Improved Metabolic Health Alters Host Metabolism in Parallel with Changes in Systemic Xeno-Metabolites of Gut Origin

Novel plasma metabolite patterns reflective of improved metabolic health (insulin sensitivity, fitness, reduced body weight) were identified before and after a 14–17 wk weight loss and exercise intervention in sedentary, obese insulin-resistant women. To control for potential confounding effects of diet- or microbiome-derived molecules on the systemic metabolome, sampling was during a tightly-controlled feeding test week paradigm. Pairwise and multivariate analysis revealed intervention- and insulin-sensitivity associated: (1) Changes in plasma xeno-metabolites (“non-self” metabolites of dietary or gut microbial origin) following an oral glucose tolerance test (e.g. higher post-OGTT propane-1,2,3-tricarboxylate [tricarballylic acid]) or in the overnight-fasted state (e.g., lower γ-tocopherol); (2) Increased indices of saturated very long chain fatty acid elongation capacity; (3) Increased post-OGTT α-ketoglutaric acid (α-KG), fasting α-KG inversely correlated with Matsuda index, and altered patterns of malate, pyruvate and glutamine hypothesized to stem from improved mitochondrial efficiency and more robust oxidation of glucose. The results support a working model in which improved metabolic health modifies host metabolism in parallel with altering systemic exposure to xeno-metabolites. This highlights that interpretations regarding the origins of peripheral blood or urinary “signatures” of insulin resistance and metabolic health must consider the potentially important contribution of gut-derived metabolites toward the host''s metabolome.     

Intracellular location regulates calcium-calmodulin-dependent activation of organelle-restricted eNOS

Mislocalization of endothelial nitric oxide (NO) synthase (eNOS) in response to oxidized low-density lipoprotein, cholesterol depletion, elevated blood pressure, and bound eNOS interacting protein/NOS traffic inducer is associated with reduced NO release via unknown mechanisms. The proper targeting of eNOS to the plasma membrane or intracellular organelles is an important regulatory step controlling enzyme activity. Previous studies have shown that plasma membrane eNOS is constitutively phosphorylated on serine 1179 and highly active. In contrast, the activity of eNOS targeted to intracellular organelles is more complex. The cis-Golgi eNOS is fully activated by Akt-dependent phosphorylation. However, eNOS targeted to the trans-Golgi is decidedly less active in response to all modes of activation, including mutation to the phosphomimetic aspartic acid. In this study, we establish that when expressed within other intracellular organelles, such as the mitochondria and nucleus, the activity of eNOS is also greatly reduced. To address the mechanisms underlying the impaired catalytic activity of eNOS within these locations, we generated subcellular-targeted constructs that express a calcium-independent NOS isoform, iNOS. With the use of organelle specific (plasma membrane, cis- vs. trans-Golgi, plasma membrane, and Golgi, nucleus, and mitochondria) targeting motifs fused to the wild-type iNOS, we measured NO release from intact cells. With the exception of the Golgi lumen, our results showed no impairment in the ability of targeted iNOS to synthesize NO. Confirmation of correct targeting was obtained through confocal microscopy using identical constructs fused to the green fluorescent protein. We conclude that the reduced activation of eNOS within discrete cytoplasmic regions of the Golgi, the mitochondria and the nucleus is primarily due to insufficient access to calcium-calmodulin. nitric oxide; Akt; Golgi     

Specific Strains of Escherichia coli Are Pathogenic for the Endometrium of Cattle and Cause Pelvic Inflammatory Disease in Cattle and Mice

Background

Escherichia coli are widespread in the environment and pathogenic strains cause diseases of mucosal surfaces including the female genital tract. Pelvic inflammatory disease (PID; metritis) or endometritis affects ∼40% of cattle after parturition. We tested the expectation that multiple genetically diverse E. coli from the environment opportunistically contaminate the uterine lumen after parturition to establish PID.

Methodology/Principal Findings

Distinct clonal groups of E. coli were identified by Random Amplification of Polymorphic DNA (RAPD) and Multilocus sequence typing (MLST) from animals with uterine disease and these differed from known diarrhoeic or extra-intestinal pathogenic E. coli. The endometrial pathogenic E. coli (EnPEC) were more adherent and invasive for endometrial epithelial and stromal cells, compared with E. coli isolated from the uterus of clinically unaffected animals. The endometrial epithelial and stromal cells produced more prostaglandin E₂ and interleukin-8 in response to lipopolysaccharide (LPS) purified from EnPEC compared with non-pathogenic E. coli. The EnPEC or their LPS also caused PID when infused into the uterus of mice with accumulation of neutrophils and macrophages in the endometrium. Infusion of EnPEC was only associated with bacterial invasion of the endometrium and myometrium. Despite their ability to invade cultured cells, elicit host cell responses and establish PID, EnPEC lacked sixteen genes commonly associated with adhesion and invasion by enteric or extraintestinal pathogenic E. coli, though the ferric yersiniabactin uptake gene (fyuA) was present in PID-associated EnPEC. Endometrial epithelial or stromal cells from wild type but not Toll-like receptor 4 (TLR4) null mice secreted prostaglandin E₂ and chemokine (C-X-C motif) ligand 1 (CXCL1) in response to LPS from EnPEC, highlighting the key role of LPS in PID.

Conclusions/Significance

The implication arising from the discovery of EnPEC is that development of treatments or vaccines for PID should focus specifically on EnPEC and not other strains of E. coli.     

Angiopoietin-1 inhibits endothelial cell apoptosis via the Akt/survivin pathway

A productive angiogenic response must couple to the survival machinery of endothelial cells to preserve the integrity of newly formed vessels. Angiopoietin-1 (Ang-1) is an endothelium-specific ligand essential for embryonic vascular stabilization, branching morphogenesis, and post-natal angiogenesis, but its contribution to endothelial cell survival has not been completely elucidated. Here we show that Ang-1 acting via the Tie 2 receptor induces phosphorylation of the survival serine-threonine kinase, Akt (or protein kinase B). This is associated with up-regulation of the apoptosis inhibitor, survivin, in endothelial cells and protection of endothelium from death-inducing stimuli. Moreover, dominant negative survivin negates the ability of Ang-1 to protect cells from undergoing apoptosis. The activation of anti-apoptotic pathways mediated by Akt and survivin in endothelial cells may contribute to Ang-1 stabilization of vascular structures during angiogenesis, in vivo.     

Phosphorylation of threonine 497 in endothelial nitric-oxide synthase coordinates the coupling of L-arginine metabolism to efficient nitric oxide production

There is evidence that endothelial nitric-oxide synthase (eNOS) is regulated by reciprocal dephosphorylation of Thr497 and phosphorylation of Ser1179. To examine the interrelationship between these sites, cells were transfected with wild-type (WT), T497A, T497D, S1179D, and T497A/S1179D eNOS and activity, NO release and eNOS localization were assessed. Although eNOS T497A, S1179D and T497A/S1179D eNOS had greater enzymatic activity than did WT eNOS in lysates, basal production of NO from cells was markedly reduced in cells transfected with T497A and T497A/S1179D eNOS but augmented in cells transfected with S1179D eNOS. Stimulating cells with ATP or ionophore normalized the loss of function seen with T497A and T497A/S1179D eNOS to levels observed with WT and S1179D eNOS, respectively. Despite these functional differences, the localization of eNOS mutants were similar to WT. Because both T497A and T497A/S1179D eNOS exhibited higher enzyme activity but reduced production of NO, we examined whether these mutations were "uncoupling" NO synthesis. T497A and T497A/S1179D eNOS generated 2-3 times more superoxide anion than WT eNOS, and both basal and stimulated interactions of T497A/S1179D eNOS with hsp90 were reduced in co-immunoprecipitation experiments. Thus, the phosphorylation/dephosphorylation of Thr497 may be an intrinsic switch mechanism that determines whether eNOS generates NO versus superoxide in cells.     

Differential mechanisms of adenosine- and ATPγS-induced microvascular endothelial barrier strengthening

Maintenance of the endothelial cell (EC) barrier is critical to vascular homeostasis and a loss of barrier integrity results in increased vascular permeability. While the mechanisms that govern increased EC permeability have been under intense investigation over the past several decades, the processes regulating the preservation/restoration of the EC barrier remain poorly understood. Herein we show that the extracellular purines, adenosine (Ado) and adenosine 5′-[γ-thio]-triphosphate (ATPγS) can strengthen the barrier function of human lung microvascular EC (HLMVEC). This ability involves protein kinase A (PKA) activation and decreases in myosin light chain 20 (MLC20) phosphorylation secondary to the involvement of MLC phosphatase (MLCP). In contrast to Ado, ATPγS-induced PKA activation is accompanied by a modest, but significant decrease in cyclic adenosine monophosphate (cAMP) levels supporting the existence of an unconventional cAMP-independent pathway of PKA activation. Furthermore, ATPγS-induced EC barrier strengthening does not involve the Rap guanine nucleotide exchange factor 3 (EPAC1) which is directly activated by cAMP but is instead dependent upon PKA-anchor protein 2 (AKAP2) expression. We also found that AKAP2 can directly interact with the myosin phosphatase-targeting protein MYPT1 and that depletion of AKAP2 abolished ATPγS-induced increases in transendothelial electrical resistance. Ado-induced strengthening of the HLMVEC barrier required the coordinated activation of PKA and EPAC1 in a cAMP-dependent manner. In summary, ATPγS-induced enhancement of the EC barrier is EPAC1-independent and is instead mediated by activation of PKA which is then guided by AKAP2, in a cAMP-independent mechanism, to activate MLCP which dephosphorylates MLC20 resulting in reduced EC contraction and preservation.