Insulin and exercise differentially regulate PI3-kinase and glycogen synthase in human skeletal muscle.

The purpose of this study was to determine the separate and combined effects of exercise and insulin on the activation of phosphatidylinositol 3-kinase (PI3-kinase) and glycogen synthase in human skeletal muscle in vivo. Seven healthy men performed three trials in random order. The trials included 1) ingestion of 2 g/kg body wt carbohydrate in a 10% solution (CHO); 2) 75 min of semirecumbent cycling exercise at 75% of peak O(2) consumption; followed by 5 x 1-min maximal sprints (Ex); and 3) Ex, immediately followed by ingestion of the carbohydrate solution (ExCHO). Plasma glucose and insulin were increased (P < 0.05) at 15 and 30 (Post-15 and Post-30) min after the trial during CHO and ExCHO, although insulin was lower for ExCHO. Hyperinsulinemia during recovery in CHO and ExCHO led to an increase (P < 0.001) in PI3-kinase activity at Post-30 compared with basal, although the increase was lower (P < 0. 004) for ExCHO. Furthermore, PI3-kinase activity was suppressed (P < 0.02) immediately after exercise (Post-0) during Ex and ExCHO. Area under the insulin response curve for all trials was positively associated with PI3-kinase activity (r = 0.66, P < 0.001). Glycogen synthase activity did not increase during CHO but was increased (P < 0.05) at Post-0 and Post-30 during Ex and ExCHO. Ingestion of the drink increased (P < 0.05) carbohydrate oxidation during CHO and ExCHO, although the increase after ExCHO was lower (P < 0.05) than CHO. Carbohydrate oxidation was directly correlated with PI3-kinase activity for all trials (r = 0.63, P < 0.001). In conclusion, under resting conditions, ingestion of a carbohydrate solution led to activation of the PI3-kinase pathway and oxidation of the carbohydrate. However, when carbohydrate was ingested after intense exercise, the PI3-kinase response was attenuated and glycogen synthase activity was augmented, thus facilitating nonoxidative metabolism or storage of the carbohydrate. Activation of glycogen synthase was independent of PI3-kinase.     

Impaired substrate oxidation in healthy elderly men after eccentric exercise.

The metabolic response to eccentric exercise in healthy older adults is unknown. Therefore, substrate metabolism was examined in the basal state and after sustained hyperglycemia (180 min, 10 mM) in eight healthy, sedentary older [66 +/- 2 yr; body mass index (BMI) of 25.5 +/- 1.2 kg/m] and nine younger (23 +/- 1 yr; BMI of 23.6 +/- 1.7 kg/m) men, under control conditions and 48 h after eccentric exercise. Indirect calorimetry was performed to evaluate carbohydrate and lipid oxidation (C(ox) and L(ox), respectively). Eccentric exercise caused muscle soreness and increased plasma creatine kinase in both groups of men (P < 0.02). Although a similar level of hyperglycemia was maintained in the two groups, glucose infusion rates were lower (P < 0.001) in the older men. Compared with basal levels, hyperglycemia stimulated an increase in C(ox) and a decrease in L(ox) during the control and exercise trials in the younger group (P < 0.03), but only during the control trial in the older subjects (P < 0.007). C(ox) was unchanged after eccentric exercise in the younger men [4.00 +/- 0.30 vs. 3.54 +/- 0.44 mg x kg fat-free mass (FFM)(-1) x min(-1); exercise vs. control] but was suppressed by 20% in the older group (3.37 +/- 0.37 vs. 4.21 +/- 0.23 mg x kg FFM(-1) x min(-1); P < 0.04). Moreover, L(ox) was reduced by 38% in the younger subjects (0.47 +/- 0.09 vs. 0.76 +/- 0.10 mg x kg FFM(-1) x min(-1); P< 0.03) but was augmented by 89% in the older group (0.68 +/- 0.11 vs. 0.36 +/- 0.08 mg x kg FFM(-1) x min(-1); P < 0.04). In addition, hyperglycemia-stimulated C(ox), L(ox), and respiratory exchange ratio responses to eccentric exercise were related to abdominal adiposity (r = -0.57, P < 0.04, r = 0.68, P < 0.02 and r = -0.60, P < 0.02, respectively). Despite normal glucose tolerance and the absence of obesity per se, older men experience a reduction in carbohydrate oxidation in response to hyperglycemia after eccentric exercise.     

Proof-of-concept human laboratory study for protracted abstinence in alcohol dependence: effects of gabapentin

There is a need for safe medications that can effectively support recovery by treating symptoms of protracted abstinence that may precipitate relapse in alcoholics, e.g. craving and disturbances in sleep and mood. This proof-of-concept study reports on the effectiveness of gabapentin 1200 mg for attenuating these symptoms in a non-treatment-seeking sample of cue-reactive, alcohol-dependent individuals. Subjects were 33 paid volunteers with current Diagnostic and Statistical Manual of Mental Disorders-IV alcohol dependence and a strength of craving rating 1 SD or greater for alcohol than water cues. Subjects were randomly assigned to gabapentin or placebo for 1 week and then participated in a within-subjects trial where each was exposed to standardized sets of pleasant, neutral and unpleasant visual stimuli followed by alcohol or water cues. Gabapentin was associated with significantly greater reductions than placebo on several measures of subjective craving for alcohol as well as for affectively evoked craving. Gabapentin was also associated with significant improvement on several measures of sleep quality. Side effects were minimal, and gabapentin effects were not found to resemble any major classes of abused drugs. Results suggest that gabapentin may be effective for treating the protracted abstinence phase in alcohol dependence and that a randomized clinical trial would be an appropriate next step. The study also suggests the value of cue-reactivity studies as proof-of-concept screens for potential antirelapse drugs.     

Identification of a Unique “Stability Control Region” that Controls Protein Stability of Tropomyosin: A Two-stranded α-Helical Coiled-coil

Nine recombinant chicken skeletal α-tropomyosin proteins were prepared, eight C-terminal deletion constructs and the full length protein (1-81, 1-92, 1-99, 1-105, 1-110, 1-119, 1-131, 1-260 and 1-284) and characterized by circular dichroism spectroscopy and analytical ultracentrifugation. We identified for the first time, a stability control region between residues 97 and 118. Fragments of tropomyosin lacking this region (1-81, 1-92, and 1-99) still fold into two-stranded α-helical coiled-coils but are significantly less stable (T_m between 26-28.5 °C) than longer fragments containing this region (1-119, 1-131, 1-260 and 1-284) which show a large increase in their thermal midpoints (T_m 40-43 °C) for a ΔT_m of 16-18 °C between 1-99 and 1-119. We further investigated two additional fragments that ended between residues 99 and 119, that is fragments 1-105 and 1-110. These fragments were more stable than 1-99 and less stable than 1-119, and showed that there were three separate sites that synergistically contribute to the large jump in protein stability (electrostatic clusters 97-104 and 112-118, and a hydrophobic interaction from Leu 110). All the residues involved in these stabilizing interactions are located outside the hydrophobic core a and d positions that have been shown to be the major contributor to coiled-coil stability. Our results show clearly that protein stability is more complex than previously thought and unique sites can synergistically control protein stability over long distances.     

SEPALLATA3: the 'glue' for MADS box transcription factor complex formation

Background  

Plant MADS box proteins play important roles in a plethora of developmental processes. In order to regulate specific sets of target genes, MADS box proteins dimerize and are thought to assemble into multimeric complexes. In this study a large-scale yeast three-hybrid screen is utilized to provide insight into the higher-order complex formation capacity of the Arabidopsis MADS box family. SEPALLATA3 (SEP3) has been shown to mediate complex formation and, therefore, special attention is paid to this factor in this study.     

Transmission of Stability Information through the N-domain of Tropomyosin Is Interrupted by a Stabilizing Mutation (A109L) in the Hydrophobic Core of the Stability Control Region (Residues 97–118)

Tropomyosin (Tm) is an actin-binding, thin filament, two-stranded α-helical coiled-coil critical for muscle contraction and cytoskeletal function. We made the first identification of a stability control region (SCR), residues 97–118, in the Tm sequence that controls overall protein stability but is not required for folding. We also showed that the individual α-helical strands of the coiled-coil are stabilized by Leu-110, whereas the hydrophobic core is destabilized in the SCR by Ala residues at three consecutive d positions. Our hypothesis is that the stabilization of the individual α-helices provides an optimum stability and allows functionally beneficial dynamic motion between the α-helices that is critical for the transmission of stabilizing information along the coiled-coil from the SCR. We prepared three recombinant (rat) Tm(1–131) proteins, including the wild type sequence, a destabilizing mutation L110A, and a stabilizing mutation A109L. These proteins were evaluated by circular dichroism (CD) and differential scanning calorimetry. The single mutation L110A destabilizes the entire Tm(1–131) molecule, showing that the effect of this mutation is transmitted 165 Å along the coiled-coil in the N-terminal direction. The single mutation A109L prevents the SCR from transmitting stabilizing information and separates the coiled-coil into two domains, one that is ∼9 °C more stable than wild type and one that is ∼16 °C less stable. We know of no other example of the substitution of a stabilizing Leu residue in a coiled-coil hydrophobic core position d that causes this dramatic effect. We demonstrate the importance of the SCR in controlling and transmitting the stability signal along this rodlike molecule.     

Prevalence of neutralising antibodies against SARS-CoV-2 in acute infection and convalescence: A systematic review and meta-analysis

BackgroundIndividuals infected with SARS-CoV-2 develop neutralising antibodies. We investigated the proportion of individuals with SARS-CoV-2 neutralising antibodies after infection and how this proportion varies with selected covariates.Methodology/Principal findingsThis systematic review and meta-analysis examined the proportion of individuals with SARS-CoV-2 neutralising antibodies after infection and how these proportions vary with selected covariates. Three models using the maximum likelihood method assessed these proportions by study group, covariates and individually extracted data (protocol CRD42020208913). A total of 983 reports were identified and 27 were included. The pooled (95%CI) proportion of individuals with neutralising antibodies was 85.3% (83.5–86.9) using the titre cut off >1:20 and 83.9% (82.2–85.6), 70.2% (68.1–72.5) and 54.2% (52.0–56.5) with titres >1:40, >1:80 and >1:160, respectively. These proportions were higher among patients with severe COVID-19 (e.g., titres >1:80, 84.8% [80.0–89.2], >1:160, 74.4% [67.5–79.7]) than those with mild presentation (56.7% [49.9–62.9] and 44.1% [37.3–50.6], respectively) and lowest among asymptomatic infections (28.6% [17.9–39.2] and 10.0% [3.7–20.1], respectively). IgG and neutralising antibody levels correlated poorly.Conclusions/Significance85% of individuals with proven SARS-CoV-2 infection had detectable neutralising antibodies. This proportion varied with disease severity, study setting, time since infection and the method used to measure antibodies.     

SorCS2 Controls Functional Expression of Amino Acid Transporter EAAT3 and Protects Neurons from Oxidative Stress and Epilepsy-Induced Pathology

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