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OBJECTIVE - Type 2 diabetes mellitus (T2DM) reduces exercise capacity, but the mechanisms are incompletely understood. We probed the impact of ischemic stress on skeletal muscle metabolite signatures and T2DM-related vascular dysfunction.
METHODS - we recruited 38 subjects (18 healthy, 20 T2DM), placed an antecubital intravenous catheter, and performed ipsilateral brachial artery reactivity testing. Blood samples for plasma metabolite profiling were obtained at baseline and immediately upon cuff release after 5 min of ischemia. Brachial artery diameter was measured at baseline and 1 min after cuff release.
RESULTS - as expected, flow-mediated vasodilation was attenuated in subjects with T2DM (P<0.01). We confirmed known T2DM-associated baseline differences in plasma metabolites, including homocysteine, dimethylguanidino valeric acid and β-alanine (all P<0.05). Ischemia-induced metabolite changes that differed between groups included 5-hydroxyindoleacetic acid (healthy: -27%; DM +14%), orotic acid (healthy: +5%; DM -7%), trimethylamine-N-oxide (healthy: -51%; DM +0.2%), and glyoxylic acid (healthy: +19%; DM -6%) (all P<0.05). Levels of serine, betaine, β-aminoisobutyric acid and anthranilic acid were associated with vessel diameter at baseline, but only in T2DM (all P<0.05). Metabolite responses to ischemia were significantly associated with vasodilation extent, but primarily observed in T2DM, and included enrichment in phospholipid metabolism (P<0.05).
CONCLUSIONS - our study highlights impairments in muscle and vascular signaling at rest and during ischemic stress in T2DM. While metabolites change in both healthy and T2DM subjects in response to ischemia, the relationship between muscle metabolism and vascular function is modified in T2DM, suggesting that dysregulated muscle metabolism in T2DM may have direct effects on vascular function.
© 2020 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.
Lecithin:cholesterol acyltransferase (LCAT) catalyzes a critical step of reverse cholesterol transport by esterifying cholesterol in high density lipoprotein (HDL) particles. LCAT is activated by apolipoprotein A-I (ApoA-I), which forms a double belt around HDL, however the manner in which LCAT engages its lipidic substrates and ApoA-I in HDL is poorly understood. Here, we used negative stain electron microscopy, crosslinking, and hydrogen-deuterium exchange studies to refine the molecular details of the LCAT-HDL complex. Our data are consistent with LCAT preferentially binding to the edge of discoidal HDL near the boundary between helix 5 and 6 of ApoA-I in a manner that creates a path from the lipid bilayer to the active site of LCAT. Our results provide not only an explanation why LCAT activity diminishes as HDL particles mature, but also direct support for the anti-parallel double belt model of HDL, with LCAT binding preferentially to the helix 4/6 region.
The specific matrix used in matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) can have an effect on the molecules ionized from a tissue sample. The sensitivity for distinct classes of biomolecules can vary when employing different MALDI matrices. Here, we compare the intensities of various lipid subclasses measured by Fourier transform ion cyclotron resonance (FT-ICR) IMS of murine liver tissue when using 9-aminoacridine (9AA), 5-chloro-2-mercaptobenzothiazole (CMBT), 1,5-diaminonaphthalene (DAN), 2,5-Dihydroxyacetophenone (DHA), and 2,5-dihydroxybenzoic acid (DHB). Principal component analysis and receiver operating characteristic curve analysis revealed significant matrix effects on the relative signal intensities observed for different lipid subclasses and adducts. Comparison of spectral profiles and quantitative assessment of the number and intensity of species from each lipid subclass showed that each matrix produces unique lipid signals. In positive ion mode, matrix application methods played a role in the MALDI analysis for different cationic species. Comparisons of different methods for the application of DHA showed a significant increase in the intensity of sodiated and potassiated analytes when using an aerosol sprayer. In negative ion mode, lipid profiles generated using DAN were significantly different than all other matrices tested. This difference was found to be driven by modification of phosphatidylcholines during ionization that enables them to be detected in negative ion mode. These modified phosphatidylcholines are isomeric with common phosphatidylethanolamines confounding MALDI IMS analysis when using DAN. These results show an experimental basis of MALDI analyses when analyzing lipids from tissue and allow for more informed selection of MALDI matrices when performing lipid IMS experiments.
© 2019 John Wiley & Sons, Ltd.
Our goal was to investigate the neuroprotective effects of galantamine in a mouse model of blast-induced indirect traumatic optic neuropathy (bITON). Galantamine is an FDA-approved acetylcholinesterase inhibitor used to treat mild-moderate Alzheimer's disease. We exposed one eye of an anesthetized mouse to repeat bursts of over-pressurized air to induce traumatic optic neuropathy. Mice were given regular or galantamine-containing water (120 mg/L) ad libitum, beginning immediately after blast and continuing for one month. Electroretinograms and visual evoked potentials were performed just prior to endpoint collection. Histological and biochemical assessments were performed to assess activation of sterile inflammation, axon degeneration, and synaptic changes. Galantamine treatment mitigated visual function deficits induced by our bITON model via preservation of the b-wave of the electroretinogram and the N1 of the visual evoked potential. We also observed a reduction in axon degeneration in the optic nerve as well as decreased rod bipolar cell dendritic retraction. Galantamine also showed anti-inflammatory and antioxidant effects. Galantamine may be a promising treatment for blast-induced indirect traumatic optic neuropathy as well as other optic neuropathies.
Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.
OBJECTIVES - Cardiopulmonary bypass-induced endothelial dysfunction has been inferred by changes in pulmonary vascular resistance, alterations in circulating biomarkers, and postoperative capillary leak. Endothelial-dependent vasomotor dysfunction of the systemic vasculature has never been quantified in this setting. The objective of the present study was to quantify acute effects of cardiopulmonary bypass on endothelial vasomotor control and attempt to correlate these effects with postoperative cytokines, tissue edema, and clinical outcomes in infants.
DESIGN - Single-center prospective observational cohort pilot study.
SETTING - Pediatric cardiac ICU at a tertiary children's hospital.
PATIENTS - Children less than 1 year old requiring cardiopulmonary bypass for repair of a congenital heart lesion.
INTERVENTION - None.
MEASUREMENTS AND MAIN RESULTS - Laser Doppler perfusion monitoring was coupled with local iontophoresis of acetylcholine (endothelium-dependent vasodilator) or sodium nitroprusside (endothelium-independent vasodilator) to quantify endothelial-dependent vasomotor function in the cutaneous microcirculation. Measurements were obtained preoperatively, 2-4 hours, and 24 hours after separation from cardiopulmonary bypass. Fifteen patients completed all laser Doppler perfusion monitor (Perimed, Järfälla, Sweden) measurements. Comparing prebypass with 2-4 hours postbypass responses, there was a decrease in both peak perfusion (p = 0.0006) and area under the dose-response curve (p = 0.005) following acetylcholine, but no change in responses to sodium nitroprusside. Twenty-four hours after bypass responsiveness to acetylcholine improved, but typically remained depressed from baseline. Conserved endothelial function was associated with higher urine output during the first 48 postoperative hours (R = 0.43; p = 0.008).
CONCLUSIONS - Cutaneous endothelial dysfunction is present in infants immediately following cardiopulmonary bypass and recovers significantly in some patients within 24 hours postoperatively. Confirmation of an association between persistent endothelial-dependent vasomotor dysfunction and decreased urine output could have important clinical implications. Ongoing research will explore the pattern of endothelial-dependent vasomotor dysfunction after cardiopulmonary bypass and its relationship with biochemical markers of inflammation and clinical outcomes.
APOA1 is the most abundant protein in HDL. It modulates interactions that affect HDL's cardioprotective functions, in part via its activation of the enzyme, LCAT. On nascent discoidal HDL, APOA1 comprises 10 α-helical repeats arranged in an anti-parallel stacked-ring structure that encapsulates a lipid bilayer. Previous chemical cross-linking studies suggested that these APOA1 rings can adopt at least two different orientations, or registries, with respect to each other; however, the functional impact of these structural changes is unknown. Here, we placed cysteine residues at locations predicted to form disulfide bonds in each orientation and then measured APOA1's ability to adopt the two registries during HDL particle formation. We found that most APOA1 oriented with the fifth helix of one molecule across from fifth helix of the other (5/5 helical registry), but a fraction adopted a 5/2 registry. Engineered HDLs that were locked in 5/5 or 5/2 registries by disulfide bonds equally promoted cholesterol efflux from macrophages, indicating functional particles. However, unlike the 5/5 registry or the WT, the 5/2 registry impaired LCAT cholesteryl esterification activity ( < 0.001), despite LCAT binding equally to all particles. Chemical cross-linking studies suggest that full LCAT activity requires a hybrid epitope composed of helices 5-7 on one APOA1 molecule and helices 3-4 on the other. Thus, APOA1 may use a reciprocating thumbwheel-like mechanism to activate HDL-remodeling proteins.
Bexarotene is a pleiotropic molecule that has been proposed as an amyloid-β (Aβ)-lowering drug for the treatment of Alzheimer's disease (AD). It acts by upregulation of an apolipoprotein E (apoE)-mediated Aβ clearance mechanism. However, whether bexarotene induces removal of Aβ plaques in mouse models of AD has been controversial. Here, we show by NMR and CD spectroscopy that bexarotene directly interacts with and stabilizes the transmembrane domain α-helix of the amyloid precursor protein (APP) in a region where cholesterol binds. This effect is not mediated by changes in membrane lipid packing, as bexarotene does not share with cholesterol the property of inducing phospholipid condensation. Bexarotene inhibited the intramembrane cleavage by γ-secretase of the APP C-terminal fragment C99 to release Aβ in cell-free assays of the reconstituted enzyme in liposomes, but not in cells, and only at very high micromolar concentrations. Surprisingly, in vitro, bexarotene also inhibited the cleavage of Notch1, another major γ-secretase substrate, demonstrating that its inhibition of γ-secretase is not substrate specific and not mediated by acting via the cholesterol binding site of C99. Our data suggest that bexarotene is a pleiotropic molecule that interfere with Aβ metabolism through multiple mechanisms.
The pharmacology of the M muscarinic acetylcholine receptor (mAChR) is the least understood of the five mAChR subtypes due to a historic lack of selective small molecule tools. To address this shortcoming, we have continued the optimization effort around the prototypical M positive allosteric modulator (PAM) ML380 and have discovered and optimized a new series of M PAMs based on a chiral N-(indanyl)piperidine amide core with robust SAR, human and rat M PAM EC values <100 nM and rat brain/plasma K values of ∼0.40. Interestingly, unlike M and M PAMs with unprecedented mAChR subtype selectivity, this series of M PAMs displayed varying degrees of PAM activity at the other two natively G-coupled mAChRs, M and M, yet were inactive at M and M.
Highly selective positive allosteric modulators (PAMs) of the M subtype of muscarinic acetylcholine receptor have emerged as an exciting new approach for improving cognitive function in patients suffering from Alzheimer's disease and schizophrenia. However, excessive activation of M is known to induce seizure activity and have actions in the prefrontal cortex (PFC) that could impair cognitive function. We now report a series of pharmacological, electrophysiological, and behavioral studies in which we find that recently reported M PAMs, PF-06764427 and MK-7622, have robust agonist activity in cell lines and agonist effects in the mouse PFC, and have the potential to overactivate the M receptor and disrupt PFC function. In contrast, structurally distinct M PAMs (VU0453595 and VU0550164) are devoid of agonist activity in cell lines and maintain activity dependence of M activation in the PFC. Consistent with the previously reported effect of PF-06764427, the ago-PAM MK-7622 induces severe behavioral convulsions in mice. In contrast, VU0453595 does not induce behavioral convulsions at doses well above those required for maximal efficacy in enhancing cognitive function. Furthermore, in contrast to the robust efficacy of VU0453595, the ago-PAM MK-7622 failed to improve novel object recognition, a rodent assay of cognitive function. These findings suggest that in vivo cognition-enhancing efficacy of M PAMs can be observed with PAMs lacking intrinsic agonist activity and that intrinsic agonist activity of M PAMs may contribute to adverse effects and reduced efficacy in improving cognitive function.
Cholinergic regulation of dopaminergic inputs into the striatum is critical for normal basal ganglia (BG) function. This regulation of BG function is thought to be primarily mediated by acetylcholine released from cholinergic interneurons (ChIs) acting locally in the striatum. We now report a combination of pharmacological, electrophysiological, optogenetic, chemogenetic, and functional magnetic resonance imaging studies suggesting extra-striatal cholinergic projections from the pedunculopontine nucleus to the substantia nigra pars reticulata (SNr) act on muscarinic acetylcholine receptor subtype 4 (M) to oppose cAMP-dependent dopamine receptor subtype 1 (D) signaling in presynaptic terminals of direct pathway striatal spiny projections neurons. This induces a tonic inhibition of transmission at direct pathway synapses and D-mediated activation of motor activity. These studies provide important new insights into the unique role of M in regulating BG function and challenge the prevailing hypothesis of the centrality of striatal ChIs in opposing dopamine regulation of BG output.
Copyright © 2017 Elsevier Inc. All rights reserved.