Adenosine increases LPS-induced nuclear factor kappa B activation in smooth muscle cells via an intracellular mechanism and modulates it via actions on adenosine receptors.

AIM: In inflamed and damaged cardiovascular tissues, local extracellular adenosine concentrations increase coincidentally with activation of the transcription factor nuclear factor kappa B (NFκB). To investigate whether adenosine influences NFκB activation in vascular smooth muscle cells (VSMCs) and, if so, to examine the role of its receptors. METHODS: VSMCs were isolated from NFκB-luciferase reporter mice, cultured and then treated by lipopolysaccharide (LPS) to activate NFκB signalling. Adenosine, adenosine receptor agonists and antagonists, adenosine deaminase and uptake inhibitors were used together with LPS to evaluate the role of adenosine and its receptors on NFκB activation, which was assessed by luciferase activity and NFκB target gene expression. RESULTS: Adenosine potentiated LPS-induced NFκB activation. This was dependent on adenosine uptake and enhanced by an adenosine deaminase inhibitor, suggesting that intracellular adenosine plays an important role. Non-selective adenosine receptor agonists (2Cl-Ado and NECA) inhibited NFκB activation induced by LPS. Selective A1 or A2A antagonist given alone could not completely antagonize the NECA effect, indicating that the inhibitory effect was due to multiple adenosine receptors. The activation of the A3 receptor further increased LPS-induced NFκB activation. CONCLUSIONS: Adenosine increases LPS-induced nuclear factor kappa B activation in smooth muscle cells via an intracellular mechanism and decreases it via actions on A1 and A2A receptors. These results provide novel insights into the role of adenosine as a regulator of inflammation-induced NFκB activation.

Protecting the heart through delivering DNA encoding for heme oxygenase-1 into skeletal muscle.

AIM: To evaluate if remote gene delivery of HMOX-1 prior to myocardial infarction can prevent cardiac remodeling and preserve function, without causing general angiogenesis. MAIN METHODS: Right quadriceps muscles of mice were treated with DNA encoding for HMOX-1 or empty vector (pcDNA) and electroporated to enhance nuclear uptake, while a third group received saline. Transfection efficacy was evaluated by real time PCR and situ hybridization in transfected muscle, contralateral muscle, and heart. Seven days after transfection baseline echocardiography was performed. Myocardial infarction was induced by ligation of the left coronary artery. Six weeks later heart function was reassessed by echocardiography. Hearts were extracted for evaluation of infarct size. Immunoflorescent staining was used to evaluate angiogenesis using the endothelial marker CD31 in cross-sections of the transfected quadriceps muscle, the untreated muscle, and hearts. KEY FINDINGS: Gene delivery of HMOX-1 leads to a local expression of HMOX-1 in the treated muscle, but not in any other organ. HMOX-1 treated mice had reduced infarct size (p=0.03) and improved function evident as higher ejection fraction (p=0.001), improved fractional shortening (p<0.0001) and higher stroke volume (p=0.002). HMOX-1 did not cause angiogenesis in the heart or skeletal muscle. SIGNIFICANCE: Remote delivery of DNA encoding for HMOX-1 was cardioprotective, as evidenced by preserved cardiac structure and function. Angiogenesis was not induced by HMOX-1 treatment.

Expression of perilipins in human skeletal muscle in vitro and in vivo in relation to diet, exercise and energy balance.

The perilipin proteins enclose intracellular lipid droplets. We describe the mRNA expression of the five perilipins in human skeletal muscle in relation to fatty acid supply, exercise and energy balance. We observed that all perilipins were expressed in skeletal muscle biopsies with the highest mRNA levels of perilipin 2, 4 and 5. Cultured myotubes predominantly expressed perilipin 2 and 3. In vitro, incubation of myotubes with fatty acids enhanced mRNA expression of perilipin 1, 2 and 4. In vivo, low fat diet increased mRNA levels of perilipin 3 and 4. Endurance training, but not strength training, enhanced the expression of perilipin 2 and 3. Perilipin 1 mRNA correlated positively with body fat mass, whereas none of the perilipins were associated with insulin sensitivity. In conclusion, all perilipins mRNAs were expressed in human skeletal muscle. Diet as well as endurance exercise modulated the expression of perilipins.

Altered expression of genes in adipose tissues associated with reduced fat mass in patients with pancreatic cancer.

Loss of adipose tissue in patients with pancreatic cancer may involve altered gene expression. Peri-operative mRNA levels of 44 genes were analysed by RT-PCR in intra-abdominal (IAAT) and subcutaneous adipose tissue (SCAT) sampled from pancreatic ductal adenocarcinoma (PDAC) patients undergoing tumour resection (n = 20), and control patients without cancer (n = 11). Peri- and post-operative IAAT and SCAT masses were measured by computerized tomography. PDAC patients displayed 2.6- and 1.7-fold higher Zn-α2-glycoprotein (AZGP1) mRNA levels than controls in IAAT and SCAT, respectively (P < 0.01), but expression was not correlated with post-operative changes in fat masses. IAAT mass changes correlated with genes in lipid metabolism, inflammation and apoptosis: e.g. stearoyl-Coenzyme A desaturase 1 (SCD), tumour necrosis factor (TNF) and chemokine (C-C motif) ligand 2 (CCL2; MCP-1). Patients with PDAC displayed increased AZGP1 mRNA levels in both IAAT and SCAT, but expression of other genes may predict IAAT loss.

Inhibition by insulin of resistin gene expression in 3T3-L1 adipocytes.

Expression of the gene encoding resistin, a low molecular weight protein secreted from adipose tissue postulated to link obesity and type II diabetes, was examined in 3T3-L1 adipocytes. Resistin mRNA was detected in 3T3-L1 cells by day 3 following induction of differentiation into adipocytes; by day 4 the level of resistin mRNA peaked and remained high. The PPARgamma activators, rosiglitazone or darglitazone, reduced the level of resistin mRNA. Dexamethasone upregulated resistin mRNA level, but no effect was observed with the beta(3)-adrenoceptor agonist, BRL 37344. A substantial reduction in resistin mRNA level was observed with insulin, which induced decreases at physiological concentrations. Insulin may be a major inhibitor of resistin production, and this does not support a role for resistin in insulin resistance.

Reduction of leptin gene expression by dietary polyunsaturated fatty acids.

Supplementation with n-3 polyunsaturated fatty acids (PUFA) for 6 weeks did not alter plasma leptin concentrations in male smokers. Changes in dietary intake of saturated fatty acids (FA) correlated positively, whereas changes in the intake of PUFA correlated negatively to changes in plasma leptin levels. A 3-week n-3 PUFA-enriched diet, as compared with a 3-week lard-enriched diet, induced lower plasma leptin concentration and reduced leptin mRNA expression in rat epididymal adipose tissue. In the human trophoblast cell line (BeWo), n-3 PUFA had a dose- and time-dependent effect on leptin expression. One mM of eicosapentaenoic acid or docosahexaenoic acid (DHA) reduced leptin expression by 71% and 78%, respectively, as compared with control, after 72 h. There was no effect on expression of the signal transducing form of the leptin receptor. In BeWo cells transfected with the human leptin promoter, we found that n-3 PUFA reduced leptin promoter activity; in contrast saturated and monounsaturated FA had no effect on leptin promoter activity. The transcription factors peroxysomal proliferator activated receptor gamma and sterol regulatory element binding protein-1 mRNAs were reduced after incubation with n-3 PUFA, whereas the expression of CCAAT/enhancer binding protein alpha was unchanged. DHA-reduced leptin expression was abolished in BeWo cells grown in cholesterol-free medium. In conclusion, n-3 FA decreased leptin gene expression both in vivo and in vitro. The direct effects of PUFA on leptin promoter activity indicate a specific regulatory action of FA on leptin expression.

Evidence for double helical kappa-carrageenan in aqueous LiI-solution and model for iodide binding.

NMR experiments with a 21412 Redfield observation pulse were carried out on a kappa-carrageenan aqueous solution containing 0.5M LiI. A detected hydrogen-bonded proton resonance at approximately 17.6 ppm, is in accordance with an ordered carrageenan structure in the solution similar to the double, parallel-stranded helix with interstrand hydrogen-bonds found in the condensed state by R.P. Millane, R. Chandrasekaran, S. Arnott, and I.C.M. Dea (Carbohydr. Res. 198, 1 1988). Starting with the published iota-carrageenan coordinates obtained from the Brookhaven Databank, we modelled this kappa-carrageenan structure by the molecular modelling software INSIGHTH-II. The kappa-carrageenan double-helix has a wide interior that can accommodate iodide. Conversely, the iota-carrageenan double-helix has a narrow interior not suited for a large ion like iodide. We propose that the detected selective iodide binding sites in the ordered kappa-carrageenan structure (H. Grasdalen, and O. Smidsrød, Macromolecules 14, 1842 1981) could very well be in the interior of the double-helix.

Kinetics and specificity of alginate lyases: Part I, A case study.

Purified preparations of alginate lyase from Klebsiella aerogenes and Haliotis sp. were investigated for activity and degradation patterns with alginate and alginate fragments having different compositions and sequences. With fragments approaching homopolymers of guluronate and mannuronate, Michaelis-Menten kinetics were obeyed and kinetic parameters could be obtained. Degradation of alginates containing all four possible linkages in various proportions, followed by isolation of the fragments and identification of the end groups by n.m.r. spectroscopy, indicated that the enzyme preparations can attack more than one type of linkage. The results are discussed with reference to the concept of specificity for enzymes with copolymeric substrates having non-regular distributions of units.