The BCKDH kinase inhibitor BT2 promotes BCAA disposal and mitochondrial proton leak in both insulin-sensitive and insulin-resistant C2C12 myotubes.

Elevated circulating branched-chain amino acids (BCAAs) have been correlated with the severity of insulin resistance, leading to recent investigations that stimulate BCAA metabolism for the potential benefit of metabolic diseases. BT2 (3,6-dichlorobenzo[b]thiophene-2-carboxylic acid), an inhibitor of branched-chain ketoacid dehydrogenase kinase, promotes BCAA metabolism by enhancing BCKDH complex activity. The purpose of this report was to investigate the effects of BT2 on mitochondrial and glycolytic metabolism, insulin sensitivity, and de novo lipogenesis both with and without insulin resistance. C2C12 myotubes were treated with or without low or moderate levels of BT2 with or without insulin resistance. Western blot and quantitative real-time polymerase chain reaction were used to assess protein and gene expression, respectively. Mitochondrial, nuclei, and lipid content were measured using fluorescent staining and microscopy. Cell metabolism was assessed via oxygen consumption and extracellular acidification rate. Liquid chromatography-mass spectrometry was used to quantify BCAA media content. BT2 treatment consistently promoted mitochondrial uncoupling following 24-h treatment, which occurred largely independent of changes in expressional profiles associated with mitochondrial biogenesis, mitochondrial dynamics, BCAA catabolism, insulin sensitivity, or lipogenesis. Acute metabolic studies revealed a significant and dose-dependent effect of BT2 on mitochondrial proton leak, suggesting BT2 functions as a small-molecule uncoupler. Additionally, BT2 treatment consistently and dose-dependently reduced extracellular BCAA levels without altering expression of BCAA catabolic enzymes or pBCKDHa activation. BT2 appears to act as a small-molecule mitochondrial uncoupler that promotes BCAA utilization, though the interplay between these two observations requires further investigation.

Valsartan Rescues Suppressed Mitochondrial Metabolism during Insulin Resistance in C2C12 Myotubes.

Elevated circulating branched-chain amino acids (BCAA) have been linked with the severity of insulin resistance across numerous populations, implicating heightened BCAA metabolism as a potential therapy for insulin resistance. Recently, the angiotensin II type 1 receptor (AT1R) inhibitor Valsartan (VAL) was identified as a potent inhibitor of branched-chain alpha-keto acid dehydrogenase kinase (BCKDK), a negative regulator of BCAA metabolism. This work investigated the effect of VAL on myotube metabolism and insulin sensitivity under both insulin sensitive and insulin resistant conditions. C2C12 myotubes were treated with or without VAL at 8 µM for 24 h, both with and without hyperinsulinemic-induced insulin resistance. Oxygen consumption and extracellular acidification were used to measure mitochondrial and glycolytic metabolism, respectively. Gene expression was assessed via qRT-PCR, and insulin sensitivity was assessed via Western blot. Insulin resistance significantly reduced both basal and peak mitochondrial function which were rescued to control levels by concurrent VAL. Changes in mitochondrial function occurred without substantial changes in mitochondrial content or related gene expression. Insulin sensitivity and glycolytic metabolism were unaffected by VAL, as was lipogenic signaling and lipid content. Additionally, both VAL and insulin resistance depressed Bckdha expression. Interestingly, an interaction effect was observed for extracellular isoleucine, valine, and total BCAA (but not leucine), suggesting VAL may alter BCAA utilization in an insulin sensitivity-dependent manner. Insulin resistance appears to suppress mitochondrial function in a myotube model which can be rescued by VAL. Further research will be required to explore the implications of these findings in more complex models.

Effect of mTORC Agonism via MHY1485 with and without Rapamycin on C2C12 Myotube Metabolism.

The mechanistic target of rapamycin complex (mTORC) regulates protein synthesis and can be activated by branched-chain amino acids (BCAAs). mTORC has also been implicated in the regulation of mitochondrial metabolism and BCAA catabolism. Some speculate that mTORC overactivation by BCAAs may contribute to insulin resistance. The present experiments assessed the effect of mTORC activation on myotube metabolism and insulin sensitivity using the mTORC agonist MHY1485, which does not share structural similarities with BCAAs. METHODS: C2C12 myotubes were treated with MHY1485 or DMSO control both with and without rapamycin. Gene expression was assessed using qRT-PCR and insulin sensitivity and protein expression by western blot. Glycolytic and mitochondrial metabolism were measured by extracellular acidification rate and oxygen consumption. Mitochondrial and lipid content were analyzed by fluorescent staining. Liquid chromatography-mass spectrometry was used to assess extracellular BCAAs. RESULTS: Rapamycin reduced p-mTORC expression, mitochondrial content, and mitochondrial function. Surprisingly, MHY1485 did not alter p-mTORC expression or cell metabolism. Neither treatment altered indicators of BCAA metabolism or extracellular BCAA content. CONCLUSION: Collectively, inhibition of mTORC via rapamycin reduces myotube metabolism and mitochondrial content but not BCAA metabolism. The lack of p-mTORC activation by MHY1485 is a limitation of these experiments and warrants additional investigation.

5-Aza-2'-deoxycytidine-mediated DNA hypomethylation with and without concurrent insulin resistance suppresses myotube mitochondrial capacity.

Type 2 diabetes is characterized by elevated blood glucose and reduced insulin sensitivity in target tissues. Moreover, reduced mitochondrial metabolism and expressional profile of genes governing mitochondrial metabolism (such as peroxisome proliferator-activated receptor gamma coactivator 1-alpha [PGC-1α]) are also reduced during insulin resistance. Epigenetic regulation via DNA methylation of genes including PGC-1α may contribute to diminished mitochondrial capacity, while hypomethylation of PGC-1α (such as that invoked by exercise) has been associated with increased PGC-1α expression and favorable metabolic outcomes. The purpose of the present report is to characterize the effects of DNA hypomethylation on myotube metabolism and expression of several related metabolic targets. C2C12 myotubes were treated with 5-Aza-2'-deoxycytidine (5-Aza) for either 24 or 72 h both with and without hyperinsulinemic-induced insulin resistance. Mitochondrial and glycolytic metabolism were measured via oxygen consumption and extracellular acidification rate, respectively. Metabolic gene and protein expression were assessed via quantitative real time polymerase chain reaction and western blot analysis, respectively. Though expression of PGC-1α and other related targets remained unaltered, insulin resistance and 5-Aza treatment significantly reduced mitochondrial metabolism. Similarly, peak glycolytic metabolism was diminished by 5-Aza-treated cells, while basal glycolytic metabolism was unaltered. 5-Aza also reduced the expression of branched-chain amino acid (BCAA) catabolic components, however BCAA utilization was enhanced during insulin resistance with 5-Aza treatment. Together the present work provides proof-of-concept evidence of the potential role of DNA methylation in the regulation of mitochondrial metabolism and the potential interactions with insulin resistance in a model of skeletal muscle.

A Systematic Review of the Balloon Analogue Risk Task (BART) in Alcohol Research.

Risk-taking propensity has been crucial to the investigation of alcohol use and consequences. One measure, the balloon analogue risk task (BART), has been used consistently over the past two decades. However, it is unclear how this measure is related to alcohol outcomes. This paper systematically reviews the literature on the BART and alcohol outcomes. First, direct associations between the BART and alcohol use are reviewed including correlations, group comparisons, the BART's prediction of alcohol outcomes and BART performance after consuming alcohol. Then, potential moderators that explain when and for whom the BART is related to alcohol outcomes are reviewed. Finally, potential mechanisms that explain how the BART and alcohol outcomes are related are reviewed. This review reveals patterns in the BART suggesting risk-taking propensity may be related to changes in alcohol use over time; however, there is little evidence to suggest BART scores increase after consuming alcohol. Yet, additional research suggests adjusted average pump scores may be too simplistic for the amount of information the BART captures and understanding individual's patterns of responses on the BART is important for investigating its relation to alcohol outcomes. Finally, this review opens up several future directions for research to understand how risk-taking propensity is related to alcohol outcomes.

Fructose Reduces Mitochondrial Metabolism and Increases Extracellular BCAA during Insulin Resistance in C2C12 Myotubes.

Fructose is a commonly consumed monosaccharide implicated in developing several metabolic diseases. Previously, elevated branched-chain amino acids (BCAA) have been correlated with the severity of insulin resistance. Most recently, the effect of fructose consumption on the downregulation of BCAA catabolic enzymes was observed. Thus, this mechanistic study investigated the effects of physiologically attainable levels of fructose, both with and without concurrent insulin resistance, in a myotube model of skeletal muscle. METHODS: C2C12 mouse myoblasts were treated with fructose at a concentration of 100 µM (which approximates physiologically attainable concentrations in peripheral circulation) both with and without hyperinsulinemic-mediated insulin resistance. Gene expression was assessed by qRT-PCR, and protein expression was assessed by Western blot. Oxygen consumption rate and extracellular acidification rate were used to assess mitochondrial oxidative and glycolytic metabolism, respectively. Liquid chromatography-mass spectrometry was utilized to analyze leucine, isoleucine and valine concentration values. RESULTS: Fructose significantly reduced peak glycolytic and peak mitochondrial metabolism without altering related gene or protein expression. Similarly, no effect of fructose on BCAA catabolic enzymes was observed; however, fructose treatment resulted in elevated total extracellular BCAA in insulin-resistant cells. DISCUSSION: Collectively, these observations demonstrate that fructose at physiologically attainable levels does not appear to alter insulin sensitivity or BCAA catabolic potential in cultured myotubes. However, fructose may depress peak cell metabolism and BCAA utilization during insulin resistance.

Workforce Planning Models for Oral Health Care: A Scoping Review.

BACKGROUND: For health care services to address the health care needs of populations and respond to changes in needs over time, workforces must be planned. This requires quantitative models to estimate future workforce requirements that take account of population size, oral health needs, evidence-based approaches to addressing needs, and methods of service provision that maximize productivity. The aim of this scoping review was to assess whether and how these 4 elements contribute to existing models of oral health workforce planning. METHODS: A scoping review was conducted. MEDLINE, Embase, HMIC, and EconLit were searched, all via OVID. Additionally, gray literature databases were searched and key bodies and policy makers contacted. Workforce planning models were included if they projected workforce numbers and were specific to oral health. No limits were placed on country. A single reviewer completed initial screening of abstracts; 2 independent reviewers completed secondary screening and data extraction. A narrative synthesis was conducted. RESULTS: A total of 4,009 records were screened, resulting in 42 included articles detailing 47 models. The workforce planning models varied significantly in their use of data on oral health needs, evidence-based services, and provider productivity, with most models relying on observed levels of service utilization and demand. CONCLUSIONS: This review has identified quantitative workforce planning models that aim to estimate future workforce requirements. Approaches to planning the oral health workforce are not always based on deriving workforce requirements from population oral health needs. In many cases, requirements are not linked to population needs, while in models where needs are included, they are constrained by the existence and availability of the required data. It is critical that information systems be developed to effectively capture data necessary to plan future oral health care workforces in ways that relate directly to the needs of the populations being served. KNOWLEDGE TRANSFER STATEMENT: Policy makers can use the results of this study when making decisions about the planning of oral health care workforces and about the data to routinely collect within health services. Collection of suitable data will allow for the continual improvement of workforce planning, leading to a responsive health service and likely future cost savings.

Social Network Changes and Disclosure Responses after Sexual Assault.

Social support after sexual assault is important for recovery, but violence and recovery may also challenge relationships. We examined functional and structural social support changes following sexual assault and their association with mental health. College women (N=544) with and without a sexual assault history completed a cross-sectional survey assessing current and past egocentric social networks. Functional support (perceived global support, assault disclosure, perceived helpfulness of responses) and structural support (network density, size, retention) were examined. Multilevel models revealed that, relative to non-survivors, survivors reported smaller, less dense past networks, but similarly sized current networks. Survivors retained less of their networks than non-survivors, and network members who provided unhelpful responses to disclosure were less likely to be retained. Structural equation modeling revealed that, among survivors, perceived unhelpful responses to disclosure and a greater loss of network members were associated with worse mental health. Findings suggest that survivors may experience a restructuring of social networks following sexual assault, especially when network members respond in unhelpful ways to disclosure. Although survivors appeared to build new relationships, this restructuring was associated with more mental health problems. It is possible that interventions to improve post-assault social network retention may facilitate recovery.

Enamel Caries Detection and Diagnosis: An Analysis of Systematic Reviews.

Detection and diagnosis of caries-typically undertaken through a visual-tactile examination, often with supporting radiographic investigations-is commonly regarded as being broadly effective at detecting caries that has progressed into dentine and reached a threshold where restoration is necessary. With earlier detection comes an opportunity to stabilize disease or even remineralize the tooth surface, maximizing retention of tooth tissue and preventing a lifelong cycle of restoration. We undertook a formal comparative analysis of the diagnostic accuracy of different technologies to detect and inform the diagnosis of early caries using published Cochrane systematic reviews. Forming the basis of our comparative analysis were 5 Cochrane diagnostic test accuracy systematic reviews evaluating fluorescence, visual or visual-tactile classification systems, imaging, transillumination and optical coherence tomography, and electrical conductance or impedance technologies. Acceptable reference standards included histology, operative exploration, or enhanced visual assessment (with or without tooth separation) as appropriate. We conducted 2 analyses based on study design: a fully within-study, within-person analysis and a network meta-analysis based on direct and indirect comparisons. Nineteen studies provided data for the fully within-person analysis and 64 studies for the network meta-analysis. Of the 5 technologies evaluated, the greatest pairwise differences were observed in summary sensitivity points for imaging and all other technologies, but summary specificity points were broadly similar. For both analyses, the wide 95% prediction intervals indicated the uncertainty of future diagnostic accuracy across all technologies. The certainty of evidence was low, downgraded for study limitations, inconsistency, and indirectness. Summary estimates of diagnostic accuracy for most technologies indicate that the degree of certitude with which a decision is made regarding the presence or absence of disease may be enhanced with the use of such devices. However, given the broad prediction intervals, it is challenging to predict their accuracy in any future "real world" context.

Engineering plant membranes using droplet interface bilayers.

Droplet interface bilayers (DIBs) have become widely recognised as a robust platform for constructing model membranes and are emerging as a key technology for the bottom-up assembly of synthetic cell-like and tissue-like structures. DIBs are formed when lipid-monolayer coated water droplets are brought together inside a well of oil, which is excluded from the interface as the DIB forms. The unique features of the system, compared to traditional approaches (e.g., supported lipid bilayers, black lipid membranes, and liposomes), is the ability to engineer multi-layered bilayer networks by connecting multiple droplets together in 3D, and the capability to impart bilayer asymmetry freely within these droplet architectures by supplying droplets with different lipids. Yet despite these achievements, one potential limitation of the technology is that DIBs formed from biologically relevant components have not been well studied. This could limit the reach of the platform to biological systems where bilayer composition and asymmetry are understood to play a key role. Herein, we address this issue by reporting the assembly of asymmetric DIBs designed to replicate the plasma membrane compositions of three different plant species; Arabidopsis thaliana, tobacco, and oats, by engineering vesicles with different amounts of plant phospholipids, sterols and cerebrosides for the first time. We show that vesicles made from our plant lipid formulations are stable and can be used to assemble asymmetric plant DIBs. We verify this using a bilayer permeation assay, from which we extract values for absolute effective bilayer permeation and bilayer stability. Our results confirm that stable DIBs can be assembled from our plant membrane mimics and could lead to new approaches for assembling model systems to study membrane translocation and to screen new agrochemicals in plants.

Feasibility study: assessing the efficacy and social acceptability of using dental hygienist-therapists as front-line clinicians.

Background The oral health of the adult population has been improving in the United Kingdom decade upon decade. Over half of dental service activity in the National Health Service (NHS) is limited to a check-up without any further treatment. This raises a question as to whether check-ups could be provided by dental hygienist-therapists, rather than general dental practitioners. The aim of this study was to assess the feasibility of a definitive trial to evaluate the costs and effects of using dental hygienist-therapists to undertake the check-up.Methods/design Adult NHS patients were randomised into three arms in two dental practices: patients who only saw dental hygienist-therapists for a check-up, those that saw the general dental practitioner and dental hygienist-therapist alternatively and a control, where patients only saw the general dental practitioner for their check-up. The study ran for 15 months. The primary outcome measures of the study were patient recruitment, retention and fidelity. A parallel and embedded qualitative study was undertaken which recorded the views of participating patients to determine the social acceptability of the intervention.Results Sixty patients participated in the study. The initial recruitment rate for the study was 33.7%. This figure increased to over 82.1% when telephone calls or face-to-face recruitment was utilised. The retention rates were 60% for both the dental hygienist-therapist only group and the alternate general dental practitioner and dental hygienist-therapist group, compared to 70% for the general dental practitioner only group. Fifteen patients were interviewed in the qualitative study and supported a team approach to the provision of check-ups in the NHS. Conclusion This study demonstrates the feasibility of a definitive trial to evaluate the costs and effects of using dental-hygienist-therapists to undertake the check-up.

A method to distinguish between pore and carrier kinetics applied to urea transport across the erythrocyte membrane.

Permeability coefficients (P) measured at various penetrant concentrations (C) by the perturbation method can be plotted to distinguish simple diffusion, simple pore kinetics and simple carrier kinetics as follows: for simple diffusion, 1/P = constant; for a simple pore, 1/P = 1/Po + 1/Po[1/Kin + 1/Ko]C; for a simple carrier, 1/P = 1/Po + 1/Po[1/Kin + 1/Ko]C + 1/Po[1/(K3K4)] C2 where Po is the maximal permeability at zero penetrant concentration and the K's are combinations of kinetic constants defining each of the transport steps. (Kin and Ko are the half-saturation constant for zero-trans efflux and influx, respectively; K3 is the half-saturation constant for equilibrium exchange, and K4 is related to the mobility of the free carrier). In human erythrocytes, permeability coefficients for diethylene glycol were constant suggesting simple diffusion. For glucose, a plot of 1/P versus concentration was nonlinear indicating carrier kinetics. Plots of 1/P versus penetrant concentrations gave straight lines with positive slopes for urea in human and bovine erythrocytes and for methylurea in human red cells, indicating these penetrants follow simple pore kinetics or simple carrier kinetics in which K4 is very large.

Temperature- and concentration-dependence of urea permeability of human erythrocytes determined by NMR.

The temperature- and concentration-dependence of [13C]urea self-exchange across the human red cell membrane has been determined by NMR measurements of T1 (spin-lattice) relaxation times. T1 for intracellular label is 17 s, which is much longer than the urea exchange time across the cell membrane (about 0.5 s). T1 for urea in extracellular solution is quenched with 17 mM of impermeable Mn2+ in less than 2 ms. Hence the observed T1 (corrected for intracellular decay) is a measure of urea exchange across the cell membrane. The method is tested by showing both PCMBS and increasing concentrations of urea lengthen T1. Urea exchange permeability, defined as Purea = flux/conc, can be described by Purea = Vmax/(K1/2 + conc). Studies of temperature-dependence showed that activation energies were strongly dependent on both temperature and concentration. However, this apparently anomalous behavior was resolved into two well-behaved functions, K1/2 and Vmax, with linear Arrhenius plots and apparent 'activation energies' of 15.5 and 12.4 kcal/mol, respectively. These were used to construct an equation for calculating Purea at any concentration and temperature. Assuming a simple channel model with single binding, K1/2 becomes the dissociation equilibrium constant for the site with delta H degree = 15.5 kcal/mol and delta S degree = 51.8 cal/(mol.deg); dissociation is entropically driven.

Shape and stability changes in human erythrocyte membranes induced by metal cations.

Asymmetric human erythrocyte ghost membranes behave as bilayer couple and exhibit a radius of curvature preference depending on the state of expansion or contraction of each side of the bilayer. The inside-out preference in the absence of added metal cations is gradually reduced as the K+ concentration is raised to 200 mM until a slight right-side-out preference may be exhibited Divalent cations (denoted 72+; Ca2+, Mg2+ and Mn2+) induce inside-out curvature at very low concentrations, right-side-out curvature at intermediate concentrations, and inside-out curvature again at high ones. This "triphasic" response is attributed to changes in the packing of acidic phospholipid (PL-) pairs in the A-face as a function of M2+ : PL- binding stoichiometry: 0 : 1 (PL- electrostatic repulsion and A-face expansion), 1 : 2 (PL2M crossbridging an contraction), and 1 : 1 (PLM+ repulsion and expansion). Generally increasing parent vesicle size is associated with higher cation concentrations. This is distinguished from the internal and external membrane blebbing preferred at different concentrations in accord with sidedness preference. Parent vesicle size was interpreted to be most closely associated with cation stabilisation (resistance to fragmentation) of the membrane, while sidedness and size (radius of curvature) of blebs were most closely correlated with packing of lipid molecules in the bilayer.

Anion exchange in human erythrocytes has a large activation volume.

Sulfate equilibrium exchange in human red cells has an activation volume of +150 +/- 20 cm3/mol over the pressure range 0.1 to 83 MPa (15 to 12000 lb/in2) at 30 degrees C. This value greatly exceeds the expected contribution from sulfate binding to the anion exchanger. We suggest that the activation volume reflects conformational changes during the transport cycle.

Diffusional water permeability of red cells. Independence on osmolality.

The osmotic permeability coefficient (Lp) for human red cells has been reported to depend on the osmolality of the suspending solution. These results are also consistent with the view that the value of Lp depends on flow rectification. In this report an NMR method is used to measure the dependence of water exchange times at constant cell volume on osmolality. Our results indicate that the diffusion water permeability is constant over a large range of osmolality (300-1000 mosM) produced by the permeable solutes urea, methanol, ethanol, and glycerol. The results support the view that the apparent dependence of Lp on osmolality is due to flow rectification.

Erythrocyte membrane potentials determined by hydrogen ion distribution.

If the extracellular fluid is left unbuffered, dynamic membrane potential changes in the red blood cell may be determined from external pH readings. For some types of experiments it is necessary to accelerate H+ equilibration by adding minute amounts of hydrogen carriers. The method is independent of hematocrit over a wide range of membrane potential changes. Membrane potential jumps produced by permeability changes or by changes in ionic composition may be measured. The method provides a convenient means of measuring parameters of both the conductive and non-conductive anion pathways in the red cell.

Determination of the electric potential at the external and internal bilayer-aqueous interfaces of the human erythrocyte membrane using spin probes.

Partitioning of oppositely charged amphipathic spin probes indicates that the electric potential at the external bilayer-aqueous interface of the human erythrocyte is insignificant, and that protruding sialic acids do not contribute to this potential. This potential at the surface is distinguished from the electrokinetic potential due to all charged groups within the hydrodynamic surface of shear. By contrast, using inside-out erythrocyte membrane vesicles, a substantial potential is observed at the cytoplasmic membrane surface. This can be attributed to the asymmetric distribution of acidic phospholipids on the two sides of the erythrocyte membrane bilayer.

Hemoglobin polymerization in sickle cells studied by circular polarized light scattering.

We have studied intracellular polymerization of hemoglobin S in suspensions of small populations of sickle cells using circular polarized light scattering. We argue that the preferential scattering of right circular polarized light (as expressed by measurements of the S14 Mueller scattering matrix element) directly reflects the amount of polymer inside cells. This technique has made it possible to investigate the effect of oxygen tension, cell density and osmotic stress on intracellular hemoglobin polymerization. Using S14 to determine hemoglobin polymer, we show that the polymer increases with deoxyhemoglobin concentration, that cells containing higher hemoglobin concentrations show significantly more polymer than cells containing less hemoglobin, and that polymerization occurs in sickle-trait cells in hypertonic solutions as the oxygen tension in the suspension is reduced. We also present kinetic measurements of polymerization, including that induced by osmotic shock. Finally, we demonstrate that the total light scattered (S11 Mueller scattering matrix element) that is routinely measured simultaneously with S14 can be used to estimate the percent of reduced (deoxy) Hb in the sample. These experiments demonstrate the potential of this technique to monitor hemoglobin polymerization simultaneously with oxygen dissociation under a wide variety of physiological conditions.