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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.

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.

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. Significance Statement: The angiotensin II type 1 receptor (AT1R) inhibitor Valsartan rescued reduced mitochondrial function during insulin resistance in a myotube model of skeletal muscle. These findings provide proof‐of‐concept data suggesting Valsartan may provide metabolic benefits beyond blood pressure lowering effects. [ABSTRACT FROM AUTHOR]

The article describes two case scenarios in which liraglutide demonstrated glucose lowering similar to agents categorized as having very high efficacy. A 63-year-old Black man was referred to an outpatient clinic's in-house pharmacist-run diabetes education and management service while a 63-year-old White man was referred to the same clinical pharmacy service. The cases show the potential for robust A1C lowering with the use of liraglutide in patients with type 2 diabetes who overeat.

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. Significance statement: Epigenetics are important in the regulation of muscle metabolism. DNA methylation status of vital metabolic targets are often altered in the muscle of those with insulin resistance. We demonstrate nonspecific DNA hypomethylation via 5‐Aza reduces myotube metabolism as well as expression of branched‐chain amino acid catabolic enzymes in both insulin sensitive and insulin resistant myotubes. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Fifth generation (5G) cellular standards are set to utilize millimeter wave (mmWave) frequencies, which enable data speeds greater than 10 Gbps and sub-centimeter localization accuracy. These capabilities rely on accurate estimates of the channel parameters, which we define as the angle of arrival, angle of departure, and path distance for each path between the transmitter and receiver. Estimating the channel parameters in a computationally efficient manner poses a challenge because it requires estimation of parameters from a high-dimensional measurement - particularly for multi-carrier systems since each subcarrier must be estimated separately. Additionally, channel parameter estimation must be able to handle hybrid beamforming, which uses a combination of digital and analog beamforming to reduce the number of required analog to digital converters. This paper introduces a channel parameter estimation technique based on the multilinear singular value decomposition (MSVD), a Tucker form tensor analog of the singular value decomposition, for massive multiple input multiple output (MIMO) multi-carrier systems with hybrid beamforming. The MSVD tensor estimation approach is more computationally efficient than methods such as the canonical polyadic decomposition (CPD) and the Tucker form of the MSVD enables paths to be extracted based on signal energy. The algorithms performance is compared to the CPD method and shown to closely match the Cramer-Rao bound (CRB) of channel parameter estimates through simulations. Additionally, limitations of channel parameter estimation and communication waveform effects are studied.

Abstract Millimeter wave (mmWave) technology is expected to be a major component of 5G wireless networks. Ultra-wide bandwidths of mmWave signals and the possibility of utilizing large number of antennas at the transmitter and the receiver allow accurate identification of multipath components in temporal and angular domains, making mmWave systems advantageous for localization applications. In this paper, we analyze the performance of a two-step mmWave localization approach that can utilize time-of-arrival, angle-of-arrival, and angle-of-departure from multiple nodes in an urban environment with both line-of-sight (LOS) and non-LOS (NLOS) links. Networks with/without radio-environmental mapping (REM) are considered, where a network with REM is able to localize nearby scatterers. Estimation of a UE location is challenging due to large numbers of local optima in the likelihood function. To address this problem, a gradient-assisted particle filter (GAPF) estimator is proposed to accurately estimate a user equipment (UE) location as well as the locations of nearby scatterers. Monte-Carlo simulations show that the GAPF estimator performance matches the Cramer-Rao bound (CRB). The estimator is also used to create a REM. It is seen that significant localization gains can be achieved by increasing beam directionality or by utilizing REM.

We have investigated the permeability of the human red blood cell to four di-hydroxy alcohols, 1,2PD (1,2 propanediol), 1,3PD (1.3 propanediol), 1,4BD (1,4 butanediol), and 2,3BD (2,3 butanediol), and to water by using a recently developed ESR stopped-flow method which is free from artifacts found in light scattering methods. Numerical solutions of the Kedem-Katchalsky equations fit to experimental data yielded the following permeability coefficients: P1,2PD = 3.17 x 10(-5) cm sec-1, P1,3PD = 1.75 x 10(-5) cm sec-1, P1,4BD = 2.05 x 10(-5) cm sec-1, P2,3BD = 7.32 x 10(-5) cm sec-1. Reflection coefficients (sigma) were evaluated by comparing data fit with assumed values of sigma = 0.6, 0.8 and 1.0. In all four cases the best fit was obtained with sigma = 1.0. Treatment of cells with PCMBS (para-chloro mercuri-benzene-sulfonate) was followed by a large (> 10-fold) decrease in water permeability with virtually no change in alcohol permeability. We conclude that these alcohols do not permeate the water channels to any significant extent, and discuss some of the problems in light scattering measurements of reflection coefficients that could lead to erroneous values for sigma. [ABSTRACT FROM AUTHOR]

In this paper a variety of mercurials, including a pCMB-nitroxide analogue, were used to study urea transport in human red cell ghosts. It was determined that the rate of inhibition for pCMBS, pCMB, pCMB-nitroxide, and chlormerodrin extended over four orders of magnitude consistent with their measured oil/water partition coefficients. From these results, we concluded that a significant hydrophobic barrier limits access to the urea inhibition site, suggesting that the urea site is buried in the bilayer or in a hydrophobic region of the transporter. In contrast, the rate of water inhibition by the mercurials ranged by only a factor of four and did not correlate with their hydrophobicities. Thus, the water inhibition site may be more directly accessible via the aqueous phase. Under conditions that leave water transport unaffected, we determined that < or = 32,000 labeled sites per cell corresponded to complete inhibition of urea transport. This rules out major transmembrane proteins such as band 3, the glucose carrier, and CHIP28 as candidates for the urea transporter. In contrast, this result is consistent with the Kidd (Jk) antigen being the urea transporter with an estimated 14,000 copies per cell. From the experimental number of urea sites, a turnover number between 2-6 x 10(6) sec-1 at 22 degrees C is calculated suggesting a channel mechanism. [ABSTRACT FROM AUTHOR]

Red cell water volumes were measured using ESR methods during transient osmotic perturbation, and under equilibrium conditions. Cell water contents were determined using the spin label Tempone (2,2,6,6-tetramethyl piperidine-N-oxyl) and the membrane impermeable quencher potassium chromium oxalate. With appropriate corrections for intracellular viscosity and changes in cavity sensitivity, equilibrium cell water measured both by electron spin resonance (ESR) and wet minus dry weight methods gave excellent agreement in solutions from 243-907 mOsm. Intracellular viscosities determined from the Tempone correlation times in the same cells gave values ranging from 9-47 centipoise at 21 degrees C. Osmotically induced transient volume changes were measured using Tempone and an ESR stopped-flow configuration. The Tempone response time was estimated at 17 msec compared to 250-350 msec for normal water relaxations. Nonlinear least square solutions to the Kedem-Katchalsky equations including a correction for the finite Tempone permeability gave 0.029 and 0.030 cm/sec for the osmotic permeability of RBCs in swell and shrink experiments, respectively. In stopped-flow experiments accurate water flux data are obtained very soon after challenging cells and do not require baseline subtractions. These results represent significant improvements over conventional light scattering techniques which necessitate corrections for long lasting optical artifacts (200-300 msec), and baseline drifts. [ABSTRACT FROM AUTHOR]

Permeabilities for an homologous series of amine nitroxide spin probes were measured in liposomes of varying composition by an electron paramagnetic resonance (EPR) method. Results show that the rate-limiting step in permeation is not adsorption/desorption at the aqueous/membrane interface for two probes in phosphatidylcholine/phosphatidic acid liposomes and for one probe in phosphatidylcholine/cholesterol/phosphatidic acid liposomes. Accordingly, we interpret observed selectivity patterns for the entire series of probes in liposomes and red cells in terms of the properties of the bilayer interior. Results are inconsistent with simple applications of either free volume or hydrocarbon sheet models of nonelectrolyte permeation. In the former case, it was found that liposomes do not select against these probes on the basis of molecular volume. In the latter case, probe permeabilities are all much lower than would be predicted for a sheet of bulk hydrocarbon and the polarity of the rate-limiting region is shown to be greater than bulk hydrocarbon. Together with the results of previous studies of spin-labeled solutes in membranes, as well as studies of lipid dynamics in membranes, these latter results suggest that the rate-limiting region in nonelectrolyte permeation is not in the center of the bilayer, but in the relatively ordered acyl chain segments near the glycerol backbone. [ABSTRACT FROM AUTHOR]

Permeabilities for a homologous series of amine and carboxylate nitroxide spin probes were measured in human red blood cells by an electron paramagnetic resonance (EPR) method. Permeabilities determined in this study are much lower than would be predicted for a sheet of bulk hydrocarbon and the polarity of the rate-limiting region is shown to be greater than bulk hydrocarbon. This suggests that the rate-limiting region for permeation of these nonelectrolytes is somewhere in the membrane periphery rather than in the center of the membrane. The red cell membrane does not discriminate between these probes on the basis of molecular volume, as might be predicted by a simple free-volume theory of membrane permeation. [ABSTRACT FROM AUTHOR]

Aliphatic alcohols are found to stimulate the transmembrane fluxes of a hydrophobic cation (tetraphenylarsonium, TPA) and anion (AN-12) 5-20 times in red blood cells. The results are analyzed using the Born-Parsegian equation (Parsegian, A., 1969, Nature (London) 221:844-846), together with the Clausius-Mossotti equation to calculate membrane dielectric energy barriers. Using established literature values of membrane thickness, native membrane dielectric constant, TPA ionic radius, and alcohol properties (partition coefficient, molar volume, dielectric constant), the TPA permeability data is predicted remarkably well by theory. If the radius of AN-12 is taken as 1.9 A, its permeability in the presence of butanol is also described by our analysis. Further, the theory quantitatively accounts for the data of Gutknecht and Tosteson (Gutknecht, J., Tosteson, D.C., 1970, J. Gen. Physiol. 55:359-374) covering alcohol-induced conductivity changes of 3 orders of magnitude in artificial bilayers. Other explanations including perturbations of membrane fluidity, surface charge, membrane thickness, and dipole potential are discussed. However, the large magnitude of the stimulation, the more pronounced effect on smaller ions, and the acceleration of both anions and cations suggest membrane dielectric constant change as the primary basis of alcohol effects. [ABSTRACT FROM AUTHOR]

A gas-free high-pressure cell has been developed to measure planar bilayer conductances induced by hydrophobic ions and ionophores as a function of hydrostatic pressure. Plots of log conductance versus pressure for valinomycin and nonactin-mediated potassium transport in egg phosphatidyl cholinedecane membranes are essentially linear over a pressure range of 1 to 818 atm. Calculated activation volumes give similar results for both nonactin and valinomycin yielding values of + 48 and + 42 cc/mole, respectively. The valinomycin activation volume agrees reasonably well with the results obtained by Johnson and Miller (Biochim. Biophys. Acta 375:286-291, 1975) for K+-valinomycin transport in liposomes. In contrast to the activation volumes for nonactin and valinomycin, relaxation measurements of tetraphenyl boron (TPB) and dipicrylamine (DPA) give very small values of less than 5 cc/mole for the translocation rate constant, ki. Similarly, steady-state conductance measurements on tetraphenyl arsonium (TPA) and carbonylcyanide m-chlorophenylhydrazone (CCCP), give small values of 6 and 7 cc/mole, respectively. These low figures do not support transport theories based on the formation of bilayer holes or kinks (H. Träuble, J. Membrane Biol. 4:193-208, 1971). The low values for TPB and TPA are especially interesting because their cross-sectional areas are not much different than those of valinomycin and nonactin. Pressure-induced changes in membrane dielectric constant and thickness which lower the bilayer electrostatic barrier could explain the low values for the hydrophobic ions. Additionally, larger activation volumes might be expected for carriers such as nonactin and valinomycin that undergo significant rearrangement and change in hydration during surface complexation of cations. [ABSTRACT FROM AUTHOR]

The permeability and partition coefficients of tetraphenylarsonium (TPA) and several other organic cations were studied in the human erythrocyte using an ion-selective electrode. The permeability constant for the different cations could be explained quite well by differences in oil/water partition coefficients. No evidence for facilitated transport could be found. Binding of the organic ions occurred to both the cell membrane and to intracellular contents. Partitioning to the membrane remained relatively constant despite variation from ion intracellular binding with blood samples from different donors. TPA flux is stimulated by substoichiometric amounts of tetraphenylboron and other organic anions, suggesting an ion-pairing mechanism. [ABSTRACT FROM AUTHOR]

The relation of osmotic to diffusional water permeability of human red blood cells was compared after treating the cells with different concentrations of PCMBS (p-chloromercuribenzene sulfonate). After subtracting the PCMBS-insensitive permeability (presumably the water permeability of the lipid bilayer) from each, the ratio of osmotic to diffusional permeability remains invariant (approximately equal to 11) as more and more water channels are inhibited by increasing concentrations of PCMBS. This result implies that the channels close in an all-or-none way and suggests a two-state model. Analysis of the dependence of osmotic water permeability on PCMBS concentration in terms of the model reveals a 1:1 stoichiometry and a dissociation constant for the PCMBS/membrane receptor complex of about 0.019 mM at 37 degrees C. Temperature dependence studies show that the reaction is entropically driven (delta H degrees approximately equal to 25 kcal/mol, delta S degrees approximately equal to 100 cal/mol-deg) and suggest the involvement of hydrophobic interactions. [ABSTRACT FROM AUTHOR]