Prostaglandin E2 Exerts Multiple Regulatory Actions on Human Obese Adipose Tissue Remodeling,Inflammation, Adaptive Thermogenesis and Lipolysis

Obesity induces white adipose tissue (WAT) dysfunction characterized by unremitting inflammation and fibrosis, impaired adaptive thermogenesis and increased lipolysis. Prostaglandins (PGs) are powerful lipid mediators that influence the homeostasis of several organs and tissues. The aim of the current study was to explore the regulatory actions of PGs in human omental WAT collected from obese patients undergoing laparoscopic bariatric surgery. In addition to adipocyte hypertrophy, obese WAT showed remarkable inflammation and total and pericellular fibrosis. In this tissue, a unique molecular signature characterized by altered expression of genes involved in inflammation, fibrosis and WAT browning was identified by microarray analysis. Targeted LC-MS/MS lipidomic analysis identified increased PGE₂ levels in obese fat in the context of a remarkable COX-2 induction and in the absence of changes in the expression of terminal prostaglandin E synthases (i.e. mPGES-1, mPGES-2 and cPGES). IPA analysis established PGE₂ as a common top regulator of the fibrogenic/inflammatory process present in this tissue. Exogenous addition of PGE₂ significantly reduced the expression of fibrogenic genes in human WAT explants and significantly down-regulated Col1α1, Col1α2 and αSMA in differentiated 3T3 adipocytes exposed to TGF-β. In addition, PGE₂ inhibited the expression of inflammatory genes (i.e. IL-6 and MCP-1) in WAT explants as well as in adipocytes challenged with LPS. PGE₂ anti-inflammatory actions were confirmed by microarray analysis of human pre-adipocytes incubated with this prostanoid. Moreover, PGE₂ induced expression of brown markers (UCP1 and PRDM16) in WAT and adipocytes, but not in pre-adipocytes, suggesting that PGE₂ might induce the trans-differentiation of adipocytes towards beige/brite cells. Finally, PGE₂ inhibited isoproterenol-induced adipocyte lipolysis. Taken together, these findings identify PGE₂ as a regulator of the complex network of interactions driving uncontrolled inflammation and fibrosis and impaired adaptive thermogenesis and lipolysis in human obese visceral WAT.     

Cigarette smoke concentrate increases 8-epi-PGF2alpha and TGFbeta1 secretion in rat mesangial cells

Epidemiological studies have shown that cigarette smoke, an oxidant agent, is a risk factor for the development of diabetic nephropathy (DN), in which pathogenesis transforming growth factor beta(1) (TGFbeta(1)) plays a key role. In our experimental model we exposed mesangial cell cultures to cigarette smoke concentrate (CSC) to study the effect of smoking on the pathogenesis of DN. Thus, we analyzed the effect of CSC on TGFbeta(1) and lipid peroxidation (8-epi-PGF(2alpha)) in rat mesangial cells. Furthermore, since the protein kinase C (PKC) pathway appears to be a key factor for the enhanced production of TGFbeta(1), we also analyzed the effect of the selective PKCbeta inhibitor LY379196 on TGFbeta(1) response to CSC. CSC induced an increase of both TGFbeta(1) and 8-epi-PGF(2) compared to basal conditions (5 mM glucose). The CSC-induced increase in TGFbeta(1) secretion was significantly suppressed by LY379196. These data suggest that smoking could increase TGFbeta(1) production, probably due to oxidative stress and PKCbeta activation. This finding supports the concept that smoking is a risk factor for DN development.     

Resolvin D1 and its precursor docosahexaenoic acid promote resolution of adipose tissue inflammation by eliciting macrophage polarization toward an M2-like phenotype

We recently demonstrated that ω-3-polyunsaturated fatty acids ameliorate obesity-induced adipose tissue inflammation and insulin resistance. In this study, we report novel mechanisms underlying ω-3-polyunsaturated fatty acid actions on adipose tissue, adipocytes, and stromal vascular cells (SVC). Inflamed adipose tissue from high-fat diet-induced obese mice showed increased F4/80 and CD11b double-positive macrophage staining and elevated IL-6 and MCP-1 levels. Docosahexaenoic acid (DHA; 4 μg/g) did not change the total number of macrophages but significantly reduced the percentage of high CD11b/high F4/80-expressing cells in parallel with the emergence of low-expressing CD11b/F4/80 macrophages in the adipose tissue. This effect was associated with downregulation of proinflammatory adipokines in parallel with increased expression of IL-10, CD206, arginase 1, resistin-like molecule α, and chitinase-3 like protein, indicating a phenotypic switch in macrophage polarization toward an M2-like phenotype. This shift was confined to the SVC fraction, in which secretion of Th1 cytokines (IL-6, MCP-1, and TNF-α) was blocked by DHA. Notably, resolvin D1, an anti-inflammatory and proresolving mediator biosynthesized from DHA, markedly attenuated IFN-γ/LPS-induced Th1 cytokines while upregulating arginase 1 expression in a concentration-dependent manner. Resolvin D1 also stimulated nonphlogistic phagocytosis in adipose SVC macrophages by increasing both the number of macrophages containing ingested particles and the number of phagocytosed particles and by reducing macrophage reactive oxygen species production. No changes in adipocyte area and the phosphorylation of hormone-sensitive lipase, a rate-limiting enzyme regulating adipocyte lipolysis, were observed. These findings illustrate novel mechanisms through which resolvin D1 and its precursor DHA confer anti-inflammatory and proresolving actions in inflamed adipose tissue.     

Dehalogenation of dichloroethene in a contaminated soil: fatty acids and alcohols as electron donors and an apparent requirement for tetrachloroethene

Environmental soil contamination at an industrial site in Marion, Ohio (USA) with tetrachloroethene (perchloroethene, PCE) resulted in residual cis-1, 2-dichloroethene (DCE) contamination that had not declined after more than 15 years. Microcosm slurries containing 2.6% soil from this site were supplemented with different electron donors, i.e., individual fatty acids or alcohols. None of the microcosms supported complete DCE dechlorination, unless PCE was added to the microcosm at initiation. The addition of fresh PCE resulted in the dehalogenation of PCE to DCE in the microcosms supplemented with fatty acids having an even number of carbon atoms (acetate, butyrate, and caproate), but not in those with an odd number of carbon atoms (formate, propionate, and valerate), where negligible or no activity was detected. No significant further DCE degradation was observed in any of the microcosms supplied with fatty acids as electron donors. Microcosms supplemented with freshly added PCE bioconverted PCE to DCE and completely dehalogenated both the ex-novo and soil-supplied DCE within 60 days, but only if alcohols having an even number of carbon atoms (ethanol or butanol) were also added as electron donors. Odd-numbered alcohols either did not produce dehalogenation (as with methanol) or only dehalogenated PCE to DCE (as with propanol).     

Coordinate Functional Regulation between Microsomal Prostaglandin E Synthase-1 (mPGES-1) and Peroxisome Proliferator-activated Receptor γ (PPARγ) in the Conversion of White-to-brown Adipocytes

Peroxisome proliferator-activated receptor γ (PPARγ) is a ligand-activated nuclear receptor and a master regulator of adipogenesis. Microsomal prostaglandin E (PGE) synthase-1 (mPGES-1) is an inducible enzyme that couples with cyclooxygenase-2 for the biosynthesis of PGE₂. In this study we demonstrate the existence of a coordinate functional interaction between PPARγ and mPGES-1 in controlling the process of pre-adipocyte differentiation in white adipose tissue (WAT). Adipocyte-specific PPARγ knock-out mice carrying an aP2 promoter-driven Cre recombinase transgene showed a blunted response to the adipogenic effects of a high fat diet. Pre-adipocytes from these knock-out mice showed loss of PPARγ and were resistant to rosiglitazone-induced WAT differentiation. In parallel, WAT from these mice showed increased expression of uncoupling protein 1, a mitochondrial enzyme that dissipates chemical energy as heat. Adipose tissue from mice lacking PPARγ also showed mPGES-1 up-regulation and increased PGE₂ levels. In turn, PGE₂ suppressed PPARγ expression and blocked rosiglitazone-induced pre-adipocyte differentiation toward white adipocytes while directly elevating uncoupling protein 1 expression and pre-adipocyte differentiation into mature beige/brite adipocytes. Consistently, pharmacological mPGES-1 inhibition directed pre-adipocyte differentiation toward white adipocytes while suppressing differentiation into beige/brite adipocytes. This browning effect was reproduced in knockdown experiments using a siRNA directed against mPGES-1. The effects of PGE₂ on pre-adipocyte differentiation were not seen in mice lacking PPARγ in adipose tissue and were not mirrored by other eicosanoids (i.e. leukotriene B₄). Taken together, these findings identify PGE₂ as a key regulator of white-to-brown adipogenesis and suggest the existence of a coordinate regulation of adipogenesis between PPARγ and mPGES-1.     

Chromosome length and perinuclear attachment constrain resolution of DNA intertwines

To allow chromosome segregation, topoisomerase II (topo II) must resolve sister chromatid intertwines (SCI) formed during deoxynucleic acid (DNA) replication. How this process extends to the full genome is not well understood. In budding yeast, the unique structure of the ribosomal DNA (rDNA) array is thought to cause late SCI resolution of this genomic region during anaphase. In this paper, we show that chromosome length, and not the presence of rDNA repeats, is the critical feature determining the time of topo II–dependent segregation. Segregation of chromosomes lacking rDNA also requires the function of topo II in anaphase, and increasing chromosome length aggravates missegregation in topo II mutant cells. Furthermore, anaphase Stu2-dependent microtubule dynamics are critical for separation of long chromosomes. Finally, defects caused by topo II or Stu2 impairment depend on attachment of telomeres to the nuclear envelope. We propose that topological constraints imposed by chromosome length and perinuclear attachment determine the amount of SCI that topo II and dynamic microtubules resolve during anaphase.     

Regulatory effects of arachidonate 5-lipoxygenase on hepatic microsomal TG transfer protein activity and VLDL-triglyceride and apoB secretion in obese mice

As 5-lipoxygenase (5-LO) is an emerging target in obesity and insulin resistance, we have investigated whether this arachidonate pathway is also implicated in the progression of obesity-related fatty liver disease. Our results show that 5-LO activity and 5-LO-derived product levels are significantly elevated in the liver of obese ob/ob mice with respect to wild-type controls. Treatment of ob/ob mice with a selective 5-LO inhibitor exerted a remarkable protection from hepatic steatosis as revealed by decreased oil red-O staining and reduced hepatic triglyceride (TG) concentrations. In addition, 5-LO inhibition in ob/ob mice downregulated genes involved in hepatic fatty acid uptake (i.e., L-FABP and FAT/CD36) and normalized peroxisome proliferator-activated receptor alpha (PPARalpha) and acyl-CoA oxidase expression, whereas the expression of lipogenic genes [i.e., fatty acid synthase (FASN) and SREBP-1c] remained unaltered. Furthermore, 5-LO inhibition restored hepatic microsomal TG transfer protein (MTP) activity in parallel with a stimulation of hepatic VLDL-TG and apoB secretion in ob/ob mice. Consistent with these findings, 5-LO products directly inhibited MTP activity and triggered cytosolic TG accumulation in CC-1 cells, a murine hepatocyte cell line. Taken together, these findings identify a novel steatogenic role for 5-LO in the liver through mechanisms involving the regulation of hepatic MTP activity and VLDL-TG and apoB secretion.     

Hepatocytes are a rich source of novel aspirin-triggered 15-epi-lipoxin A4

Novel aspirin (ASA)-triggered 15-epi-lipoxins (ATL) comprise newpotent bioactive eicosanoids that may contribute to the therapeutic effect of this drug. ATL biosynthesis is initiated by ASA acetylation of cyclooxygenase (COX)-2 and was originally identified during theinteraction of leukocytes with either endothelial or epithelial cells.Here, we examined ATL biosynthesis in rat hepatocytes either alone orin coincubation with nonparenchymal liver cells (NPC) and in liverhomogenates from ASA-treated rats. Rat hepatocytes and CC-1 cells, arat hepatocyte cell line, displayed COX-1 but not COX-2 mRNA expressionand predominantly produced thromboxane A₂(TXA₂) and15-hydroxyeicosatetraenoic acid (15-HETE). In these cells, ASA shiftedthe arachidonic acid metabolism fromTXA₂ to 15-HETE in aconcentration-dependent manner. In contrast, neither indomethacin,ibuprofen, valeryl salicylate, nor nimesulide was able to trigger15-HETE biosynthesis. SKF-525A, a cytochromeP-450 inhibitor, significantly reducedthe effect of ASA on 15-HETE biosynthesis. Furthermore, phenobarbital,a potent inducer of cytochrome P-450activity, further increased ASA-induced 15-HETE production. ASAtreatment of hepatocyte-NPC coincubations resulted in the generation ofsignificant amounts of ATL. In addition, in vivo experimentsdemonstrated augmented hepatic levels of 15-epi-lipoxin A₄ in ASA-treated rats. Takentogether and considering that ASA is hydrolyzed on its first passthrough the portal circulation, these data indicate that, during ASA'sconsumption, liver tissue generates biologically relevant amounts ofATL by COX-2-independent mechanisms.

     

Rapid identification of single nucleotide polymorphisms by fluorescence-based capillary electrophoresis

We describe the application of two different fluorescence-based techniques (ddNTP primer extension and single-strand conformation polymorphism (SSCP)) to the detection of single nucleotide polymorphisms (SNPs) by capillary electrophoresis. The ddNTP primer extension technique is based on the extension, in the presence of fluorescence-labeled dideoxy nucleotides (ddNTP, terminators), of an unlabeled oligonucleotide primer that binds to the complementary template immediately adjacent to the mutant nucleotide position. Given that there are no unlabeled dNTPs, a single ddNTP is added to its 3' end, resulting in a fluorescence-labeled primer extension product which is readily separated by capillary electrophoresis. On the other hand, the non-radioisotopic version of SSCP established in this study uses fluorescent dye to label the PCR products, which are also analyzed by capillary electrophoresis. These procedures were used to identify a well-defined SNP in exon 7 of the human p53 gene in DNA samples isolated from two human cell lines (CEM and THP-1 cells). The results revealed a heterozygous single-base transition (G to A) at nucleotide position 14071 in CEM cells, proving that both fluorescence-based ddNTP primer extension and SSCP are rapid, simple, robust, specific and with no ambiguity in interpretation for the detection of well-defined SNPs.     

A Cross-sectional Serological Study of Cysticercosis,Schistosomiasis, Toxocariasis and Echinococcosis in HIV-1 Infected People in Beira,Mozambique

Background

Helminthic infections are highly endemic in Mozambique, due to limited access to healthcare and resources for disease prevention. Data on the subclinical prevalence of these diseases are scarce due to the fact that an immunological and imaging diagnosis is not often available in endemic areas. We conducted a cross-sectional study on HIV1⁺ patients from Beira city in order to determine the seroprevalence of cysticercosis, schistosomiasis, toxocariasis and echinoccocosis and its possible interaction with HIV infection.

Methodology/Principal Findings

Patients (601) were voluntarily recruited at the Ponta Gea Health Center and their demographic and clinical data were recorded (including CD4⁺ cell count and antiretroviral regimen). Mean age was 39.7 years, 378 (62.9%) were women and 223 (37.1%) were men. Four hundred seventy-five (475) patients (79%) were already on highly active antiretroviral therapy (HAART), and 90 started therapy after being enrolled in the study. For serological testing we used a Multiplex Western Blot IgG from LDBIO Diagnostics. The overall seroprevalence was 10.2% for cysticercosis, 23% for schistosomiasis, 7.3% for toxocariasis and 17.3% for echinococcosis.

Conclusions/Significance

Neither age nor the CD4⁺ count were significantly associated with the seroprevalence of the helminths studied. However, patients with CD4⁺ between 200–500/µl had a higher seroprevalence to all helminths than those with less than 200/µl cells/and those with more than 500 cells/µl. Female gender was significantly associated with cysticercosis and schistosomiasis, and being in HAART with toxocariasis. Headache was significantly associated with cysticercosis and toxocariasis. There was no association between epilepsy and seropositivity to any of the parasites. The study concluded that a clear understanding of the prevalence and manifestations of these coinfections, how best to diagnose subclinical cases, and how to manage diseases with concomitant antiretroviral therapy is needed.