Restoring leptin signaling reduces hyperlipidemia and improves vascular stiffness induced by chronic intermittent hypoxia

Chronic intermittent hypoxia (IH) during sleep can result from obstructive sleep apnea (OSA), a disorder that is particularly prevalent in obesity. OSA is associated with high levels of circulating leptin, cardiovascular dysfunction, and dyslipidemia. Relationships between leptin and cardiovascular function in OSA and chronic IH are poorly understood. We exposed lean wild-type (WT) and obese leptin-deficient ob/ob mice to IH for 4 wk, with and without leptin infusion, and measured cardiovascular indices including aortic vascular stiffness, endothelial function, cardiac myocyte morphology, and contractile properties. At baseline, ob/ob mice had decreased vascular compliance and endothelial function vs. WT mice. We found that 4 wk of IH decreased vascular compliance and endothelial relaxation responses to acetylcholine in both WT and leptin-deficient ob/ob animals. Recombinant leptin infusion in both strains restored IH-induced vascular abnormalities toward normoxic WT levels. Cardiac myocyte morphology and function were unaltered by IH. Serum cholesterol and triglyceride levels were significantly decreased by leptin treatment in IH mice, as was hepatic stearoyl-Coenzyme A desaturase 1 expression. Taken together, these data suggest that restoring normal leptin signaling can reduce vascular stiffness, increase endothelial relaxation, and correct dyslipidemia associated with IH.     

Studies on kidney sialidase in normal and diabetic rats

Rat kidney cortex sialidase was studied using alpha-sialyl-(2----3)-[3H]lactitol and alpha-sialyl-(2----6)-[3H]lactitol as substrates. The enzyme was found mainly in the lysosomal fraction. Only 23% of the sialidase activity of this fraction could be solubilized by a combination of freezing-thawing, sonication and Triton X-100 treatment. The optimal pH for the lysosomal enzyme activity was 4.2 and the enzyme's Km values for alpha-sialyl-(2----3)-lactitol and alpha-sialyl-(2----6)-lactitol were 0.28 and 0.41 mM, respectively. The specific activity was twice as high with the former substrate than with the latter. Sialidase activities in dialyzed kidney cortex homogenates of streptozotocin-diabetic rats and of age-matched control rats were compared. The specific activity was found to be significantly increased in the diabetic animals when using both substrates 5950 +/- 720 (S.E.) dpm/h per mg protein (n = 7) vs. 3970 +/- 370 in the controls (n = 8) with alpha-sialyl-(2----3)-lactitol (P less than 0.025) and 2870 +/- 300 vs. 1820 +/- 170 with alpha-sialyl-(2----6)-lactitol (P less than 0.02). The activities were also found to be increased when expressed per whole kidney cortex (P less than 0.005 and P less than 0.001, respectively). The elevated sialidase activity in diabetic kidney cortex may be related to the reported decrease in sialic acid content of the glomerular basement membrane, which lowers its negative charges and which may contribute to an increased permeability to proteins.     

Identification of Bacterial Infection in Neotropical Primates

Emerging infectious diseases usually arise from wild animal populations. In the present work, we performed a screening for bacterial infection in natural populations of New World primates. The blood cell bulk DNAs from 181 individuals of four Platyrrhini genera were PCR screened for eubacterial 16S rRNA genes. Bacteria were detected and identified in 13 distinct individuals of Alouatta belzebul, Alouatta caraya, and Cebus apella monkeys from geographically distant regions in the states of Mato Grosso and Pará, Brazil. Sequence analyses showed that these Platyrrhini bacteria are closely related not only to human pathogens Pseudomonas spp. but also to Pseudomonas simiae and sheep-Acari infecting Pseudomonas spp. The identified Pseudomonas possibly represents a group of bacteria circulating in natural monkey populations.     

The renal urate transporter SLC17A1 locus: confirmation of association with gout

Introduction

Two major gout-causing genes have been identified, the urate transport genes SLC2A9 and ABCG2. Variation within the SLC17A1 locus, which encodes sodium-dependent phosphate transporter 1, a renal transporter of uric acid, has also been associated with serum urate concentration. However, evidence for association with gout is equivocal. We investigated the association of the SLC17A1 locus with gout in New Zealand sample sets.

Methods

Five variants (rs1165196, rs1183201, rs9358890, rs3799344, rs12664474) were genotyped across a New Zealand sample set totaling 971 cases and 1,742 controls. Cases were ascertained according to American Rheumatism Association criteria. Two population groups were studied: Caucasian and Polynesian.

Results

At rs1183201 (SLC17A1), evidence for association with gout was observed in both the Caucasian (odds ratio (OR) = 0.67, P = 3.0 × 10^-6) and Polynesian (OR = 0.74, P = 3.0 × 10^-3) groups. Meta-analysis confirmed association of rs1183201 with gout at a genome-wide level of significance (OR = 0.70, P = 3.0 × 10^-8). Haplotype analysis suggested the presence of a common protective haplotype.

Conclusion

We confirm the SLC17A1 locus as the third associated with gout at a genome-wide level of significance.     

Theoretical studies of the role of C-terminal cysteines in the process of <Emphasis Type="Italic">S</Emphasis>-nitrosylation of human Src kinases

Src tyrosine kinases are a family of non-receptor proteins that are responsible for the growth process, cellular proliferation, differentiation and survival. Lack of Src kinase control has been associated with the development of certain human cancers. This family of proteins is constituted of four domains, with SH1 being the kinase or catalytic domain. SH1 also presents three important regulatory sites. Two residues, Tyr416 and Tyr527, are responsible for important phosphorylation processes that lead to, respectively, activation and deactivation of these kinases. More recently, however, a set of four cysteine residues located near the C-terminus-Cys483, Cys487, Cys496 and Cys498-has been associated with the activation of the Src kinases through S-nitrosylation reactions. Particularly, the Cys498 has been specified as a fundamental residue when considering this regulatory mechanism. Aiming to understand the role of these four cysteines in S-nitrosylation, theoretical studies of electrostatic, steric and hydrophobic properties were performed with a sequence of 20 amino acids, enclosing the four cysteine residues under study, extracted from the PDB coordinates of the crystal obtained from the inactive state of Src kinase. Results indicate that Cys498 is buried deeply in the protein, in hydrophobic surroundings in which NO is more likely to suffer decomposition into the electrophilic intermediates known to be responsible for S-nitrosylation reactions. Electronic calculated properties, such as punctual atomic charges, electrostatic potentials and molecular orbital energy, also demonstrated the good nucleophilic potential of Cys498.

Open image in new window

Graphical Abstract Structure of Src kinase with zoomed area representing the 20 amino acids comprising the CC motif extracted from the whole protein structure. Right upper panel Electrostatic potential map, right lower panel hydrophilic map in anterior view

     

In Vivo Evaluation of Blood Based and Reference Tissue Based PET Quantifications of [11C]DASB in the Canine Brain

This first-in-dog study evaluates the use of the PET-radioligand [¹¹C]DASB to image the density and availability of the serotonin transporter (SERT) in the canine brain. Imaging the serotonergic system could improve diagnosis and therapy of multiple canine behavioural disorders. Furthermore, as many similarities are reported between several human neuropsychiatric conditions and naturally occurring canine behavioural disorders, making this tracer available for use in dogs also provide researchers an interesting non-primate animal model to investigate human disorders. Five adult beagles underwent a 90 minutes dynamic PET scan and arterial whole blood was sampled throughout the scan. For each ROI, the distribution volume (V_T), obtained via the one- and two- tissue compartment model (1-TC, 2-TC) and the Logan Plot, was calculated and the goodness-of-fit was evaluated by the Akaike Information Criterion (AIC). For the preferred compartmental model BP_ND values were estimated and compared with those derived by four reference tissue models: 4-parameter RTM, SRTM2, MRTM2 and the Logan reference tissue model. The 2-TC model indicated in 61% of the ROIs a better fit compared to the 1-TC model. The Logan plot produced almost identical V_T values and can be used as an alternative. Compared with the 2-TC model, all investigated reference tissue models showed high correlations but small underestimations of the BP_ND-parameter. The highest correlation was achieved with the Logan reference tissue model (Y = 0.9266 x + 0.0257; R² = 0.9722). Therefore, this model can be put forward as a non-invasive standard model for future PET-experiments with [¹¹C]DASB in dogs.     

Evidence for Direct Physical Association between a K+ Channel (Kir6.2) and an ATP-Binding Cassette Protein (SUR1) Which Affects Cellular Distribution and Kinetic Behavior of an ATP-Sensitive K+ Channel

Structurally unique among ion channels, ATP-sensitive K⁺ (K_ATP) channels are essential in coupling cellular metabolism with membrane excitability, and their activity can be reconstituted by coexpression of an inwardly rectifying K⁺ channel, Kir6.2, with an ATP-binding cassette protein, SUR1. To determine if constitutive channel subunits form a physical complex, we developed antibodies to specifically label and immunoprecipitate Kir6.2. From a mixture of Kir6.2 and SUR1 in vitro-translated proteins, and from COS cells transfected with both channel subunits, the Kir6.2-specific antibody coimmunoprecipitated 38- and 140-kDa proteins corresponding to Kir6.2 and SUR1, respectively. Since previous reports suggest that the carboxy-truncated Kir6.2 can form a channel independent of SUR, we deleted 114 nucleotides from the carboxy terminus of the Kir6.2 open reading frame (Kir6.2ΔC37). Kir6.2ΔC37 still coimmunoprecipitated with SUR1, suggesting that the distal carboxy terminus of Kir6.2 is unnecessary for subunit association. Confocal microscopic images of COS cells transfected with Kir6.2 or Kir6.2ΔC37 and labeled with fluorescent antibodies revealed unique honeycomb patterns unlike the diffuse immunostaining observed when cells were cotransfected with Kir6.2-SUR1 or Kir6.2ΔC37-SUR1. Membrane patches excised from COS cells cotransfected with Kir6.2-SUR1 or Kir6.2ΔC37-SUR1 exhibited single-channel activity characteristic of pancreatic K_ATP channels. Kir6.2ΔC37 alone formed functional channels with single-channel conductance and intraburst kinetic properties similar to those of Kir6.2-SUR1 or Kir6.2ΔC37-SUR1 but with reduced burst duration. This study provides direct evidence that an inwardly rectifying K⁺ channel and an ATP-binding cassette protein physically associate, which affects the cellular distribution and kinetic behavior of a K_ATP channel.     

Mapping hypoxia-induced bioenergetic rearrangements and metabolic signaling by 18O-assisted 31P NMR and 1H NMR spectroscopy

Brief hypoxia or ischemia perturbs energy metabolism inducing paradoxically a stress-tolerant state, yet metabolic signals that trigger cytoprotection remain poorly understood. To evaluate bioenergetic rearrangements, control and hypoxic hearts were analyzed with 18O-assisted 31P NMR and 1H NMR spectroscopy. The 18O-induced isotope shift in the 31P NMR spectrum of CrP, betaADP and betaATP was used to quantify phosphotransfer fluxes through creatine kinase and adenylate kinase. This analysis was supplemented with determination of energetically relevant metabolites in the phosphomonoester (PME) region of 31P NMR spectra, and in both aromatic and aliphatic regions of 1H NMR spectra. In control conditions, creatine kinase was the major phosphotransfer pathway processing high-energy phosphoryls between sites of ATP consumption and ATP production. In hypoxia, creatine kinase flux was dramatically reduced with a compensatory increase in adenylate kinase flux, which supported heart energetics by regenerating and transferring beta- and gamma-phosphoryls of ATP. Activation of adenylate kinase led to a build-up of AMP, IMP and adenosine, molecules involved in cardioprotective signaling. 31P and 1H NMR spectral analysis further revealed NADH and H+ scavenging by alpha-glycerophosphate dehydrogenase (alphaGPDH) and lactate dehydrogenase contributing to maintained glycolysis under hypoxia. Hypoxia-induced accumulation of alpha-glycerophosphate and nucleoside 5'-monophosphates, through alphaGPDH and adenylate kinase reactions, respectively, was mapped within the increased PME signal in the 31P NMR spectrum. Thus, 18O-assisted 31P NMR combined with 1H NMR provide a powerful approach in capturing rearrangements in cardiac bioenergetics, and associated metabolic signaling that underlie the cardiac adaptive response to stress.     

Defective metabolic signaling in adenylate kinase AK1 gene knock-out hearts compromises post-ischemic coronary reflow

Matching blood flow to myocardial energy demand is vital for heart performance and recovery following ischemia. The molecular mechanisms responsible for transduction of myocardial energetic signals into reactive vasodilatation are, however, elusive. Adenylate kinase, associated with AMP signaling, is a sensitive reporter of the cellular energy state, yet the contribution of this phosphotransfer system in coupling myocardial metabolism with coronary flow has not been explored. Here, knock out of the major adenylate kinase isoform, AK1, disrupted the synchrony between inorganic phosphate P(i) turnover at ATP-consuming sites and gamma-ATP exchange at ATP synthesis sites, as revealed by (18)O-assisted (31)P NMR. This reduced energetic signal communication in the post-ischemic heart. AK1 gene deletion blunted vascular adenylate kinase phosphotransfer, compromised the contractility-coronary flow relationship, and precipitated inadequate coronary reflow following ischemia-reperfusion. Deficit in adenylate kinase activity abrogated AMP signal generation and reduced the vascular adenylate kinase/creatine kinase activity ratio essential for the response of metabolic sensors. The sarcolemma-associated splice variant AK1beta facilitated adenosine production, a function lost in the absence of adenylate kinase activity. Adenosine treatment bypassed AK1 deficiency and restored post-ischemic flow to wild-type levels, achieving phenotype rescue. AK1 phosphotransfer thus transduces stress signals into adequate vascular response, providing linkage between cell bioenergetics and coronary flow.