Discovery of an intravenous hepatoselective glucokinase activator for the treatment of inpatient hyperglycemia

Glucokinase (hexokinase IV) continues to be a compelling target for the treatment of type 2 diabetes given the wealth of supporting human genetics data and numerous reports of robust clinical glucose lowering in patients treated with small molecule allosteric activators. Recent work has demonstrated the ability of hepatoselective activators to deliver glucose lowering efficacy with minimal risk of hypoglycemia. While orally administered agents require a considerable degree of passive permeability to promote suitable exposures, there is no such restriction on intravenously delivered drugs. Therefore, minimization of membrane diffusion in the context of an intravenously agent should ensure optimal hepatic targeting and therapeutic index. This work details the identification a hepatoselective GKA exhibiting the aforementioned properties.     

ACTH- and Cortisol-Associated Neutrophil Modulation in Coronary Artery Disease Patients Undergoing Stent Implantation

Background

Psychosocial stress and activation of neutrophil granulocytes are increasingly recognized as major risk factors of coronary artery disease (CAD), but the possible relationship of these two factors in CAD patients is largely unexplored. Activation of neutrophils was reported to be associated with stenting; however, the issue of neutrophil state in connection with percutaneous coronary intervention (PCI) is incompletely understood from the aspect of stress and its hypothalamic-pituitary-adrenal axis (HPA) background. Thus, we aimed to study cortisol- and ACTH-associated changes in granulocyte activation in patients undergoing PCI.

Methodology/Principal Findings

Blood samples of 21 stable angina pectoris (SAP) and 20 acute coronary syndrome (ACS) patients were collected directly before (pre-PCI), after (post-PCI) and on the following day of PCI (1d-PCI). Granulocyte surface L-selectin, CD15 and (neutrophil-specific) lactoferrin were analysed by flow cytometry. Plasma cortisol, ACTH, and lactoferrin, IL-6 were also assayed. In both groups, pre- and post-PCI ratios of lactoferrin-bearing neutrophils were relatively high, these percentages decreased substantially next day; similarly, 1d-PCI plasma lactoferrin was about half of the post-PCI value (all p≤0.0001). Post-PCI ACTH was reduced markedly next day, especially in ACS group (SAP: p<0.01, ACS: p≤0.0001). In ACS, elevated pre-PCI cortisol decreased considerably a day after stenting (p<0.01); in pre-PCI samples, cortisol correlated with plasma lactoferrin (r∼0.5, p<0.05). In 1d-PCI samples of both groups, ACTH showed negative associations with the ratio of lactoferrin-bearing neutrophils (SAP: r = −0.601, p<0.005; ACS: r = −0.541, p<0.05) and with plasma lactoferrin (SAP: r = −0.435, p<0.05; ACS: r = −0.609, p<0.005).

Conclusions/Significance

Pre- and post-PCI states were associated with increased percentage of activated/degranulated neutrophils indicated by elevated lactoferrin parameters, the 1d-PCI declines of which were associated with plasma ACTH in both groups. The correlation of plasma cortisol with plasma lactoferrin in the extremely stressed ACS before stenting, however, suggests an association of cortisol with neutrophil activation.     

Diversity of extremophilic bacteria in the sediment of high-altitude lakes located in the mountain desert of Ojos del Salado volcano,Dry-Andes

Ojos del Salado, the highest volcano on Earth is surrounded by a special mountain desert with extreme aridity, great daily temperature range, intense solar radiation, and permafrost from 5000 meters above sea level. Several saline lakes and permafrost derived high-altitude lakes can be found in this area, often surrounded by fumaroles and hot springs. The aim of this study was to gain information about the bacterial communities inhabiting the sediment of high-altitude lakes of the Ojos del Salado region located between 3770 and 6500 m. Altogether 11 sediment samples from 4 different altitudes were examined with 16S rRNA gene based denaturing gradient gel electrophoresis and clone libraries. Members of 17 phyla or candidate divisions were detected with the dominance of Proteobacteria, Acidobacteria, Actinobacteria and Bacteroidetes. The bacterial community composition was determined mainly by the altitude of the sampling sites; nevertheless, the extreme aridity and the active volcanism had a strong influence on it. Most of the sequences showed the highest relation to bacterial species or uncultured clones from similar extreme environments.     

Regulation of the muscle-specific AMP-activated protein kinase alpha2beta2gamma3 complexes by AMP and implications of the mutations in the gamma3-subunit for the AMP dependence of the enzyme

The AMP-activated protein kinase is an evolutionarily conserved heterotrimer that is important for metabolic sensing in all eukaryotes. The muscle-specific isoform of the regulatory gamma-subunit of the kinase, AMP-activated protein kinase gamma3, has a key role in glucose and fat metabolism in skeletal muscle, as suggested by metabolic characterization of humans, pigs and mice harboring substitutions in the AMP-binding Bateman domains of gamma3. We demonstrate that AMP-activated protein kinase alpha2beta2gamma3 trimers are allosterically activated approximately three-fold by AMP with a half-maximal stimulation (A(0.5)) at 1.9 +/- 0.5 or 2.6 +/- 0.3 microm, as measured for complexes expressed in Escherichia coli or mammalian cells, respectively. We show that mutations in the N-terminal Bateman domain of gamma3 (R225Q, H306R and R307G) increased the A(0.5) values for AMP, whereas the fold activation of the enzyme by 200 microm AMP remained unchanged in comparison to the wild-type complex. The mutations in the C-terminal Bateman domain of gamma3 (H453R and R454G), on the other hand, substantially reduced the fold stimulation of the complex by 200 microm AMP, and resulted in AMP dependence curves similar to those of the double mutant, R225Q/R454G. A V224I mutation in gamma3, known to result in a reduced glycogen content in pigs, did not affect the fold activation or the A(0.5) values for AMP. Importantly, we did not detect any increase in phosphorylation of Thr172 of alpha2 by the upstream kinases in the presence of increasing concentrations of AMP. Taken together, the data show that different mutations in gamma3 exert different effects on the allosteric regulation of the alpha2beta2gamma3 complex by AMP, whereas we find no evidence for their role in regulating the level of phosphorylation of alpha2 by upstream kinases.     

The '3Rs' model and the concept of alternatives in animal research: a questionnaire survey

It has been 45 years since Russell and Burch first proposed the concept of the '3Rs', yet it remains unclear how those individuals involved in animal research view and implement these concepts. The authors used a questionnaire survey to determine how well-known experts judged issues related to the 3Rs.     

Pharmacological and functional biochemical properties of D-Ala2-D-Nle5-enkephalin-Arg-Phe

Tyr-D-Ala-Gly-Phe-D-Nle-Arg-Phe (DADN) a synthetic analogue of the endogenous Met-enkephalin-Arg-Phe (Tyr-Gly-Gly-Phe-Met-Arg-Phe; MERF), was investigated in radioligand binding assays, [(35)S]GTPgammaS stimulation experiments as well as in in vivo algesiometric tests. Binding properties of [(3)H]DADN were measured in crude membrane fractions of rat spinal cord tissues and in homogenates of Chinese hamster ovary (CHO) cells selectively expressing delta-, kappa-or micro-opioid receptors. The highest affinity for [(3)H]DADN binding was observed in membranes from CHO cells transfected with micro-opioid receptors confirming the micro-selectivity of the peptide. Unlabeled DADN was also investigated in functional biochemical experiments by measuring opioid receptor-mediated G-protein activation in rat brain membrane fractions. The peptide stimulated the activity of the regulatory G-proteins in a concentration dependent manner, and the stimulation was efficiently inhibited in the presence of micro-receptor specific antagonist ligands further supporting the selectivity profile of DADN. Intrathecally administered DADN produced a dose-related, naloxone-reversible antinociception in rat hot water tail-flick tests. Among the selective opioid antagonists tested, the delta-selective naltrindole (NTI) and the kappa-specific norbinaltorphimine (norBNI) showed only slight blocking effects compared with naloxone. The results obtained in the in vitro agonist-stimulated [(35)S]GTPgammaS binding assays are in good agreement with the opioid agonist effect seen in the in vivo pain test.     

Therapy-induced antibodies against the antiviral and antiproliferative effects of interferons in patients with chronic hepatitis C virus infection

Sera from 86 patients with chronic hepatitis C virus (HCV) infection treated with recombinant interferons-alpha (rIFN-alpha) were screened for IFN-binding and antiviral effect-neutralizing antibodies. Out of the 61 patients treated with rIFN-alpha2b, 46% had binding and 28% had neutralizing antibodies. 44% of the 25 patients treated with rIFN-alpha2a developed binding antibodies and 24% had neutralizing antibodies. Contradictory data were observed concerning the appearance of anti-IFN antibodies and the outcome of IFN therapy. A significantly higher number of the patients with a sustained response to rIFN-alpha2b therapy formed antibodies than the number among the non-responder patients. At the same time, in the patients treated with rIFN-alpha2a, opposite data were found. The activity of the antibodies in some sera was studied against the antiproliferative effect of IFNs on Daudi cells by measuring the [3H]thymidine incorporation. The binding antibodies without neutralization of the antiviral effect of the IFNs inhibited the antiproliferative activity of the rIFNs, similarly to antibodies having both IFN-binding and antiviral effect-neutralizing capacities. At the same time, the antiproliferative effect of the natural IFN was less affected. It is suggested that the antiproliferative assay is more sensitive than the antiviral method for demonstration of the presence of antibodies exerting an inhibitory effect on the biological activities of IFN.     

Positive health: connecting well-being with biology

Two key types of well-being, eudaimonic and hedonic, are reviewed. The first addresses ideas of self-development, personal growth and purposeful engagement, while the second is concerned with positive feelings such as happiness and contentment. How well-being varies by age and socio-economic standing is briefly summarized, followed by examination of its biological correlates (neuroendocrine, immune, cardiovascular, rapid eye movement (REM) sleep). Preliminary findings on a sample of ageing women showed that those with higher levels of eudaimonic well-being had lower levels of daily salivary cortisol, pro-inflammatory cytokines, cardiovascular risk, and longer duration REM sleep compared with those showing lower levels of eudaimonic well-being. Hedonic well-being, however, showed minimal linkage to biomarker assessments. Future research directions building on these initial findings are discussed.     

Effects of hyperoxia on VEGF,its receptors,and HIF-2alpha in the newborn rat lung

Signaling through the hypoxia inducible factor (HIF)-VEGF-VEGF receptor system (VEGF signaling system) leads to angiogenesis and epithelial cell proliferation and is a key mechanism regulating alveolarization in lungs of newborn rats. Hyperoxia exposure (>95% O2 days 4-14) arrests lung alveolarization and may do so through suppression of the VEGF signaling system. Lung tissue mRNA levels of HIF-2alpha and VEGF increased from days 4-14 in normoxic animals, but hyperoxia suppressed these increases. Levels of HIF-2alpha and VEGF mRNA were correlated in the air but not the O2-treated group, suggesting that the low levels of HIF-2alpha observed at high O2 concentrations are not stimulating VEGF expression. VEGF164 protein levels increased with developmental age, and with hyperoxia to day 9, but continuing hyperoxia decreased levels by day 12. VEGFR1 and VEGFR2 mRNA expression also increased in air-exposed animals, and these, too, were significantly decreased by hyperoxia by day 9 and day 12, respectively. Receptor protein levels did not increase with development; however, O2 did decrease protein to less than air values. Hyperoxic suppression of VEGF signaling from days 9-14 may be one mechanism by which alveolarization is arrested.