Search

The continued drive towards increased zones of laminar flow on aircraft flying surfaces requires the research community to improve its ability to understand and predict factors causing instability leading to transition and turbulent flow. One of the factors that arise in trying to develop laminar wings is surface finish and the influence played by various types of surface imperfections. It turns out that small protuberances, or depressions, on the surface can interact with either sound waves, or existing instability waves, to generate additional instability disturbances through the receptivity process. There are many possible ways of tackling this problem and predicting the disturbances created by the interaction of, say, a sound wave and a small surface bump. Colleagues who work with DNS methods assure me that this problem can be tackled effectively by DNS, but as we are mainly considering very small acoustic excitation so that linearised equations are appropriate. High order methods commonly employed can deal with very weak perturbations with adequate accuracy. Until these solutions are available for the parameters of the current project we cannot gauge the value of the method. It is also possible to further simplify the computing by linearising the boundary conditions on the excrescence to plate values on the bounding surface. This is certainly permissible for very shallow imperfections but may not be suitable for larger ones. Triple deck methods have also successfully solved these problems see [1].

One of the possible forcing mechanisms for inducing transition from laminar to turbulent flow is freestream turbulence. Among the different transition mechanisms, freestream turbulence influences the cross-flow instability which occurs on swept wings. In order to understand better the physical interaction between freestream turbulence and boundary layer, this work analyses how the turbulence is distorted approaching a leading edge and whether it is amplified approaching a swept leading edge. The paper contains a brief literature review of both the two-dimensional stagnation point and three-dimensional attachment-line flows. This reveals that freestream turbulence amplification has been investigated for two-dimensional leading edge flows, but there is a lack of research on the swept leading edge. In addition, the paper presents some experimental results on a circular cylinder at various sweep angles. The results show that the phenomenon observed at the two-dimensional stagnation point also occurs at a swept attachment-line, although some differences are identified.

An experimental investigation was carried out in a low-turbulence wind tunnel to study the early development of artificially initiated turbulent spots in a laminar boundary layer over a flat plate. The reproducibility of the experiments allowed us to observe fine structural details that have not been observed previously. Initial velocity disturbances quickly developed into hairpin-like structures that multiplied downstream, which increased the width, length and height of the incipient turbulent spots. Only those disturbances that were greater than a threshold value developed into turbulent spots while the others decayed. The rate of development was also affected by the duration of the initial disturbances. We found that the behaviour of turbulence generation within a turbulent spot is similar to the burst events in the turbulent boundary layer, where ejection events are followed by sweep events.

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 at plate model. The freestream 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 30�m < jhj < 750�m (equivalent to 0:025 < jhj=��B < 0:630 in relative roughness height to the local unperturbed Blasius boundary layer displacement thickness). These depths are designed to cover both the predicted linear and non-linear 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 �m (jhj=��B � 0:126). The departure from the linear behaviour is, however, dependent on the geometry of the surface imperfection. For cavities of signi�cant depth, the non-linear 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.

Cultured human myotubes offer a unique model to distinguish between primary and environmental factors in the aetiology of insulin resistance in human skeletal muscle. The objective of this review was to summarize our and other group studies on insulin resistance in human myotubes established from lean, obese and type 2 diabetes (T2D) subjects. Overall, studies of human myotubes established from lean, obese and T2D subjects clearly show that part of the diabetic phenotype observed in vivo is preserved in diabetic myotubes. Diabetic myotubes express a primary coordinated impairment of lipid oxidation, oxidative phosphorylation (OXPHOS) and insulin-stimulated glucose metabolism. Currently, both the responsible molecular mechanisms as well as the extent to which these alterations depend on genetic and/or epigenetic alterations have yet to be identified. Based on the data, it is hypothesized that the impaired insulin-mediated glucose metabolism, impaired OXPHOS and reduced lipid oxidation observed in diabetic myotubes are caused by the reduced peroxisome proliferator-activated receptor gamma coactivator-1α (PGC1α) expression.

A decrease in skeletal muscle lipolysis and hormone sensitive-lipase (HSL) expression has been linked to insulin resistance in obesity. The purpose of this study was to identify potential intrinsic defects in lipid turnover and lipolysis in myotubes established from obese and type 2 diabetic subjects. Lipid trafficking and lipolysis were measured by pulse-chase assay with radiolabeled substrates in myotubes from non-obese/non-diabetic (lean), obese/non-diabetic (obese) and obese/diabetic (T2D) subjects. Lipolytic protein content and level of Akt phosphorylation were measured by Western blot. HSL was overexpressed by adenovirus-mediated gene delivery. Myotubes established from obese and T2D subjects had lower lipolysis (-30-40%) when compared to lean, using oleic acid as precursor. Similar observations were also seen for labelled glycerol. Incorporation of oleic acid into diacylglycerol (DAG) and free fatty acid (FFA) level was lower in T2D myotubes, and acetate incorporation into FFA and complex lipids was also lower in obese and/or T2D subjects. Both protein expression of HSL (but not ATGL) and changes in DAG during lipolysis were markedly lower in cells from obese and T2D when compared to lean subjects. Insulin-stimulated glycogen synthesis (-60%) and Akt phosphorylation (-90%) were lower in myotubes from T2D, however, overexpression of HSL in T2D myotubes did not rescue the diabetic phenotype. In conclusion, intrinsic defects in lipolysis and HSL expression co-exist with reduced insulin action in myotubes from obese T2D subjects. Despite reductions in intramyocellular lipolysis and HSL expression, overexpression of HSL did not rescue defects in insulin action in skeletal myotubes from obese T2D subjects.

Polycystic ovary syndrome (PCOS) is associated with insulin resistance and increased risk of type 2 diabetes. Skeletal muscle is the major site of insulin mediated glucose disposal and the skeletal muscle tissue is capable to synthesize, convert and degrade androgens. Insulin sensitivity is conserved in cultured myotubes (in vitro) from patients with PCOS, but the effect of testosterone on this insulin sensitivity is unknown. We investigated the effect of 7days testosterone treatment (100nmol/l) on glucose transport and gene expression levels of hormone receptors and enzymes involved in the synthesis and conversion of testosterone (HSD17B1, HSD17B2, CYP19A1, SRD5A1-2, AR, ER-α, HSD17B6 and AKR1-3) in myotubes from ten patients with PCOS and ten matched controls. Testosterone treatment significantly increased aromatase and androgen receptor gene expression levels in patients and controls. Glucose transport in myotubes was comparable in patients with PCOS vs. controls and was unchanged by testosterone treatment (p=0.21 PCOS vs. controls). These results suggest that testosterone treatment of myotubes increases the aromatase and androgen receptor gene expression without affecting insulin sensitivity and if testosterone is implicated in muscular insulin resistance in PCOS, this is by and indirect mechanism. Polycystic ovary syndrome (PCOS) is associated with insulin resistance and increased risk of type 2 diabetes. Skeletal muscle is the major site of insulin mediated glucose disposal and the skeletal muscle tissue is capable to synthesize, convert and degrade androgens. Insulin sensitivity is conserved in cultured myotubes (in vitro) from patients with PCOS, but the effect of testosterone on this insulin sensitivity is unknown. We investigated the effect of 7 days testosterone treatment (100 nmol/l) on glucose transport and gene expression levels of hormone receptors and enzymes involved in the synthesis and conversion of testosterone (HSD17B1, HSD17B2, CYP19A1, SRD5A1-2, AR, ER-α, HSD17B6 and AKR1-3) in myotubes from ten patients with PCOS and ten matched controls. Testosterone treatment significantly increased aromatase and androgen receptor gene expression levels in patients and controls. Glucose transport in myotubes was comparable in patients with PCOS vs. controls and was unchanged by testosterone treatment (p = 0.21 PCOS vs. controls). These results suggest that testosterone treatment of myotubes increases the aromatase and androgen receptor gene expression without affecting insulin sensitivity and if testosterone is implicated in muscular insulin resistance in PCOS, this is by and indirect mechanism.

The paper describes research work that has been carried out to reduce the viscous drag of transport aircraft wings by controlling the turbulent contamination on the attachment line. Laminar leading edges will reduce the boundary layer thickness downstream and thus reduce drag. This scenario was postulated by M. Gaster in a paper presented to the AIAA Seattle conference in 2008. The effect of maintaining a laminar attachment line on the overall flow on a wing has been modeled and drag reductions calculated. These values are compared with wind tunnel measurements of viscous drag of a wing with both laminar and turbulent attachment lines.

SummaryDiabetes, obesity, and cancer affect upward of 15% of the world’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 “selective partial agonists,” 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.

BACKGROUND AND PURPOSE The sarcoplasmic reticulum Ca2+-ATPase (SERCA) plays a role in thermogenesis. The exogenous compound capsaicin increased SERCA-mediated ATP hydrolysis not coupled to Ca2+ transport. Here, we have sought to identify endogenous compounds that may function as SERCA uncoupling agents. EXPERIMENTAL APPROACH Using isolated SR vesicles from rabbits, we have screened for endogenous compounds that uncouple SERCA. We have also studied their ability to deplete cytoplasmic ATP from human skeletal muscle cells in culture. KEY RESULTS Studies on SR vesicles showed that the endogenous lipid metabolite N-arachidonoyl dopamine (NADA) was a potent stimulator of SERCA uncoupling. NADA stabilized an E1-like pump conformation that had a lower dephosphorylation rate, low affinity for Ca2+ at the luminal sites and a specific proteinase K cleavage pattern involving protection of the C-terminal p83C fragment from further cleavage. Moreover, we found a significantly decreased cytoplasmic ATP levels following treatment of skeletal muscle cells with 100 nM NADA. This effect was dependent on the presence of glucose and abolished by pretreatment with the specific SERCA inhibitor thapsigargin, regardless of the presence of glucose. CONCLUSIONS AND IMPLICATIONS NADA is an endogenous molecule that may function as SERCA uncoupling agent in vivo. Members of the endocannabinoid family exert concerted actions on several Ca2+-handling proteins. Uncoupling of SERCA by exogenous compounds could be a novel post-mitochondrial strategy for reduction of cellular ATP levels. In addition, signalling networks leading to SERCA uncoupling can be explored to study the importance of this ion pump in pathophysiological conditions related to metabolism.

The diabetic phenotype is complex, requiring elucidation of key initiating defects. Recent research has shown that diabetic myotubes express a primary reduced tricarboxylic acid (TCA) cycle flux. A reduced TCA cycle flux has also been shown both in insulin resistant offspring of T2D patients and exercising T2D patients in vivo. This review will discuss the latest advances in the understanding of the molecular mechanisms regulating the TCA cycle with focus on possible underlying mechanism which could explain the impaired TCA flux in insulin resistant human skeletal muscle in type 2 diabetes. A reduced TCA is both a marker and a maker of the diabetic phenotype.

The perilipin proteins enclose intracellular lipid droplets. We describe the mRNA expression of the five perilipins in human skeletal muscle in relation to fatty acid supply, exercise and energy balance. We observed that all perilipins were expressed in skeletal muscle biopsies with the highest mRNA levels of perilipin 2, 4 and 5. Cultured myotubes predominantly expressed perilipin 2 and 3. In vitro, incubation of myotubes with fatty acids enhanced mRNA expression of perilipin 1, 2 and 4. In vivo, low fat diet increased mRNA levels of perilipin 3 and 4. Endurance training, but not strength training, enhanced the expression of perilipin 2 and 3. Perilipin 1 mRNA correlated positively with body fat mass, whereas none of the perilipins were associated with insulin sensitivity. In conclusion, all perilipins mRNAs were expressed in human skeletal muscle. Diet as well as endurance exercise modulated the expression of perilipins.

BACKGROUND: Accumulating preclinical and clinical evidence indicates that human mesenchymal stromal cells (MSCs) are good candidates for cell therapy. For clinical applications of MSCs extensive in vitro expansion is required to obtain an adequate number of cells. It is evident that the pursuit for cell quantity must not affect quality, but it is also a fact that in vitro culture conditions affect MSC phenotype. One possible variable is the degree of cell confluence during expansion. METHODS: We investigate the influence of cell density on homogeneity and differentiation during culture expansion of un-stimulated MSCs isolated from the bone marrow in DMEM and fetal bovine serum (FBS). MSC morphology, phenotype and differentiation were investigated weekly during 5 weeks culture expansion using electron microscopy, flow cytometry, immunocytochemistry, qualitative RT-PCR and quantitative Q-PCR. RESULTS: The morphological observation and the phenotypic analyses showed that MSCs after 3 weeks cultivation constituted a phenotypically homogenous MSC cell population, which at low levels expressed genes for different cell lineages, confirming their multilineage plasticity, without actual differentiation. This phenotype persisted independent of increasing cell densities. DISCUSSION: These data demonstrate that MSC characteristics and plasticity can be maintained during culture expansion from bone marrow mononuclear cells to MSCs and that a homogeneous phenotype of undifferentiated MSCs which persists independent of cell density can be used for clinical therapies.

N EAR-STALL three-dimensional separation patterns, which are also referred to as “stall cells,” can appear on the suction side of certain airfoils. An early report on this phenomenon was provided by Gregory et al. [1]. Detailed oilflow visualizations of stall cells were obtained by, e.g., Winkelmann and Barlow [2], Bippes et al. [3], and Schewe [4]. Yon and Katz [5] sampled the wall pressure in the separated flow region and observed large-amplitude but lowfrequency fluctuations at the beginning of the range of the angle of attack over which the stall cells occurred. Outside this range, the amplitude of the fluctuations was much lower and the low-frequency signal was absent. Broeren and Bragg [6] investigated several airfoils with different stalling characteristics. Stall for airfoils with trailingedge separation was characterized by the appearance of stall cells. The stall cells that were observed for angles of attack at or above stall were generally steady. Stall for airfoils with leading-edge separation (i.e., thin-airfoil stall) displayed low-frequency oscillations near stall but no stall cells. Stall cells can appear suddenly and, therefore, have a profound effect on the performance, maneuverability, and safety of aircraft. An improved understanding of the criteria determining the appearance of stall cellswouldmake flight at near-stall conditionsmore predictable, and thus safer. In addition, the mechanism responsible for the formation of stall cells may be exploited for high angle-of-attack flow control applications. Weihs and Katz [7] proposed a physical mechanism for the appearance of stall cells based on the Crow instability [8]. At stall, in the time mean, a spanwise vortex can be associatedwith the recirculating flow region on the suction side of the airfoil. Because this vortex is located close to the airfoil surface, a reflected image of the vortex with the opposite sense of rotation can be imagined. As shown by Crow [8], this steady vortex pair is unstable with respect to a symmetric mode. The growth of the symmetric mode may result in the breakup of the steady vortex and the formation of stall cells. Rodriguez and Theofilis [9] argued that stall cells are the consequence of a global instability. The linear superposition of the two-dimensional flow around a stalled airfoil with the leading stationary three-dimensional eigenmode resulted in skin-friction lines that were strongly reminiscent of stall cells. In the present Note, an explanation for the appearance of stall cells based on lifting-line theory is offered and a simple formula for estimating the stall cell spacing is derived.

Context: Polycystic ovary syndrome (PCOS) affects 5–8% of fertile women and is often accompanied by insulin resistance, leading to increased risk of developing type 2 diabetes. Skeletal muscle from insulin-resistant PCOS subjects display reduced expression of nuclear encoded genes involved in mitochondrial oxidative metabolism. Objective: We aimed to investigate whether there was a primary mitochondrial dysfunction or difference in mitochondria content that might contribute to the in vivo detected insulin resistance. Design: The ATP synthesis with and without ATP use and the mitochondrial mass was determined in mitochondria isolated from myotubes established from PCOS subjects and control subjects. Patients: Myotubes were established from eight insulin-resistant PCOS subjects (verified by euglycemic hyperinsulinemic clamp) and eight healthy weight- and age-matched controls. Results: Mitochondrial mass and measurable mitochondrial ATP synthesis, with and without ATP use, were not different between PCOS subjects and control subjects. Conclusion: We found no evidence for a primary impaired mitochondrial function or content in myotubes established from PCOS subjects, and our results suggest that reduced expression of oxidative genes in PCOS subjects is an adaptive trait.

To date, it is unknown whether mitochondrial dysfunction in skeletal muscle from subjects with type 2 diabetes is based on primarily reduced mitochondrial mass and/or a primarily decreased mitochondrial ATP synthesis. Mitochondrial mass were determined in myotubes established from eight lean, eight obese and eight subjects with type 2 diabetes precultured under normophysiological conditions. Furthermore, mitochondria were isolated and ATP production was measured by luminescence at baseline and during acute insulin stimulation with or without concomitant ATP utilization by hexokinase. Mitochondrial mass and the ATP synthesis rate, neither at baseline nor during acute insulin stimulation, were not different between groups. The ratio of ATP synthesis rate at hexokinase versus ATP synthesis rate at baseline was lower in diabetic mitochondria compared to lean mitochondria. Thus the lower content of muscle mitochondria in type 2 diabetes in vivo is an adaptive trait and mitochondrial dysfunction in type 2 diabetes in vivo is based both on primarily impaired ATP synthesis and an adaptive loss of mitochondrial mass.

Corpeleijn E, Hessvik NP, Bakke SS, Levin K, Blaak EE, Thoresen GH, Gaster M, Rustan AC. Oxidation of intramyocellular lipids is dependent on mitochondrial function and the availability of extracellular fatty acids. Am J Physiol Endocrinol Metab 299: E14-E22, 2010. First published May 4, 2010; doi:10.1152/ajpendo.00187.2010.-Obesity and insulin resistance are related to both enlarged intramyocellular triacylglycerol stores and accumulation of lipid intermediates. We investigated how lipid overflow can change the oxidation of intramyocellular lipids (ICLOX) and intramyocellular lipid storage (ICL). These experiments were extended by comparing these processes in primary cultured myotubes established from healthy lean and obese type 2 diabetic (T2D) individuals, two extremes in a range of metabolic phenotypes. ICLs were prelabeled for 2 days with 100 mu M [C-14] oleic acid (OA). ICLOX was studied using a (CO2)-C-14 trapping system and measured under various conditions of extracellular OA (5 or 100 mu M) and glucose (0.1 or 5.0 mM) and the absence or presence of mitochondrial uncoupling [carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP)]. First, increased extracellular OA availability (5 vs. 100 mu M) reduced ICLOX by 37%. No differences in total lipolysis were observed between low and high OA availability. Uncoupling with FCCP restored ICLOX to basal levels during high OA availability. Mitochondrial mass was positively related to ICLOX, but only in myotubes from lean individuals. In all, a lower mitochondrial mass and lower ICLOX were related to a higher cell-associated OA accumulation. Second, myotubes established from obese T2D individuals showed reduced ICLOX. ICLOX remained lower during uncoupling (P

OBJECTIVE—Increased availability of fatty acids is important for accumulation of intracellular lipids and development of insulin resistance in human myotubes. It is unknown whether different types of fatty acids like eicosapentaenoic acid (EPA) or tetradecylthioacetic acid (TTA) influence these processes. RESEARCH DESIGN AND METHODS—We examined fatty acid and glucose metabolism and gene expression in cultured human skeletal muscle cells from control and type 2 diabetic individuals after 4 days of preincubation with EPA or TTA. RESULTS—Type 2 diabetes myotubes exhibited reduced formation of CO2 from palmitic acid (PA), whereas release of β-oxidation products was unchanged at baseline but significantly increased with respect to control myotubes after preincubation with TTA and EPA. Preincubation with TTA enhanced both complete (CO2) and β-oxidation of palmitic acid, whereas EPA increased only β-oxidation significantly. EPA markedly enhanced triacylglycerol (TAG) accumulation in myotubes, more pronounced in type 2 diabetes cells. TAG accumulation and fatty acid oxidation were inversely correlated only after EPA preincubation, and total level of acyl-CoA was reduced. Glucose oxidation (CO2 formation) was enhanced and lactate production decreased after chronic exposure to EPA and TTA, whereas glucose uptake and storage were unchanged. EPA and especially TTA increased the expression of genes involved in fatty acid uptake, activation, accumulation, and oxidation. CONCLUSIONS—Our results suggest that 1) mitochondrial dysfunction in diabetic myotubes is caused by disturbances downstream of fatty acid β-oxidation; 2) EPA promoted accumulation of TAG, enhanced β-oxidation, and increased glucose oxidation; and 3) TTA improved complete palmitic acid oxidation in diabetic myotubes, opposed increased lipid accumulation, and increased glucose oxidation.

Udgivelsesdato: 2008-Nov AIMS/HYPOTHESIS: Microarray-based studies of skeletal muscle from patients with type 2 diabetes and high-risk individuals have demonstrated that insulin resistance and reduced mitochondrial biogenesis co-exist early in the pathogenesis of type 2 diabetes independently of hyperglycaemia and obesity. It is unknown whether reduced mitochondrial biogenesis or other transcriptional alterations co-exist with impaired insulin responsiveness in primary human muscle cells from patients with type 2 diabetes. METHODS: Using cDNA microarray technology and global pathway analysis with the Gene Map Annotator and Pathway Profiler (GenMapp 2.1) and Gene Set Enrichment Analysis (GSEA 2.0.1), we examined transcript levels in myotubes established from obese patients with type 2 diabetes and matched obese healthy participants, who had been extensively metabolically characterised both in vivo and in vitro. We have previously reported reduced basal lipid oxidation and impaired insulin-stimulated glycogen synthesis and glucose oxidation in these diabetic myotubes. RESULTS: No single gene was differently expressed after correction for multiple testing, and no biological pathway was differently expressed using either method of global pathway analysis. In particular, we found no evidence for differential expression of genes involved in mitochondrial oxidative metabolism. Consistently, there was no difference in mRNA levels of genes known to mediate the transcriptional control of mitochondrial biogenesis (PPARGC1A and NRF1) or in mitochondrial mass between diabetic and control myotubes. CONCLUSIONS/INTERPRETATION: These results support the hypothesis that impaired mitochondrial biogenesis is not a primary defect in the sequence of events leading to insulin resistance and type 2 diabetes.

Udgivelsesdato: 2007-Oct AIMS/HYPOTHESIS: Liver X receptors (LXRs) play important roles in lipid and carbohydrate metabolism. The purpose of the present study was to evaluate effects of the endogenous LXR agonist 22-R-hydroxycholesterol (22-R-HC) and its stereoisomer 22-S-hydroxycholesterol (22-S-HC), in comparison with the synthetic agonist T0901317 on lipid and glucose metabolism in human skeletal muscle cells (myotubes). METHODS: Myotubes established from lean and obese control volunteers and from obese type 2 diabetic volunteers were treated with LXR ligands for 4 days. Lipid and glucose metabolisms were studied with labelled precursors, and gene expression was analysed using real-time PCR. RESULTS: Treatment with T0901317 increased lipogenesis (de novo lipid synthesis) and lipid accumulation in myotubes, this increase being more pronounced in myotubes from type 2 diabetic volunteers than from lean volunteers. Furthermore, 22-S-HC efficiently counteracted the T0901317-induced enhancement of lipid formation. Moreover, synthesis of diacylglycerol, cholesteryl ester and free cholesterol from acetate was reduced below baseline by 22-S-HC, whereas glucose uptake and oxidation were increased. Both 22-S-HC and 22-R-HC, in contrast to T0901317, decreased the expression of genes involved in cholesterol synthesis, whereas only 22-R-HC, like T0901317, increased the expression of the gene encoding the reverse cholesterol transporter ATP-binding cassette subfamily A1 (ABCA1). CONCLUSIONS/INTERPRETATION: T0901317-induced lipogenesis and lipid formation was more pronounced in myotubes from type 2 diabetic patients than from lean individuals. 22-S-HC counteracted these effects and reduced de novo lipogenesis below baseline, while glucose uptake and oxidation were increased.

Udgivelsesdato: 2006-Jul Accumulation of intramuscular long-chain acyl-CoA esters (LCACoA) has previously in animal and human models been suggested to play an important role in lipid induced insulin resistance. The aim of this study was to examine whether myotubes established from type 2 diabetic (T2D) subjects and lean controls express differences in long-chain acyl-CoA esters (LCACoA) precultured under physiological conditions and during chronic exposure to palmitate (PA) and oleic acids (OA) with/without acute insulin stimulation. No significant differences were found between diabetic and control myotubes, neither in the total amount nor among individual LCA-CoA species during basal and acute insulin stimulation. LCA-CoA accumulated during exposure to palmitic acid but not during exposure to oleic acid. During PA and OA exposure, only palmitoyl-CoA, oleoyl-CoA and total LCA-CoA change. PA exposure increased the palmitoyl-CoA, whereas oleoyl-CoA was reduced and vice versa during OA exposure. No differences were found in the LCA-CoA level between T2D and control subjects, neither in the total amount nor in the individual specific LCA-CoA species during fatty acid exposure. Chronic (24 h), high PA, but not OA exposure induced insulin resistance at the level of glycogen synthesis in control subjects. These results indicate that (1) no primary defects are responsible for LCA-CoA accumulation in diabetic subjects; (2) LCA-CoA changes in vivo are partly adaptive to changes in the PA level and possibly other saturated fatty acids; and (3) PA induced insulin resistance may be mediated through an increased level of palmitoyl-CoA.

Udgivelsesdato: 2006-Jul During exercise, triacylglycerol (TG) is recruited in skeletal muscles. We hypothesized that both muscle hormone-sensitive lipase (HSL) activity and TG recruitment would be higher in trained than in untrained subjects in response to prolonged exercise. Healthy male subjects (26 +/- 1 years, body moss index 23.3 +/- 0.5 kg m(-2)), either untrained (N = 8, VO(2max) 3.8 +/- 0.2 l min(-1)) or trained (N = 8, VO(2max) 5.1 +/- 0.1 l min(-1)), were studied. Before and after 3-h exercise (58 +/- 1% VO(2max)), a biopsy was taken. Muscle citrate synthase (32 +/- 2 vs. 47 +/- 6 mumol g(-1) min(-1) d.w.) and beta-hydroxy-acyl-CoA-dehydrogenase (38 +/- 3 vs. 52 +/- 5 mumol g(-1) min(-1) d.w.) activities were lower in untrained than in trained subjects (p < 0.05). Throughout the exercise, fat oxidation was higher in trained than in untrained subjects (p < 0.05). Muscle HSL activity was similar at rest (0.72 +/- 0.08 and 0.74 +/- 0.03 mU mg(-1) protein) and after exercise (0.71 +/- 0.1 and 0.68 +/- 0.03 mU mg(-1) protein) in untrained and trained subjects. At rest, the chemically determined muscle TG content (37 +/- 8 and 26 +/- 5 mmol g(-1) d.w.) was similar (p > 0.05), and after exercise it was unchanged in untrained and lower (p < 0.05) in trained subjects (41 +/- 9 and 10 +/- 2 mmol g((1) d.w.). Determined histochemically, TG was decreased (p < 0.05) after exercise in type I and II fibres. Depletion of TG was not different between fibre types in untrained, but tended to be higher (p = 0.07) in type I compared with type II fibres in trained muscles. In conclusion, HSL activity is similar in untrained and trained skeletal muscles both before and after prolonged exercise. However, the tendency to higher muscle TG recruitment during exercise in the trained subjects suggests a difference in the regulation of HSL or other lipases during exercise in trained compared with untrained subjects.

We recently described a primarily reduced palmitate oxidation in myotubes established from type 2 diabetic subjects, whereas triacylglycerol (TAG) accumulation seemed to be adaptive. However, it is still uncertain whether these changes are similar for saturated and unsaturated fatty acids and whether high concentrations of glucose and/or insulin may change this picture. Studies of palmitic acid and oleic acid metabolism in human myotubes established from control and type 2 diabetic subjects under conditions of acute high concentrations of insulin and/or glucose may solve these questions. Total oleic acid and palmitic acid uptake in myotubes was increased during acute insulin stimulation (P < 0.01) but not under acute, high-glucose concentrations, and no differences were found between the groups. Type 2 diabetic myotubes expressed a reduced palmitic acid oxidation to carbon dioxide (P ≤ 0.04), whereas oleic acid oxidation showed no differences between myotubes from both groups. High glucose concentrations decreased oleic acid oxidation (P ≤ 0.03). Lipid distribution was not different in diabetic and control myotubes when palmitic acid and oleic acid incorporation into cellular lipids was compared. Myotubes that were exposed to palmitic acid showed an increased palmitic acid incorporation into diacylglycerol (DAG) and TAG compared with myotubes that were exposed to oleic acid (P < 0.05) expressing an increased intracellular free fatty acid (FFA) level (P < 0.05). Lipid distribution was not affected by high glucose, whereas insulin increased FFAs, DAG, and TAG (P < 0.05). De novo lipid synthesis from glucose in both diabetic and control myotubes was of the same magnitude independent of glucose and insulin concentrations. These results indicate that palmitic acid and oleic acid are utilized in the same pattern in diabetic and control myotubes even though palmitic acid oxidation is primarily reduced in diabetic cells. Palmitic acid and oleic acid are handled differently by myotubes: Palmitic acid seems to accumulate as DAG and TAG, whereas oleic acid accumulates as intracellular FFAs. These observations indicate that oleic acid is preferable as fatty acid as it accumulates to a lesser extent as DAG and TAG than palmitic acid. Neither acute hyperglycemia nor de novo lipid synthesis from glucose seems central to the TAG accumulation in obesity or type 2 diabetes.

Udgivelsesdato: 2004-Apr Nine novel sugar transporter-like proteins have been discovered in the past 5 years. The mRNA for three of these, the glucose transporters (GLUT) GLUT8, GLUT11 and GLUT12, have been detected in human skeletal muscle. In the present study, we examined the pattern of expression and localization of the GLUT isoforms 8, 11 and 12 in human skeletal muscle using an immunohistochemical approach. Biopsies of human skeletal muscle from sedentary or trained healthy adults, from fetal muscle (24 weeks of gestation), from obese type-2 diabetic subjects, and from patients suffering from polymyositis or amyotrophic lateral sclerosis (ALS) were studied. GLUT8 and 12 immunoreactivity was below detection level in both developing and adult muscle fibres. GLUT11 immunoreactivity, however, was present in slow-twitch muscle fibres, but not in fast twitch fibres. Since, in contrast, GLUT4 was expressed in all investigated muscle fibres, the pattern of expression of GLUT11 differs from that of GLUT4, suggesting a specialized function for GLUT11 with a regulation independent from that of GLUT4. Obesity, type-2 diabetes, training, conditions of de- and reinnervation (ALS) and regeneration (polymyositis) failed to induce GLUT8 or -12 expression. Likewise, the fibre type-dependent pattern of GLUT11 immunoreactivity was unaltered. However, some slow muscle fibres lose their GLUT11 immunoreactivity under regeneration. Our results indicate that GLUT11 immunoreactivity, in contrast to that of GLUT4, is expressed exclusively in slow-twitch muscle fibres and is unaffected by physiological and pathophysiological conditions except in primary myopathy. GLUT8 and GLUT12 do not appear to be of importance in human muscle under physiological and pathophysiological conditions.

Insulin resistance in skeletal muscle in vivo is associated with reduced lipid oxidation and lipid accumulation. It is still uncertain whether changes in lipid metabolism represent an adaptive compensation at the cellular level or a direct expression of a genetic trait. Studies of palmitate metabolism in human myotubes established from control and type 2 diabetic subjects may solve this problem, as genetic defects are preserved and expressed in vitro. In this study, total uptake of palmitic acid was similar in myotubes established from both control and type 2 diabetic subjects under basal conditions and acute insulin stimulation. Myotubes established from diabetic subjects expressed a primary reduced palmitic acid oxidation to carbon dioxide with a concomitantly increased esterification of palmitic acid into phospholipids compared with control myotubes under basal conditions. Triacylglycerol (TAG) content and the incorporation of palmitic acid into diacylglycerol (DAG) and TAG at basal conditions did not vary between the groups. Acute insulin treatment significantly increased palmitate uptake and incorporation of palmitic acid into DAG and TAG in myotubes established from both study groups, but no difference was found in myotubes established from control and diabetic subjects. These results indicate that the reduced lipid oxidation in diabetic skeletal muscle in vivo may be of genetic origin; it also appears that TAG metabolism is not primarily affected in diabetic muscles under basal physiological conditions.

Udgivelsesdato: 2003-Feb We have recently reported a progressive decline in the expression of glucose transporter isoform 4 (GLUT4) from control subjects through obese non-diabetics to obese type 2 diabetic subjects, indicating that the reduced GLUT4 in slow twitch fibres could be secondary to obesity. In this study we investigate the association of GLUT4 expression with the intracellular triglyceride (TG) content in the same muscle fibres and with plasma lipid parameters. We used histochemistry and stereology to study the relationship between TG content and GLUT4 expression in muscle fibres from obese, obese type 2 diabetic subjects, and young lean controls. TG density was significantly higher in slow compared to fast fibres in all studied subjects (p0.05). Plasma TG and FFA did not correlate with GLUT4 expression in slow or fast fibres (p>0.05). In conclusion, TG content was increased in diabetic slow fibres with a reduced GLUT4 expression. The GLUT4 expression was not associated with an increased intracellular triglyceride content or with increased plasma FFA levels. Thus, intracellular TG content and circulating FFA may not influence glucose transport directly through GLUT4 expression.

Editor's suggested further reading in BioEssays: Can we do better than existing author citation metrics? Abstract and Counting citations in texts rather than reference lists to improve the accuracy of assessing scientific contribution Abstract

Udgivelsesdato: 2002-Sep In this study we examined the relationship between GLUT4 expression at the plasma membrane and muscle fibre size in fibre-typed human muscle fibres by immunocytochemistry and morphometry in order to gain further insight into the regulation of GLUT4 expression. At the site of the plasma membrane, GLUT4 was more abundantly expressed in slow as compared to fast fibres at the same fibre diameter (p < 0.01) and the GLUT4 expression increased with increasing fibre radius independently of fibre type (p < 0.01). The GLUT4 density at the surface of slow fibres of both diabetic and obese was reduced compared to control subjects at the same diameter (p < 0.001). Fast fibres in obese and type 2 diabetic subjects expressed a fibre-volume-dependent GLUT4 expression (p < 0.001), while this did not reach significance in slow fibres (obese p = 0.18 and diabetic p = 0.06). Our results show that increasing fibre volume is associated with increasing GLUT4 expression in both slow and fast fibres. Based on the possible dependency of GLUT4 expression on volume, we hypothesize that the reduced GLUT4 expression in obesity and type 2 diabetes may partly be compensated for by physical activity.

The most well-described defect in the pathophysiology of type 2 diabetes is reduced insulin-mediated glycogen synthesis in skeletal muscles. It is unclear whether this defect is primary or acquired secondary to dyslipidemia, hyperinsulinemia, or hyperglycemia. We determined the glycogen synthase (GS) activity; the content of glucose-6-phosphate, glucose, and glycogen; and the glucose transport in satellite cell cultures established from diabetic and control subjects. Myotubes were precultured in increasing insulin concentrations for 4 days and subsequently stimulated acutely by insulin. The present study shows that the basal glucose uptake as well as insulin-stimulated GS activity is reduced in satellite cell cultures established from patients with type 2 diabetes. Moreover, increasing insulin concentrations could compensate for the reduced GS activity to a certain extent, whereas chronic supraphysiological insulin concentrations induced insulin resistance in GS and glucose transport activity. Our data suggest that insulin resistance in patients with type 2 diabetes comprises at least two important defects under physiological insulin concentrations: a reduced glucose transport under basal conditions and a reduced GS activity under acute insulin stimulation, implicating a reduced glucose uptake in the fasting state and a diminished insulin-mediated storage of glucose as glycogen after a meal.

Udgivelsesdato: 2001-Nov-29 There is no consensus regarding the results from in vivo and in vitro studies on the impact of chronic high insulin and/or high glucose exposure on acute insulin stimulation of glycogen synthase (GS) kinetic parameters in human skeletal muscle. The aim of this study was to evaluate the kinetic parameters of glycogen synthase activity in human myotube cultures at conditions of chronic high insulin combined or not with high glucose exposure, before and after a subsequent acute insulin stimulation. Acute insulin stimulation significantly increased the fractional activity (FV(0.1)) of GS, increased the sensitivity of GS to the allosteric activator glucose 6-phosphate (A(0.5)) and increased the sensitivity of GS to its substrate UDPG (K(m(0.1))) when myotubes were precultured at low insulin with/without high glucose conditions. However, this effect of acute insulin stimulation was abolished in myotubes precultured at high insulin with or without high glucose. Furthermore, we found significant correlations between the fractional velocities FV(0.1) of GS and K(m(0.1)) (rho=-0.72, P

The estimation of the growth of propagating instability waves in laminar boundary layers is considered when the Reynolds number is sufficiently large for the mean flow to deviate only slightly from a truly parallel flow. An approximate solution for the linear perturbation is sought in the form of a scaled solution of the related locally parallel flow problem. The amplitude scaling is chosen so as to satisfy the full linearized perturbation equations as closely as possible by making the mean-square deviation of the remainder a minimum. By re-arranging the terms in the equations so that some of the small correction terms arising from the non-parallel mean flow are contained in the ordinary differential equation (ODE) defining the quasi-parallel flow solution, a useful simplification is obtained for the scaling function. Then a modified Orr–Sommerfeld equation defines the base solution and the differential expression for the scaling that can be integrated forms a simple conservation relation.

GLUT-4 expression in individual fibers of human skeletal muscles in younger and older adults was studied. Furthermore, the dependency of insulin-stimulated glucose uptake on fiber type distribution was investigated. Fiber type distribution was determined in cryosections of muscle biopsies from 8 younger (29 yr) and 8 older (64 yr) healthy subjects, and estimates of GLUT-4 expression in individual fibers were obtained by combining immunohistochemistry and stereology. GLUT-4 was more abundantly expressed in slow compared with fast muscle fibers in both younger ( P < 0.007) and older ( P < 0.001) subjects. A 25% reduction of GLUT-4 density in fast fibers ( P < 0.001) and an unchanged GLUT-4 density in slow fibers were demonstrated in older compared with younger subjects. Insulin-stimulated glucose uptake rates measured by hyperinsulinemic, euglycemic clamp were not correlated with the fraction of slow fibers in the young ( r = −0.45, P > 0.25) or in the elderly ( r = 0.11, P > 0.75) subjects. In conclusion, in human skeletal muscle, GLUT-4 expression is fiber type dependent and decreases with age, particularly in fast muscle fibers.

The nonlinear evolution of wavepackets in a laminar boundary layer has been observed experimentally. The packets originated from disturbances generated by a loudspeaker coupled to the boundary layer by a small hole in the plate. In a preliminary experiment two types of short-duration acoustic pulses were used, one with a positive excitation and another with a negative excitation. The experiments indicated that the packet that originated from a positive pulse displayed nonlinear behaviour at considerably lower amplitudes than that from a negative pulse. However, the preliminary experiments suggested that at some distance from the source the packets were identical in shape with a relative phase shift of 180°. Using complex-amplitude pulses it was possible to extend the experiments to include packets with other phases. This more comprehensive experiment not only showed a strong influence of the phase on the evolution of the packet, but also demonstrated that this nonlinear behaviour is not determined by the local effects of the excitation process. The observations suggested that the important parameter is the phase of the packet relative to the modulation envelope.

A new directionally sensitive velocity measurement system based upon a single oscillating hot-wire probe has been developed. The vibration of the hot-wire was effected by a piezoelectric oscillator that could be driven at different amplitudes and frequencies. The sign of the local flow velocity was determined by the phase shift between the motion and the output from the hot-wire. The system was used to measure mean and fluctuating velocities profiles of the separated flow behind a backward facing step.

Aims: Delta like 1/fetal antigen 1 (Dlk1/FA1) is a protein secreted by hormone producing cells in adult human and mice that is known to inhibit adipogenesis. Recent studies demonstrated the role of Dlk1/FA1 in inducing insulin resistance in mice. To investigate the involvement of circulating Dlk1/FA1 in insulin resistance and type 2 diabetes in human subjects, we studied the effects of chronic FA1 on the intermediary metabolism in myotubes established from lean, obese, and type 2 diabetic (T2D) subjects. Methods: Myotube cultures were established from lean and obese control subjects, and obese T2D subjects and treated with soluble FA1 for 4 days supplemented with/without palmitate (PA). Lipid- and glucose metabolism were studied with labeled precursors while quantitative expression of genes was analyzed using real-time PCR. Results: Diabetic myotubes express significantly reduced insulin stimulated glucose metabolism compared to lean myotubes and a significantly decreased basal PA oxidation. Chronic FA1 exposure did not affect the intermediary metabolism in myotubes. Insulin sensitivity of glucose and lipid metabolism was not affected by chronic FA1 exposure in myotubes established from lean, obese, and T2D subjects. Instead, chronic FA1 exposure induced pro-inflammatory cytokines expression (IL-6 and CCL2) in association with reducing adipogenic markers (ADD1, AP2, CD36, and PPARg2) in myotubes. Consistent with this observation, addition of FA1 to cultured myotubes was show to significantly inhibit their differentiation into adipocyte. Conclusion: Our results exclude direct effects of FA1 on glucose and lipid metabolism in cultured myotubes established from lean, obese, and T2D subjects. Therefore, the pathogenesis of FA1-induced IR might mainly be mediated via the FA1-induced stimulation of pro-inflammatory cytokines, which on turn inhibit adipogenesis in human myotubes.

Context Polycystic ovary syndrome (PCOS) is the most common endocrine disease among premenopausal women. A recent study found association between three single nucleotide polymorphisms (SNPs) and PCOS in a cohort of Han Chinese women. Objective To investigate the association between rs13405728 (LHCGR gene), rs13429458 (THADA gene) and rs2479106 (DENND1A gene), PCOS, hirsutism and metabolic and hormonal parameters in a well characterized cohort of Caucasian patients of Danish descendant with PCOS or hirsutism. Study design Patients underwent clinical examination, hormone analyses, oral glucose tolerance test and transvaginal ultrasound. Genetic variation was tested using allelic discrimination by real-time PCR. Patients 268 patients referred to The Department of Endocrinology, Odense University Hospital, Denmark with PCOS or hirsutism between 1997 and 2011. Two hundred and forty-eight healthy females were included as controls. Results Genotype distributions and allele frequencies of rs13405728, rs13429458, and rs2479106 were comparable in patients and controls. The rs2479106 G allele was associated with a decreased PCOS susceptibility. None of the SNPs were associated with hirsutism or increased metabolic parameters. Conclusions The rs2479106 G allele was associated with decreased PCOS susceptibility, thus confirming previously reported findings of association between rs2479106 and PCOS. Metabolic and hormonal parameters were comparable between genotypes of rs13405728 and rs2479106.

Objective To determine fasting and insulin-stimulated levels of carnitine precursors, total and free carnitine, and acylcarnitines, and evaluate the impact of pioglitazone treatment in obese patients with polycystic ovary syndrome (PCOS). Design The present study is a secondary analysis of a previously published case-control study, followed by a double-blind randomized clinical trial. Setting Academic tertiary care medical center. Patient(s) Thirty obese premenopausal patients with PCOS and 14 healthy women. Intervention(s) Sixteen weeks of blinded treatment with pioglitazone (30 mg/d) or placebo. Main Outcome Measure(s) Total and free carnitine and acylcarnitines. Result(s) Contrary to controls, patients with PCOS were characterized with slightly lower levels of fasting total and free carnitine, its precursors, and derivatives. Total and free carnitine correlated inversely to sex hormone-binding globulin (SHBG) in patients with PCOS, whereas no associations were found between acylcarnitines and androgenes. Insulin stimulation-induced changes in the levels of total and free carnitine, carnitine precursors, and acylcarnitines in the PCOS group followed the same trends as in the control group. Pioglitazone treatment significantly increased fasting levels of serum-free carnitine, propionyl carnitine, and total carnitine. The analysis of between group differences revealed significant changes in the isovaleryl carnitine levels and lipid oxidation rates after pioglitazone treatment compared with placebo. Conclusion(s) Acute insulin stimulation was associated with increased serum levels of free carnitine in both patients and healthy controls. Treatment with pioglitazone is able to redistribute free fatty acids from insulin-sensitive tissues, diminish demand for carnitine, and influence the overall carnitine turnover. Clinical Trial Registration Number NCT00145340.

Development of insulin resistance is positively associated with dietary saturated fatty acids and negatively associated with monounsaturated fatty acids. To clarify aspects of this difference we have compared the metabolism of oleic (OA, monounsaturated) and palmitic acids (PA, saturated) in human myotubes. Human myotubes were treated with 100muM OA or PA and the metabolism of [(14)C]-labeled fatty acid was studied. We observed that PA had a lower lipolysis rate than OA, despite a more than two-fold higher protein level of adipose triglyceride lipase after 24h incubation with PA. PA was less incorporated into triacylglycerol and more incorporated into phospholipids after 24h. Supporting this, incubation with compounds modifying lipolysis and reesterification pathways suggested a less influenced PA than OA metabolism. In addition, PA showed a lower accumulation than OA, though PA was oxidized to a relatively higher extent than OA. Gene set enrichment analysis revealed that 24h of PA treatment upregulated lipogenesis and fatty acid beta-oxidation and downregulated oxidative phosphorylation compared to OA. The differences in lipid accumulation and lipolysis between OA and PA were eliminated in combination with eicosapentaenoic acid (polyunsaturated fatty acid). In conclusion, this study reveals that the two most abundant fatty acids in our diet are partitioned toward different metabolic pathways in muscle cells, and this may be relevant to understand the link between dietary fat and skeletal muscle insulin resistance.

Our current knowledge on substrate oxidation in skeletal muscle in relation to insulin resistance and type 2 diabetes (T2D) originate mainly from in vivo studies. The oxidative capacity of skeletal muscle is highly influenced by physical activity, ageing, hormonal status, and fiber type composition, rendering it difficult to determine the contribution of heritable factors to the alteration in oxidative metabolism. Cultured human myotubes offer a unique model to distinguish between primary and environmental factors in the etiology of insulin resistance. The objective of this review is to summarise our studies on substrate oxidation in human myotubes established from lean, obese and T2D subjects, especially focusing on whether the increased respiratory quotient seen in T2D subjects is based on primary traits and whether changes in substrate oxidation may have a common explanation. Obtained results add further regulatory mechanism to our understanding of substrate oxidation in human skeletal muscle during normo- an pathophysiological conditions, focusing especially on the governing influence of a primary reduced TCA flux for the diabetic phenotype in skeletal muscle.

The free radical theory of aging says that increased oxidative stress and mitochondrial dysfunction are associated with old age. In the present study we have investigated the effects of cellular senescence on muscle energetic by comparing mitochondrial content and function in cultured muscle satellite cells at early and late passage numbers. We show that cultured muscle satellite cells undergoing senescence express a reduced mitochondrial mass, decreased whole cell ATP level, normal to increased mitochondrial ATP production under ATP utilization, increased mitochondrial membrane potential and increased superoxide/mitochondrial mass and hydrogen peroxide/mitochondrial mass ratios. Moreover, the increased ROS production correlates with the corresponding mitochondrial ATP production. Thus, myotubes differentiated from human myoblasts undergoing senescence have a reduced mitochondrial content, but the existent mitochondria express normal to increased functional capabilities. The present data suggest that the origin of aging lies outside the mitochondria and that a malfunction in the cell might be preceding and initiating the increase of mitochondrial ATP synthesis and concomitant ROS production in the single mitochondrion in response to decreased mitochondrial mass and reduced extra-mitochondrial energy supply. This then can lead to the increased damage of DNA, lipids and proteins of the mitochondria as postulated by the free radical theory of aging.