No evidence of direct association between GLUT4 and glycogen in human skeletal muscle

Previous studies have demonstrated that exercise increases whole body and skeletal muscle insulin sensitivity that is linked with increased GLUT4 at the plasma membrane following insulin stimulation and associated with muscle glycogen depletion. To assess the potential direct association between muscle glycogen and GLUT4, seven untrained, male subjects exercised for 60 min at ~75% VO2 peak, with muscle samples obtained by percutaneous needle biopsy immediately before and after exercise. Exercise reduced muscle glycogen content by ~43%. An ultracentrifugation protocol resulted in a ~2-3-fold enriched glycogen fraction from muscle samples for analysis. Total GLUT4 content was unaltered by exercise and we were unable to detect any GLUT4 in glycogen fractions, either with or without amylase treatment. In skinned muscle fiber segments, there was very little, if any, GLUT4 detected in wash solutions, except following exposure to 1% Triton X-100. Amylase treatment of single fibers did not increase GLUT4 in the wash solution and there were no differences in GLUT4 content between fibers obtained before or after exercise for any of the wash treatments. Our results indicate no direct association between GLUT4 and glycogen in human skeletal muscle, before or after exercise, and suggest that alterations in GLUT4 translocation associated with exercise-induced muscle glycogen depletion are mediated via other mechanisms

Exercise and GLUT4 in human subcutaneous adipose tissue

To examine the effect of acute and chronic exercise on adipose tissue GLUT4 expression, a total of 20 healthy, male subjects performed one of two studies. Ten subjects performed cycle ergometer exercise for 60 min at 73 ± 2% VO2 peak and abdominal adipose tissue samples were obtained immediately before and after exercise and after 3 h of recovery. Another 10 subjects completed 10 days of exercise training, comprising a combination of six sessions of 60 min at 75% VO2 peak and four sessions of 6 × 5 min at 90% VO2 peak, separated by 3 min at 40% VO2 peak. Abdominal adipose tissue and vastus lateralis muscle samples were obtained before training and 24 h after the last training session. A single bout of exercise did not change adipose tissue GLUT4 mRNA; however, there was a small, but significant, reduction in adipose tissue GLUT4 protein expression 3 h after exercise. There were no changes in adipose tissue GLUT4 or COX‐IV expression following exercise training. In contrast, skeletal muscle GLUT4 and COX‐IV were increased by 47% and 44%, respectively following exercise training. The exercise training‐induced increase in GLUT4 expression was similar in both type I and type IIa single muscle fibers. Our results indicate that neither a single exercise bout, nor 10 days of exercise training, increased adipose tissue GLUT4, in contrast with the increases observed in skeletal muscle GLUT4 expression

PL - 041 Effects of power resistance exercise and feeding on the expression of putative mechanosensing proteins in skeletal muscle of resistance-trained men

Objective Power resistance exercise involves high intensity (load and velocity) dynamic muscular contractions and is frequently performed by athletes to enhance performance via improved muscle function. To investigate the remodelling processes that contribute to improved muscle function, we investigated the expression of putative mechanosensing genes implicated in this process (Kojic et al., 2011): titin-linked Muscle Ankyrin Repeat Protein (MARPs) family CARP, Ankrd 2 and DARP, and the Z-disc associated muscle-LIM protein (MLP) in healthy, resistance-trained men (n = 7) following 90 min of rest (Rest) or power resistance exercise, with (Ex + Meal) or without (Ex only) feeding during recovery. Methods Percutaneous needle biopsy samples were obtained from the vastus lateralis of resistance-trained males using local anesthetic (2% Xylocaine), 3 h after performing each of the three experimental trials on separate days. Previously, we presented results from this study showing that the mRNA levels of CARP (~15-fold) and MLP (~2.5-fold) were upregulated in human skeletal muscle 3 h post power resistance exercise (Wette et al., 2012). Based on these results, we performed protein analyses on the same muscle samples to determine the protein levels of all MARPs and MLP in whole muscle homogenates after Rest, Ex only and Ex + Meal. To assess whether the exercise elicited a stress response in these resistance-trained individuals, the level of phosphorylated heat shock protein 27 at serine 15 (pHSP27-Ser15) was measured at Rest and 3 h after Ex only and Ex + Meal. The levels of pHSP27-Ser15 are typically upregulated 3 h after eccentric exercise in human skeletal muscle (Frankenberg et al., 2014). Results The 90 min exercise session consisted of 180 intermittent muscular contractions at high intensity (70-96% maximal strength). Compared to Rest, there were ~5.8- and 12.6-fold increases in pHSP27-Ser15 levels at 3 h post Ex only and Ex + Meal (both P = 0.049, one-way ANOVA) respectively. CARP protein levels were elevated ~2.7-fold after Ex only (P = 0.049, one-way ANOVA) and ~7.6-fold after Ex + Meal (P = 0.326), due to markedly higher levels (6-40-fold) in three of the seven participants. Pearson correlation analysis revealed a significant positive correlation between the levels of pHSP27-Ser-15 and CARP protein (r = 0.56, P = 0.008). Ankrd 2, DARP and MLP protein levels were unchanged (all P > 0.05) following Ex only and Ex + Meal. Conclusions These findings indicate that CARP is highly responsive to increased mechanical loading because the protein levels in skeletal muscle can be substantially increased as early as 3 h after stressful resistance exercise. This suggests a specialised role for CARP protein during the early phases of muscle remodelling that occur as a consequence of performing high intensity resistance exercise

Exercise training improves long-term memory in obese mice

Obesity has been linked to a range of pathologies, including dementia. In contrast, regular physical activity is associated with the prevention or reduced progression of neurodegeneration. Specifically, physical activity can improve memory and spatial cognition, reduce age-related cognitive decline, and preserve brain volume, but the mechanisms are not fully understood. Accordingly, we investigated whether any detrimental effects of high-fat diet (HFD)-induced obesity on cognition, motor behavior, adult hippocampal neurogenesis, and brain-derived neurotrophic factor (BDNF) could be mitigated by voluntary exercise training in male C57Bl/6 mice. HFD-induced impairment of motor function was not reversed by exercise. Importantly, voluntary wheel running improved long-term memory and increased hippocampal neurogenesis, suggesting that regular physical activity may prevent cognitive decline in obesity

Prediction of Small for Gestational Age Infants in Healthy Nulliparous Women Using Clinical and Ultrasound Risk Factors Combined with Early Pregnancy Biomarkers

Objective Most small for gestational age pregnancies are unrecognised before birth, resulting in substantial avoidable perinatal mortality and morbidity. Our objective was to develop multivariable prediction models for small for gestational age combining clinical risk factors and biomarkers at 15±1 weeks’ with ultrasound parameters at 20±1 weeks’ gestation. Methods Data from 5606 participants in the Screening for Pregnancy Endpoints (SCOPE) cohort study were divided into Training (n = 3735) and Validation datasets (n = 1871). The primary outcomes were All-SGA (small for gestational age with birthweight <10th customised centile), Normotensive-SGA (small for gestational age with a normotensive mother) and Hypertensive-SGA (small for gestational age with an hypertensive mother). The comparison group comprised women without the respective small for gestational age phenotype. Multivariable analysis was performed using stepwise logistic regression beginning with clinical variables, and subsequent additions of biomarker and then ultrasound (biometry and Doppler) variables. Model performance was assessed in Training and Validation datasets by calculating area under the curve. Results 633 (11.2%) infants were All-SGA, 465(8.2%) Normotensive-SGA and 168 (3%) Hypertensive-SGA. Area under the curve (95% Confidence Intervals) for All-SGA using 15±1 weeks’ clinical variables, 15±1 weeks’ clinical+ biomarker variables and clinical + biomarkers + biometry /Doppler at 20±1 weeks’ were: 0.63 (0.59–0.67), 0.64 (0.60–0.68) and 0.69 (0.66–0.73) respectively in the Validation dataset; Normotensive-SGA results were similar: 0.61 (0.57–0.66), 0.61 (0.56–0.66) and 0.68 (0.64–0.73) with small increases in performance in the Training datasets. Area under the curve (95% Confidence Intervals) for Hypertensive-SGA were: 0.76 (0.70–0.82), 0.80 (0.75–0.86) and 0.84 (0.78–0.89) with minimal change in the Training datasets. Conclusion Models for prediction of small for gestational age, which combine biomarkers, clinical and ultrasound data from a cohort of low-risk nulliparous women achieved modest performance. Incorporation of biomarkers into the models resulted in no improvement in performance of prediction of All-SGA and Normotensive-SGA but a small improvement in prediction of Hypertensive-SGA. Our models currently have insufficient reliability for application in clinical practice however, they have potential utility in two-staged screening tests which include third trimester biomarkers and or fetal biometry

Store-Operated Ca2+ Entry (SOCE) and Purinergic Receptor-Mediated Ca2+ Homeostasis in Murine bv2 Microglia Cells: Early Cellular Responses to ATP-Mediated Microglia Activation

Microglia activation is a neuroinflammatory response to parenchymal damage with release of intracellular metabolites, e.g., purines, and signaling molecules from damaged cells. Extracellular purines can elicit Ca(2+)-mediated microglia activation involving P2X/P2Y receptors with metabotropic (P2Y) and ionotropic (P2X) cell signaling in target cells. Such microglia activation results in increased phagocytic activity, activation of their inflammasome and release of cytokines to sustain neuroinflammatory (so-called M1/M2 polarization). ATP-induced activation of ionotropic P2X4 and P2X7 receptors differentially induces receptor-operated Ca(2+) entry (ROCE). Although store-operated Ca(2+) entry (SOCE) was identified to modulate ROCE in primary microglia, its existence and role in one of the most common murine microglia cell line, BV2, is unknown. To dissect SOCE from ROCE in BV2 cells, we applied high-resolution multiphoton Ca(2+) imaging. After depleting internal Ca(2+) stores, SOCE was clearly detectable. High ATP concentrations (1 mM) elicited sustained increases in intracellular [Ca(2+)]i whereas lower concentrations (≤100 μM) also induced Ca(2+) oscillations. These differential responses were assigned to P2X7 and P2X4 activation, respectively. Pharmacologically inhibiting P2Y and P2X responses did not affect SOCE, and in fact, P2Y-responses were barely detectable in BV2 cells. STIM1S content was significantly upregulated by 1 mM ATP. As P2X-mediated Ca(2+) oscillations were rare events in single cells, we implemented a high-content screening approach that allows to record Ca(2+) signal patterns from a large number of individual cells at lower optical resolution. Using automated classifier analysis, several drugs (minocycline, U73122, U73343, wortmannin, LY294002, AZ10606120) were tested on their profile to act on Ca(2+) oscillations (P2X4) and sustained [Ca(2+)]i increases. We demonstrate specific drug effects on purinergic Ca(2+) pathways and provide new pharmacological insights into Ca(2+) oscillations in BV2 cells. For example, minocycline inhibits both P2X7- and P2X4-mediated Ca(2+)-responses, and this may explain its anti-inflammatory action in neuroinflammatory disease. As a technical result, our novel automated bio-screening approach provides a biomedical engineering platform to allow high-content drug library screens to study neuro-inflammation in vitro

Effects of a Cognitive Behavioral Therapy Intervention Trial to Improve Disease Outcomes in Children with Inflammatory Bowel Disease:

Studies testing the efficacy of behavioral interventions to modify psychosocial sequelae of IBD in children are limited. This report presents outcomes through a six month follow up from a large RCT testing the efficacy of a cognitive-behavioral intervention for children with IBD and their parents

Molecular Surveillance of True Nontypeable Haemophilus influenzae: An Evaluation of PCR Screening Assays

BackgroundUnambiguous identification of nontypeable Haemophilus influenzae (NTHi) is not possible by conventional microbiology. Molecular characterisation of phenotypically defined NTHi isolates suggests that up to 40% are Haemophilus haemolyticus (Hh); however, the genetic similarity of NTHi and Hh limits the power of simple molecular techniques such as PCR for species discrimination.Methodology/Principal FindingsHere we assess the ability of previously published and novel PCR-based assays to identify true NTHi. Sixty phenotypic NTHi isolates, classified by a dual 16S rRNA gene PCR algorithm as NTHi (n = 22), Hh (n = 27) or equivocal (n = 11), were further characterised by sequencing of the 16S rRNA and recA genes then interrogated by PCR-based assays targeting the omp P2, omp P6, lgtC, hpd, 16S rRNA, fucK and iga genes. The sequencing data and PCR results were used to define NTHi for this study. Two hpd real time PCR assays (hpd#1 and hpd#3) and the conventional iga PCR assay were equally efficient at differentiating study-defined NTHi from Hh, each with a receiver operator characteristic curve area of 0.90 [0.83; 0.98]. The hpd#1 and hpd#3 assays were completely specific against a panel of common respiratory bacteria, unlike the iga PCR, and the hpd#3 assay was able to detect below 10 copies per reaction.Conclusions/SignificanceOur data suggest an evolutionary continuum between NTHi and Hh and therefore no single gene target could completely differentiate NTHi from Hh. The hpd#3 real time PCR assay proved to be the superior method for discrimination of NTHi from closely related Haemophilus species with the added potential for quantification of H. influenzae directly from specimens. We suggest the hpd#3 assay would be suitable for routine NTHi surveillance and to assess the impact of antibiotics and vaccines, on H. influenzae carriage rates, carriage density, and disease