Validation of endogenous controls for gene expression studies in human adipocytes and preadipocytes.

Quantitative gene expression protocols require adequate controls to monitor intersample variation. Quantitative approaches to describe relative changes in gene expression use endogenous controls--"housekeeping" genes. Given the low amounts of mRNA in fat cells, RT-PCR is the method of choice, and housekeeping genes are widely used as endogenous controls. However, literature reports suggest changes in gene expression of typical housekeeping genes (e. g. GAPDH, beta-actin, 18S rRNA) upon hormonal stimulation or during adipogenic differentiation. Thus, we tested the influence of 6 hormones and adipogenic differentiation on gene expression levels of 11 commonly used housekeeping genes in primary cultured mature human adipocytes and preadipocytes. Using the TaqMan RT-PCR technique and "Human Endogenous Control Assays" (PE Biosystems), we found several housekeeping genes with at least twice the difference in expression levels between stimulated and unstimulated cells (such as acidic ribosomal protein, beta-actin, beta(2)-microglobulin and beta-glucuronidase). Only GAPDH and transferrin receptor gene expression levels did not change under any of the stimuli tested, thus appeared best suited for gene expression studies in human adipose cells across a wide range of experimental settings.     

The HIV protease inhibitor ritonavir increases lipoprotein production and has no effect on lipoprotein clearance in mice

This study examined the effect of human immunodeficiency virus (HIV) protease inhibitor therapy on lipoprotein production and catabolism in vivo. The HIV protease inhibitor ritonavir was given to C57BL/6 mice fed either a basal low-fat diet or a Western type high-fat diet. Fasted mice were injected with Triton WR1339 followed by hourly blood collection to monitor lipoprotein production. Results showed that ritonavir increased VLDL triglyceride production by 30% over a 4 h period when mice were fed the low-fat basal diet. The ritonavir effect was more pronounced under high-fat feeding conditions, with a 2-fold increase in VLDL triglyceride production rate. Ritonavir did not alter hepatic expression levels of diacylglycerol acyltransferase or microsomal triglyceride transfer protein, but increased hepatic apolipoprotein B (apoB) secretion rates under both low- and high-fat dietary conditions. In contrast to its effect on lipoprotein production, ritonavir did not alter triglyceride-rich lipoprotein clearance from circulation under either dietary condition. Taken together, these results indicate that the hyperlipidemic effect of HIV protease inhibitors is a direct result of increased hepatic lipoprotein production. The mechanism appears to be related to their role in preventing proteasome-mediated degradation of apoB and activated sterol regulatory element binding proteins in the liver.     

The HIV aspartyl protease inhibitor ritonavir impairs planktonic growth,biofilm formation and proteolytic activity in Trichosporon spp.

This study evaluated the effect of the protease inhibitor ritonavir (RIT) on Trichosporon asahii and Trichosporon inkin. Susceptibility to RIT was assessed by the broth microdilution assay and the effect of RIT on protease activity was evaluated using azoalbumin as substrate. RIT was tested for its anti-biofilm properties and RIT-treated biofilms were assessed regarding protease activity, ultrastructure and matrix composition. In addition, antifungal susceptibility, surface hydrophobicity and biofilm formation were evaluated after pre-incubation of planktonic cells with RIT for 15 days. RIT (200 μg ml^?1) inhibited Trichosporon growth. RIT (100 μg ml^?1) also reduced protease activity of planktonic and biofilm cells, decreased cell adhesion and biofilm formation, and altered the structure of the biofilm and the protein composition of the biofilm matrix. Pre-incubation with RIT (100 μg ml^?1) increased the susceptibility to amphotericin B, and reduced surface hydrophobicity and cell adhesion. These results highlight the importance of proteases as promising therapeutic targets and reinforce the antifungal potential of protease inhibitors.     

The crystal structure of protease Sapp1p from Candida parapsilosis in complex with the HIV protease inhibitor ritonavir

Secreted aspartic proteases (Saps) are extracellular proteolytic enzymes that enhance the virulence of Candida pathogens. These enzymes therefore represent possible targets for therapeutic drug design. Saps are inhibited by nanomolar concentrations of the classical inhibitor of aspartic proteases pepstatin A and also by the inhibitors of the HIV protease, but with the K_i of micromolar values or higher. To contribute to the discussion regarding whether HIV protease inhibitors can act against opportunistic mycoses by the inhibition of Saps, we determined the structure of Sapp1p from Candida parapsilosis in complex with ritonavir (RTV), a clinically used inhibitor of the HIV protease. The crystal structure refined at resolution 2.4 Å proved binding of RTV into the active site of Sapp1p and provided the structural information necessary to evaluate the stability and specificity of the protein-inhibitor interaction.     

The crystal structure of protease Sapp1p from Candida parapsilosis in complex with the HIV protease inhibitor ritonavir

Secreted aspartic proteases (Saps) are extracellular proteolytic enzymes that enhance the virulence of Candida pathogens. These enzymes therefore represent possible targets for therapeutic drug design. Saps are inhibited by nanomolar concentrations of the classical inhibitor of aspartic proteases pepstatin A and also by the inhibitors of the HIV protease, but with the K(i) of micromolar values or higher. To contribute to the discussion regarding whether HIV protease inhibitors can act against opportunistic mycoses by the inhibition of Saps, we determined the structure of Sapp1p from Candida parapsilosis in complex with ritonavir (RTV), a clinically used inhibitor of the HIV protease. The crystal structure refined at resolution 2.4 ? proved binding of RTV into the active site of Sapp1p and provided the structural information necessary to evaluate the stability and specificity of the protein-inhibitor interaction.     

Overexpression of uncoupling protein 4 promotes proliferation and inhibits apoptosis and differentiation of preadipocytes

Uncoupling proteins are a family of mitochondrial proteins involved in energy metabolism. We previously showed that uncoupling protein 4 (UCP4) is differentially expressed in omental adipose tissue in diet-induced obese and normal rats. However, the effect of UCP4 on adipocytes is unclear. In this work, we established a stable preadipocyte cell line overexpressing UCP4 to observe the direct effect of UCP4 on adipocytes. Cells overexpressing UCP4 showed significantly attenuated differentiation of preadipocytes into adipocytes. During differentiation, expression of adipogenesis-associated markers such as fatty acid synthetase, peroxisome proliferator-activated receptor gamma, CCAAT enhancer binding protein alpha, adipocyte lipid binding protein and lipoprotein lipase were downregulated. Preadipoctes expressing UCP4 grew faster and more of them stayed in S phase compared to control cells. In addition, UCP4 overexpression protected preadipocytes from apoptosis induced by serum deprivation. Our results demonstrate that overexpression of UCP4 can promote proliferation and inhibit apoptosis and differentiation of preadipocytes.     

Induction of uncoupling protein-2 (UCP2) gene expression on the differentiation of rat preadipocytes to adipocytes in primary culture

We have examined uncoupling protein-2 (UCP2) gene expression in the adipose tissue of obese and normal rats and mice, and also in differentiated rat adipocytes in primary culture. Expression of the UCP2 gene was examined in rat and mouse adipose tissues using both RT-PCR and Northern blotting. Although the RT-PCR was not quantitative, the band corresponding to the UCP2 mRNA was stronger in white adipose tissue than in brown fat, regardless of the body weight of the rats. In agreement with the RT-PCR data, there was a higher level of UCP2 mRNA in the white adipocytes than in brown adipocytes, the level being greater in obese mice. Fibroblastic preadipocytes were obtained from the inguinal fat pad of suckling rats. Lipid droplets developed inside the cells upon differentiation and adipsin and UCP2 mRNAs were detected by Northern blotting. Both mRNAs were evident in the adipocytes at 4, 6, and 10 d after the induction of differentiation. There was no indication that the expression of UCP2 was markedly affected by the addition of leptin, dexamethasone or isoprenaline.     

Folate-related processes in human placenta: gene expression, aminothiols, proliferation and apoptosis

The paper contains short information concerning the role of folate-related processes in cell metabolism and multiple diseases which are characterized by hyperhomocysteinemia. The authors represent more detailed information about the folate-related processes in human placenta, namely about the content of aminothiols at different allelic variants of placental methylenetetrahydrofolate reductase during the course of physiological pregnancy and preeclampsia. The existing data concerning the expression and catalytic activity of corresponding enzymes are corroborated by the authors' own results that proved for the first time the functional activity of transsulfuration pathway in human placenta. This pathway is activated in placental explants in parallel with down-regulation of proliferation and up-regulation of apoptosis when hyperhomocysteinemia is imitated by high concentration of homocysteine in culture medium. On the whole the presented data point to the importance of placental folate-related processes for its normal function.     

Depot-specific gene expression profiles during differentiation and transdifferentiation of bovine muscle satellite cells, and differentiation of preadipocytes

We report a systematic study of gene expression during myogenesis and transdifferentiation in four bovine muscle tissues and of adipogenesis in three bovine fat tissues using DNA microarray analysis. One hundred hybridizations were performed and 7245 genes of known and unknown function were identified as being differentially expressed. Supervised hierarchical cluster analysis of gene expression patterns revealed the tissue specificity of genes. A close relationship in global gene expression observed for adipocyte-like cells derived from muscle and adipocytes derived from intramuscular fat suggests a common origin for these cells. The role of transthyretin in myogenesis is a novel finding. Different genes were highly induced during the transdifferentiation of myogenic satellite cells and in the adipogenesis of preadipocytes, indicating the involvement of different molecular mechanisms in these processes. Induction of CD36 and FABP4 expression in adipocyte-like cells and adipocytes may share a common pathway.     

Hypoxia-mimetic effects in the secretome of human preadipocytes and adipocytes

White adipose tissue (WAT) regulates energy metabolism by secretion of proteins with endocrine and paracrine effects. Dysregulation of the secretome of obesity-associated enlarged WAT may lead to obesity-related disorders. This can be caused by hypoxia as a result of poorly vascularized WAT. The effect of hypoxia on the secretome of human (pre)adipocytes is largely unknown. Therefore, we investigated the effect of CoCl₂, a hypoxia mimetic, on the secretome of human SGBS (pre)adipocytes by a proteomics approach combined with bioinformatic analysis. In addition, regulation of protein secretion was examined by protein turnover experiments. As such, secretome changes were particularly associated with protein down-regulation and extracellular matrix protein dysregulation. The observed up-regulation of collagens in adipocytes may be essential for cell survival while down-regulation of collagens in preadipocytes may indicate a disturbed differentiation process. These CoCl₂-induced changes reflect WAT dysfunction that ultimately may lead to obesity-associated complications. In addition, 9 novel adipocyte secreted proteins were identified from which 6 were regulated by CoCl₂. Mass spectrometry data have been deposited to the ProteomeXchange with identifier PXD000162.     

EGCG对猪前体脂肪细胞增殖和分化的作用

表没食子儿茶素没食子酸酯（Epigallocatechin-3-gallate,EGCG）是绿茶提取物EGCG的生物活性成分,为了探讨其对猪前体脂肪细胞增殖和分化的影响,以不同浓度EGCG处理猪前体脂肪细胞,MTT法测定EGCG对猪前体脂肪细胞生长的影响;油红O染色检测猪前体脂肪细胞的形态学变化;油红O染色提取法定量分析脂肪细胞充脂量的变化;半定量RT-PCR检测分化转录因子过氧化物酶体增生物激活受体佗（PPARγ2）和CCAAT／增强子结合蛋白α（C／EBPα）mRNA表达水平变化。结果显示：EGCG随着浓度的递增显著抑制猪前体脂肪细胞的增殖（P〈0．01）;低浓度的EGCG（5μmol／L）不影响脂肪细胞分化,而高浓度EGCG（200μmol／L）显著抑制猪前体脂肪细胞分化,同时下调PPARγ2和C／EBPαmRNA表达,本研究结果表明EGCG可抑制猪前体脂肪细胞的增殖和分化。     

Regulation of metallothionein gene expression and secretion in rat adipocytes differentiated from preadipocytes in primary culture.

The gene encoding metallothionein, a low mol. wt. metal binding and stress response protein, is expressed in white adipose tissue. In the present study, metallothionein (MT-1) gene expression and factors regulating metallothionein production have been examined in adipocytes induced to differentiate from fibroblastic preadipocytes in primary cell culture. On the induction of differentiation, the metallothionein-1 gene was strongly expressed in the cells and metallothionein released into the medium. A peak in metallothionein-1 mRNA level and metallothionein secretion occurred at 2 and 10 days post-differentiation, respectively, with a decrease in protein release after this time. The metallothionein-1 gene was expressed in the adipocytes prior to the adipsin and lipoprotein lipase genes, suggesting that it is an early marker of adipocyte differentiation. The addition of the glucocorticoid, dexamethasone, led to a substantial increase in metallothionein-1 mRNA in the cells and metallothionein secretion. Insulin and leptin also stimulated metallothionein production, although the effect was small. Neither noradrenaline nor the beta3-adrenoceptor agonist, BRL 37 344, altered metallothionein release but forskolin and bromo-cAMP were stimulatory, markedly increasing both metallothionein-1 level and metallothionein secretion. It is suggested that metallothionein is a novel secretory product of the differentiated white adipocyte and that its production is regulated particularly by glucocorticoids and through a cAMP-dependent pathway.     

PI3K/AKT抑制剂渥曼青霉素对猪前体脂肪细胞增殖和凋亡的影响

为探寻PI3K/AKT抑制剂渥曼青霉素(Wortmannin,WM)对猪前体脂肪细胞增殖和凋亡均无影响的适宜浓度,文章首先分离并验证了猪原代前体脂肪细胞的分化潜能,然后对不同浓度渥曼青霉素处理11 d的细胞采用Annexin V-FITC/PI双标法检测细胞凋亡,并通过凋亡相关基因的表达以及DNA损伤程度进行验证,同时利用甲烷硫代磺酸盐(Methanethiosulfonate,MTS)检测了细胞的增殖活性。结果表明,100 nmol/L渥曼青霉素对猪前体脂肪细胞的增殖和凋亡均无显著影响,而200 nmol/L的渥曼青霉素对猪原代脂肪细胞的增殖活性虽没有显著影响,但对细胞凋亡有显著促进作用。研究发现,处理后促凋亡因子caspase8和TNFR1表达显著上调,非caspase依赖促凋亡因子GZMA表达无显著性差异,而GZMB表达则显著上调,抗凋亡因子Bcl-x1表达显著上调,cFLIP表达则无显著性差异。100 nmol/L的渥曼青霉素对细胞DNA的损伤不显著。因此,100 nmol/L的渥曼青霉素对猪前体脂肪细胞的增殖和凋亡均无显著影响,是在不影响细胞生长的情况下研究PI3K通路对脂肪细胞分化的较为理想的浓度。     

The bile acid sensor FXR protects against dyslipidemia and aortic plaques development induced by the HIV protease inhibitor ritonavir in mice

Background

Although human immunodeficiency virus (HIV)–related morbidity and mortality rates in patients treated with a combination of high active antiretroviral therapy (HAART) have declined, significant metabolic/vascular adverse effects associated with the long term use of HIV protease inhibitors (PIs) have emerged as a significant side effect. Here we illustrate that targeting the bile acid sensor farnesoid X receptor (FXR) protects against dyslipidemia and vascular injury induced HIV-PIs in rodents.

Methodology/Principal Findings

Administration of the HIV PI ritonavir to wild type mice increased plasma triacylglycerols and cholesterol levels and this effect was exacerbated by dosing ritonavir to mice harbouring a disrupted FXR. Dyslipidemia induced by ritonavir associated with a shift in the liver expression of signature genes, Sterol Regulatory Element-Binding Protein (SREBP)-1 and fatty acid synthase. Treating wild type mice with the FXR agonist (chenodeoxycholic acid, CDCA) protected against development of dyslipidemia induced by ritonavir. Administration of ritonavir to ApoE^−/− mice, a strain that develop spontaneously atherosclerosis, increased the extent of aortic plaques without worsening the dyslipidemia. Treating these mice with CDCA reduced the extent of aortic plaques by 70% without changing plasma lipoproteins or the liver expression of signature genes. A beneficial effect on aortic plaques was also obtained by treating ApoE^−/− mice with gemfibrozil, a PPARα agonist. FXR activation counter-regulated induction of expression/activity of CD36 caused by HIV-PIs in circulating monocytes and aortic plaques. In macrophages cell lines, CDCA attenuated CD36 induction and uptake of acetylated LDL caused by ritonavir. Natural and synthetic FXR ligands reduced the nuclear translocation of SREBP1c caused by ritonavir.

Conclusions/Significance

Activation of the bile acid sensor FXR protects against dyslipidemia and atherosclerotic caused by ritonavir, a widely used HIV PI. From a mechanistic stand point it appears that besides reducing the liver expression of genes involved in fatty acid synthesis, FXR activation counter-regulates the expression/activity of CD36 on monocytes. FXR ligands might hold promise in the treatment dyslipidemia induced by ritonavir.