‐vascular (S‐V) cells as they differentiate into adipose cells and to identify which factors may alter cyclin D1 expression. Research Methods and Procedures: Western blot analysis was performed on cultured S‐V cells using 8% sodium dodecyl sulfate‐polyacrylamide gels, mouse monoclonal cyclin D1 antibody, and anti‐mouse IgG secondary labeled with horseradish peroxidase. For immunocytochemistry, cultures were fixed with 4% paraformaldehyde and incubated with anti‐CCAAT/enhancer binding protein (C/EBPα) and anti‐cyclin D1 together. Cyclin D1 expression was evaluated in 105‐day fetal dorsal subcutaneous tissues using paraffin sections. Results: Our results with Western blot analysis showed that cyclin D1 was found in freshly isolated S‐V cells and continued to be expressed during the first 3 days of adipose cell development with a significant increase in late development at day 9. Elevated cyclin D1 levels were colocalized with C/EBPα beginning at day 3 and remained colocalized with C/EBPα through day 9. Removing insulin from cultures resulted in a reduction in differentially elevated levels of cyclin D1. Discussion: The elevated level of cyclin D1 expression colocalized with C/EBPα expression is unexpected because differentiated adipocytes would be expected to have reduced proliferative potential. The elevated levels of cyclin D1 expression we observed in mature adipocytes depend on insulin. In addition, cyclin D1 is absent from lipid‐filled fetal adipose cells in vivo, where insulin levels are very low. The activity of cyclin D1 in differentiated adipocytes may be directed toward proteins outside of the cell cycle.     

Functional analysis of the chicken PPARγ gene 5'-flanking region and C/EBPα-mediated gene regulation

Peroxisome proliferator-activated receptor-γ (PPARγ) and CCAAT/enhancer binding protein-α (C/EBPα) are the master regulators of adipogenesis. The regulatory mechanism of PPARγ and C/EBPα gene expression is clear in mammals, however, little is known in chicken. The aim of the present study was to characterize chicken PPARγ promoter and investigate whether PPARγ could be regulated by C/EBPα in chickens. A 2-kb nucleotide sequence upstream of the start codon of chicken PPARγ gene was cloned and characterized by using bioinformatics and experimental approaches. This 2-kb promoter region exhibited strong promoter activity in DF1 cells. The reporter gene assay showed that the chicken C/EBPα could activate PPARγ gene promoter. Further study by electrophoretic mobility shift assay and mutational analysis revealed that the chicken C/EBPα could directly bind to and regulate the PPARγ gene promoter. Our results demonstrate that PPARγ can be directly regulated by C/EBPα in chickens.     

PLCγ1–PKCγ Signaling‐Mediated Hsp90α Plasma Membrane Translocation Facilitates Tumor Metastasis

The 90‐kDa heat shock protein (Hsp90α) has been identified on the surface of cancer cells, and is implicated in tumor invasion and metastasis, suggesting that it is a potentially important target for tumor therapy. However, the regulatory mechanism of Hsp90α plasma membrane translocation during tumor invasion remains poorly understood. Here, we show that Hsp90α plasma membrane expression is selectively upregulated upon epidermal growth factor (EGF) stimulation, which is a process independent of the extracellular matrix. Abrogation of EGF‐mediated activation of phospholipase (PLCγ1) by its siRNA or inhibitor prevents the accumulation of Hsp90α at cell protrusions. Inhibition of the downstream effectors of PLCγ1, including Ca²⁺ and protein kinase C (PKCγ), also blocks the membrane translocation of Hsp90α, while activation of PKCγ leads to increased levels of cell‐surface Hsp90α. Moreover, overexpression of PKCγ increases extracellular vesicle release, on which Hsp90α is present. Furthermore, activation or overexpression of PKCγ promotes tumor cell motility in vitro and tumor metastasis in vivo, whereas a specific neutralizing monoclonal antibody against Hsp90α inhibits such effects, demonstrating that PKCγ‐induced Hsp90α translocation is required for tumor metastasis. Taken together, our study provides a mechanistic basis for the role for the PLCγ1–PKCγ pathway in regulating Hsp90α plasma membrane translocation, which facilitates tumor cell motility and promotes tumor metastasis.     

Trans‐repression of NFκB pathway mediated by PPARγ improves vascular endothelium insulin resistance

Previous study has shown that thiazolidinediones (TZDs) improved endothelium insulin resistance (IR) induced by high glucose concentration (HG)/hyperglycaemia through a PPARγ‐dependent‐NFκB trans‐repression mechanism. However, it is unclear, whether changes in PPARγ expression affect the endothelium IR and what the underlying mechanism is. In the present study, we aimed to address this issue. HG‐treated human umbilical vascular endothelial cells (HUVEC) were transfected by either PPARγ‐overexpressing (Ad‐PPARγ) or PPARγ‐shRNA‐containing (Ad‐PPARγ‐shRNA) adenoviral vectors. Likewise, the rats fed by high‐fat diet (HFD) were infected by intravenous administration of Ad‐PPARγ or Ad‐PPARγ‐shRNA. The levels of nitric oxide (NO), endothelin‐1 (ET‐1) and cytokines (TNFα, IL‐6, sICAM‐1 and sVCAM‐1) and the expression levels of PPARγ, eNOS, AKT, p‐AKT, IKKα/β and p‐IKKα/β and IκBα were examined; and the interaction between PPARγ and NFκB‐P65 as well as vascular function were evaluated. Our present results showed that overexpression of PPARγ notably increased the levels of NO, eNOS, p‐AKT and IκBα as well as the interaction of PPARγ and NFκB‐P65, and decreased the levels of ET‐1, p‐IKKα/β, TNFα, IL‐6, sICAM‐1 and sVCAM‐1. In contrast, down‐expression of PPARγ displayed the opposite effects. The results demonstrate that the overexpression of PPARγ improves while the down‐expression worsens the endothelium IR via a PPARγ‐mediated NFκB trans‐repression dependent manner. The findings suggest PPARγ is a potential therapeutic target for diabetic vascular complications.     

Involvement of PPARα and PPARγ in apoptosis and proliferation of human hepatocarcinoma HepG2 cells

Peroxisome proliferator‐activated receptors (PPARs) mediate the effects of various ligands, known as peroxisome proliferators, a heterogeneous class of compounds including industrial chemicals, pharmaceuticals, and biomolecules such as fatty acids and eicosanoids. Among peroxisome proliferators, fibrate derivatives are considered specific ligands for PPARα, whereas eicosanoids, such as PGJ2, for PPARγ. The study aimed to clarify the relation between PPARs and apoptosis or proliferation on the same type of cells, using clofibrate as specific ligand of PPARα and PGJ2 as specific ligand of PPARγ. The cells used were human hepatocarcinoma HepG2 cells. The results showed that PPARα protein content increased in HepG2 cells treated with clofibrate, causing apoptosis in a time‐ and concentration‐dependent way, as evidenced by the citofluorimetric assay and determination of BAD, myc and protein phosphatase 2A protein content. It also emerged that PPARγ increased in the same cells when treated with a specific ligand of this PPAR; in this case the increase of PPARγ did not cause an increase of apoptosis, but a time‐ and concentration‐dependent inhibition of cell proliferation, evidenced by decreased cell numbers and increased number of cells in the G0/G1 phase of the cycle. It may be concluded that PPARα is chiefly related to apoptosis and PPARγ to cell proliferation. Copyright © 2010 John Wiley & Sons, Ltd.     

CD38 deficiency suppresses adipogenesis and lipogenesis in adipose tissues through activating Sirt1/PPARγ signaling pathway

It has been recently reported that CD38 was highly expressed in adipose tissues from obese people and CD38‐deficient mice were resistant to high‐fat diet (HFD)‐induced obesity. However, the role of CD38 in the regulation of adipogenesis and lipogenesis is unknown. In this study, to explore the roles of CD38 in adipogenesis and lipogenesis in vivo and in vitro, obesity models were generated with male CD38^?/? and WT mice fed with HFD. The adipocyte differentiations were induced with MEFs from WT and CD38^?/? mice, 3T3‐L1 and C3H10T1/2 cells in vitro. The lipid accumulations and the alternations of CD38 and the genes involved in adipogenesis and lipogenesis were determined with the adipose tissues from the HFD‐fed mice or the MEFs, 3T3‐L1 and C3H10T1/2 cells during induction of adipocyte differentiation. The results showed that CD38^?/? male mice were significantly resistant to HFD‐induced obesity. CD38 expressions in adipocytes were significantly increased in WT mice fed with HFD, and the similar results were obtained from WT MEFs, 3T3‐L1 and C3H10T1/2 during induction of adipocyte differentiation. The expressions of PPARγ, AP2 and C/EBPα were markedly attenuated in adipocytes from HFD‐fed CD38^?/? mice and CD38^?/? MEFs at late stage of adipocyte differentiation. Moreover, the expressions of SREBP1 and FASN were also significantly decreased in CD38^?/? MEFs. Finally, the CD38 deficiency‐mediated activations of Sirt1 signalling were up‐regulated or down‐regulated by resveratrol and nicotinamide, respectively. These results suggest that CD38 deficiency impairs adipogenesis and lipogenesis through activating Sirt1/PPARγ‐FASN signalling pathway during the development of obesity.     

Troxerutin downregulates C/EBP‐β gene expression via modulating the IFNγ‐ERK1/2 signaling pathway to ameliorate rotenone‐induced retinal neurodegeneration

Troxerutin, a natural flavonoid guards against oxidative stress and apoptosis with a high capability of passing through the blood‐brain barrier. Our aim was to investigate the role of troxerutin in experimentally induced retinal neurodegeneration by modulating the interferon‐gamma (IFNγ)‐extracellular signal‐regulated kinases 1/2 (ERK1/2)‐CCAAT enhancer‐binding protein β (C/EBP‐β) signaling pathway. Three groups of rats (10 each group) were included. Group I (control group), group II (rotenone treated group): the rats were injected subcutaneously with a single rotenone dosage of 3 mg/kg repeated every 48 hours for 60 days to trigger retinal neurodegeneration. Group III (troxerutin‐treated group): rats received troxerutin (150 mg/kg/day) by oral gavage 1 hour before rotenone administration. A real‐time polymerase chain reaction technique was applied to measure messenger RNA (mRNA) levels of retinal C/EBP‐β. Enzyme‐linked immunosorbent assay technique was utilized to assay tumor necrosis factor‐α (TNF‐α), IFNγ, and ERK1/2 levels. Finally, reactive oxygen species (ROS), as well as carbonylated protein (CP) levels, were assessed spectrophotometrically. Improved retinal neurodegeneration by downregulation of C/EBP‐β mRNA gene expression, also caused a significant reduction of TNF‐α, IFNγ, ERK1/2 as well as ROS and CP levels compared with the diseased group. These findings could hold promise for the usage of troxerutin as a protective agent against rotenone‐induced retinal neurodegeneration.     

Fenoxycarb promotes adipogenesis in 3T3‐L1 preadipocytes

Lipophilic insect hormones and their analogs affect mammalian physiology by regulating the expression of metabolic genes. Therefore, we determined the effect of fenoxycarb, a juvenile hormone analog, on lipid metabolism in adipocytes. Here, we demonstrated that fenoxycarb dose‐dependently promoted lipid accumulation in 3T3‐L1 adipocytes during adipocyte differentiation and that its lipogenic effect was comparable to that of rosiglitazone, a well‐known ligand for peroxisome proliferator‐activated receptor gamma (PPARγ). Furthermore, fenoxycarb stimulated PPARγ activity without affecting other nuclear receptors, such as liver X receptor (LXR), farnesoid X‐activated receptor (FXR) and Nur77. In addition, fenoxycarb treatment increased the expression of PPARγ and fatty acid transporter protein 1 (FATP1) in 3T3‐L1 adipocytes, suggesting that fenoxycarb may facilitate adipocyte differentiation by enhancing PPARγ signaling, the master regulator of adipogenesis. Together, our results suggest that fenoxycarb promoted lipid accumulation in adipocytes, in part, by stimulating PPARγ.