Reduced TCA Flux in Diabetic Myotubes: Determined by Single Defects?

The diabetic phenotype is complex, requiring elucidation of key initiating defects. Diabetic myotubes express a primary reduced tricarboxylic acid (TCA) cycle flux but at present it is unclear in which part of the TCA cycle the defect is localised. In order to localise the defect we studied ATP production in isolated mitochondria from substrates entering the TCA cycle at various points. ATP production was measured by luminescence with or without concomitant ATP utilisation by hexokinase in mitochondria isolated from myotubes established from eight lean and eight type 2 diabetic subjects. The ATP production of investigated substrate combinations was significantly reduced in mitochondria isolated from type 2 diabetic subjects compared to lean. However, when ATP synthesis rates at different substrate combinations were normalized to the corresponding individual pyruvate-malate rate, there was no significant difference between groups. These results show that the primary reduced TCA cycle flux in diabetic myotubes is not explained by defects in specific part of the TCA cycle but rather results from a general downregulation of the TCA cycle

The velocity field created by a shallow bump in a boundary layer

We report the results of measurements of the disturbance velocity field generated in a boundary layer by a shallow three-dimensional bump oscillating at a very low frequency on the surface of a flat plate. Profiles of the mean velocity, the disturbance velocity at the fundamental frequency and at the first harmonic are presented. These profiles were measured both upstream and downstream of the oscillating bump. Measurements of the disturbance velocity were also made at various spanwise and downstream locations at a fixed distance from the boundary of one displacement thickness. Finally, the spanwise spectrum of the disturbances at three locations downstream of the bump are presented

Acoustic excitation of Tollmien–Schlichting waves due to localised surface roughness

Experiments on the receptivity of two-dimensional boundary layers to acoustic disturbances from two-dimensional roughness strips were performed in a low-turbulence wind tunnel on a flat plate model. The free stream was subjected to a plane acoustic wave so that a Stokes layer (SL) was created on the plate, thus generating a Tollmien-Schlichting (T-S) wave through the receptivity process. An improved technique to measure the T-S component is described based on a retracting two-dimensional roughness, which allowed for phase-locked measurements at the acoustic wave frequency to be made. This improved technique enables both protuberances and cavities to be explored in the range < |h| (equivalent to 0.025 < |h|/ γ B ∗) in relative roughness height to the local unperturbed Blasius boundary layer displacement thickness). These depths are designed to cover both the predicted linear and nonlinear response of the T-S excitation. Experimentally, cavities had not previously been explored. Results show that a linear regime is identifiable for both positive and negative roughness heights up to ≈ 150 μm (|h| γ B ∗ ≈ 0.126). The departure from the linear behaviour is, however, dependent on the geometry of the surface imperfection. For cavities of significant depth, the nonlinear behaviour is found to be milder than in the case of protuberances - this is attributed to the flow physics in the near field of the surface features. Nonetheless, results for positive heights agree well with previous theoretical work which predicted a linear disturbance response for small-height perturbations

Insulin Resistance Is Not Conserved in Myotubes Established from Women with PCOS

Polycystic ovary syndrome (PCOS) is the most common endocrine disorder among premenopausal women, who often develop insulin resistance. We tested the hypothesis that insulin resistance in skeletal muscle of patients with polycystic ovary syndrome (PCOS) is an intrinsic defect, by investigating the metabolic characteristics and gene expression of in vitro differentiated myotubes established from well characterized PCOS subjects.Using radiotracer techniques, RT-PCR and enzyme kinetic analysis we examined myotubes established from PCOS subjects with or without pioglitazone treatment, versus healthy control subjects who had been extensively metabolically characterized in vivo. Results. Myotubes established from PCOS and matched control subjects comprehensively expressed all insulin-sensitive biomarkers; glucose uptake and oxidation, glycogen synthesis and lipid uptake. There were no significant differences between groups either at baseline or during acute insulin stimulation, although in vivo skeletal muscle was insulin resistant. In particular, we found no evidence for defects in insulin-stimulated glycogen synthase activity between groups. Myotubes established from PCOS patients with or without pioglitazone treatment also showed no significant differences between groups, neither at baseline nor during acute insulin stimulation, although in vivo pioglitazone treatment significantly improved insulin sensitivity. Consistently, the myotube cultures failed to show differences in mRNA levels of genes previously demonstrated to differ in PCOS patients with or without pioglitazone treatment (PLEK, SLC22A16, and TTBK).These results suggest that the mechanisms governing insulin resistance in skeletal muscle of PCOS patients in vivo are not primary, but rather adaptive.ClinicalTrials.gov NCT00145340

Impaired Cell Surface Expression of HLA-B Antigens on Mesenchymal Stem Cells and Muscle Cell Progenitors

HLA class-I expression is weak in embryonic stem cells but increases rapidly during lineage progression. It is unknown whether all three classical HLA class-I antigens follow the same developmental program. In the present study, we investigated allele-specific expression of HLA-A, -B, and -C at the mRNA and protein levels on human mesenchymal stem cells from bone marrow and adipose tissue as well as striated muscle satellite cells and lymphocytes. Using multicolour flow cytometry, we found high cell surface expression of HLA-A on all stem cells and PBMC examined. Surprisingly, HLA-B was either undetectable or very weakly expressed on all stem cells protecting them from complement-dependent cytotoxicity (CDC) using relevant human anti-B and anti-Cw sera. IFNγ stimulation for 48–72 h was required to induce full HLA–B protein expression. Quantitative real-time RT-PCR showed that IFNγ induced a 9–42 fold increase of all six HLA-A,-B,-C gene transcripts. Interestingly, prior to stimulation, gene transcripts for all but two alleles were present in similar amounts suggesting that post-transcriptional mechanisms regulate the constitutive expression of HLA-A,-B, and -C. Locus-restricted expression of HLA-A, -B and -C challenges our current understanding of the function of these molecules as regulators of CD8+ T-cell and NK-cell function and should lead to further inquiries into their expression on other cell types

Electrical Pulse Stimulation of Cultured Human Skeletal Muscle Cells as an In Vitro Model of Exercise

Background and Aims Physical exercise leads to substantial adaptive responses in skeletal muscles and plays a central role in a healthy life style. Since exercise induces major systemic responses, underlying cellular mechanisms are difficult to study in vivo. It was therefore desirable to develop an in vitro model that would resemble training in cultured human myotubes. Methods Electrical pulse stimulation (EPS) was applied to adherent human myotubes. Cellular contents of ATP, phosphocreatine (PCr) and lactate were determined. Glucose and oleic acid metabolism were studied using radio-labeled substrates, and gene expression was analyzed using real-time RT-PCR. Mitochondrial content and function were measured by live imaging and determination of citrate synthase activity, respectively. Protein expression was assessed by electrophoresis and immunoblotting. Results High-frequency, acute EPS increased deoxyglucose uptake and lactate production, while cell contents of both ATP and PCr decreased. Chronic, low-frequency EPS increased oxidative capacity of cultured myotubes by increasing glucose metabolism (uptake and oxidation) and complete fatty acid oxidation. mRNA expression level of pyruvate dehydrogenase complex 4 (PDK4) was significantly increased in EPS-treated cells, while mRNA expressions of interleukin 6 (IL-6), cytochrome C and carnitin palmitoyl transferase b (CPT1b) also tended to increase. Intensity of MitoTracker®Red FM was doubled after 48 h of chronic, low-frequency EPS. Protein expression of a slow fiber type marker (MHCI) was increased in EPS-treated cells. Conclusions Our results imply that in vitro EPS (acute, high-frequent as well as chronic, low-frequent) of human myotubes may be used to study effects of exercise.This work was funded by the University of Oslo, Oslo University College, the Norwegian Diabetes Foundation, the Freia Chocolade Fabriks Medical Foundation and the Anders Jahre’s Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Hedgehog partial agonism drives warburg-lie metabolism in muscle and brown fat

Diabetes, obesity, and cancer affect upward of 15% of the world&rsquo;s population. Interestingly, all three diseases juxtapose dysregulated intracellular signaling with altered metabolic state. Exactly which genetic factors define stable metabolic set points in vivo remains poorly understood. Here, we show that hedgehog signaling rewires cellular metabolism. We identify a cilium-dependent Smo-Ca2+-Ampk axis that triggers rapid Warburg-like metabolic reprogramming within minutes of activation and is required for proper metabolic selectivity and flexibility. We show that Smo modulators can uncouple the Smo-Ampk axis from canonical signaling and identify cyclopamine as one of a new class of &ldquo;selective partial agonists,&rdquo; capable of concomitant inhibition of canonical and activation of noncanonical hedgehog signaling. Intriguingly, activation of the Smo-Ampk axis in vivo drives robust insulin-independent glucose uptake in muscle and brown adipose tissue. These data identify multiple noncanonical endpoints that are pivotal for rational design of hedgehog modulators and provide a new therapeutic avenue for obesity and diabetes.<br /

Active control of boundary layer instabilities

Active control of spatially evolving three-dimensional instability waves in the boundary layer of a flat plate with zero pressure gradient has been investigated both numerically and experimentally. The boundary layer was artificially excited by various computer-generated perturbations in a low-turbulence wind tunnel so as to create a three-dimensional field of instability waves. Sensors were used to detect the on-coming disturbances and appropriate control signals sent to the downstream actuators to generate counteracting disturbances. The key element of this type of active control is the determination of the transfer function of the control system connecting the sensors with the actuators. Modelling by linear theory showed that the amplitudes of the disturbances downstream were significantly suppressed when the causal transfer function was applied. To simplify the control problem different strategies using approximations to the transfer function were used. It was shown that with a very simple transfer function almost the same result could be achieved as with the full causal transfer function. The modelling was validated experimentally, first by an off-line control using different control modes, i.e. different transfer functions, and then by a real-time hardware implementation using one of the modes. Good agreement between the experimental measurements and numerical predictions indicated that a simple control strategy could be developed to inhibit the growth of the three-dimensional instability waves over extensive regions of the flow