Insulin promotes glycogen synthesis in the absence of GSK3 phosphorylation in skeletal muscle

Insulin promotes dephosphorylation and activation of glycogen synthase (GS) by inactivating glycogen synthase kinase (GSK) 3 through phosphorylation. Insulin also promotes glucose uptake and glucose 6-phosphate (G-6-P) production, which allosterically activates GS. The relative importance of these two regulatory mechanisms in the activation of GS in vivo is unknown. The aim of this study was to investigate if dephosphorylation of GS mediated via GSK3 is required for normal glycogen synthesis in skeletal muscle with insulin. We employed GSK3 knockin mice in which wild-type GSK3 alpha and -beta genes are replaced with mutant forms (GSK3 alpha/beta S21A/S21A/S9A/S9A), which are nonresponsive to insulin. Although insulin failed to promote dephosphorylation and activation of GS in GSK3 alpha/beta S21A/S21A/S9A/S9A mice, glycogen content in different muscles from these mice was similar compared with wild-type mice. Basal and epinephrine-stimulated activity of muscle glycogen phosphorylase was comparable between wild-type and GSK3 knockin mice. Incubation of isolated soleus muscle in Krebs buffer containing 5.5 mM glucose in the presence or absence of insulin revealed that the levels of G-6-P, the rate of [14C]glucose incorporation into glycogen, and an increase in total glycogen content were similar between wild-type and GSK3 knockin mice. Injection of glucose containing 2-deoxy-[3H]glucose and [14C]glucose also resulted in similar rates of muscle glucose uptake and glycogen synthesis in vivo between wild-type and GSK3 knockin mice. These results suggest that insulin-mediated inhibition of GSK3 is not a rate-limiting step in muscle glycogen synthesis in mice. This suggests that allosteric regulation of GS by G-6-P may play a key role in insulin-stimulated muscle glycogen synthesis in vivo.     

Oxidative stress stimulates skeletal muscle glucose uptake through a phosphatidylinositol 3-kinase-dependent pathway

We determined the acute effects of oxidative stress on glucose uptake and intracellular signaling in skeletal muscle by incubating muscles with reactive oxygen species (ROS). Xanthine oxidase (XO) is a superoxide-generating enzyme that increases ROS. Exposure of isolated rat extensor digitorum longus (EDL) muscles to Hx/XO (Hx/XO) for 20 min resulted in a dose-dependent increase in glucose uptake. To determine whether the mechanism leading to Hx/XO-stimulated glucose uptake is associated with the production of H2O2, EDL muscles from rats were preincubated with the H2O2 scavenger catalase or the superoxide scavenger superoxide dismutase (SOD) prior to incubation with Hx/XO. Catalase treatment, but not SOD, completely inhibited the increase in Hx/XO-stimulated 2-deoxyglucose (2-DG) uptake, suggesting that H2O2 is an intermediary leading to Hx/XO-stimulated glucose uptake with incubation. Direct H2O2 also resulted in a dose-dependent increase in 2-DG uptake in isolated EDL muscles, and the maximal increase was threefold over basal levels at a concentration of 600 micromol/l H2O2. H2O2-stimulated 2-DG uptake was completely inhibited by the phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin, but not the nitric oxide inhibitor NG-monomethyl-l-arginine. H2O2 stimulated the phosphorylation of Akt Ser473 (7-fold) and Thr308 (2-fold) in isolated EDL muscles. H2O2 at 600 micromol/l had no effect on ATP concentrations and did not increase the activities of either the alpha1 or alpha2 catalytic isoforms of AMP-activated protein kinase. These results demonstrate that acute exposure of muscle to ROS is a potent stimulator of skeletal muscle glucose uptake and that this occurs through a PI3K-dependent mechanism.     

Discovery of TBC1D1 as an insulin-, AICAR-, and contraction-stimulated signaling nexus in mouse skeletal muscle

The Akt substrate of 160 kDa (AS160) is phosphorylated on Akt substrate (PAS) motifs in response to insulin and contraction in skeletal muscle, regulating glucose uptake. Here we discovered a dissociation between AS160 protein expression and apparent AS160 PAS phosphorylation among soleus, tibialis anterior, and extensor digitorum longus muscles. Immunodepletion of AS160 in tibialis anterior muscle lysates resulted in minimal depletion of the PAS band at 160 kDa, suggesting the presence of an additional PAS immunoreactive protein. By immunoprecipitation and mass spectrometry, we identified this protein as the AS160 paralog TBC1D1, an obesity candidate gene regulating GLUT4 translocation in adipocytes. TBC1D1 expression was severalfold higher in skeletal muscles compared with all other tissues and was the dominant protein detected by the anti-PAS antibody at 160 kDa in tibialis anterior and extensor digitorum longus but not soleus muscles. In vivo stimulation by insulin, contraction, and the AMP-activated protein kinase (AMPK) activator AICAR increased TBC1D1 PAS phosphorylation. Using mass spectrometry on TBC1D1 from mouse skeletal muscle, we identified several novel phosphorylation sites on TBC1D1 and found the majority were consensus or near consensus sites for AMPK. Semiquantitative analysis of spectra suggested that AICAR caused greater overall phosphorylation of TBC1D1 sites compared with insulin. Purified Akt and AMPK phosphorylated TBC1D1 in vitro, and AMPK, but not Akt, reduced TBC1D1 electrophoretic mobility. TBC1D1 is a major PAS immunoreactive protein in skeletal muscle that is phosphorylated in vivo by insulin, AICAR, and contraction. Both Akt and AMPK phosphorylate TBC1D1, but AMPK may be the more robust regulator.     

Disconnecting Mitochondrial Content from Respiratory Chain Capacity in PGC-1-Deficient Skeletal Muscle

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Gene flow in environmental Legionella pneumophila leads to genetic and pathogenic heterogeneity within a Legionnaires’ disease outbreak

Background

Legionnaires’ disease is a severe form of pneumonia caused by the environmental bacterium Legionella pneumophila. Outbreaks commonly affect people with known risk factors, but the genetic and pathogenic complexity of L. pneumophila within an outbreak is not well understood. Here, we investigate the etiology of the major Legionnaires’ disease outbreak that occurred in Edinburgh, UK, in 2012, by examining the evolutionary history, genome content, and virulence of L. pneumophila clinical isolates.

Results

Our high resolution genomic approach reveals that the outbreak was caused by multiple genetic subtypes of L. pneumophila, the majority of which had diversified from a single progenitor through mutation, recombination, and horizontal gene transfer within an environmental reservoir prior to release. In addition, we discover that some patients were infected with multiple L. pneumophila subtypes, a finding which can affect the certainty of source attribution. Importantly, variation in the complement of type IV secretion systems encoded by different genetic subtypes correlates with virulence in a Galleria mellonella model of infection, revealing variation in pathogenic potential among the outbreak source population of L. pneumophila.

Conclusions

Taken together, our study indicates previously cryptic levels of pathogen heterogeneity within a Legionnaires’ disease outbreak, a discovery that impacts on source attribution for future outbreak investigations. Furthermore, our data suggest that in addition to host immune status, pathogen diversity may be an important influence on the clinical outcome of individual outbreak infections.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-014-0504-1) contains supplementary material, which is available to authorized users.     

Repeated bouts of eccentrically biased endurance exercise stimulate salivary IgA secretion rate

To determine the salivary secretory immunoglobulin A (sIgA) response to repeated bouts of unaccustomed, downhill running (eccentrically biased) and examine potential protective immunological adaption from a repeated bout effect. Eleven active but untrained males (age: 19.7±0.4 years; VO_2peak: 47.8± 3.6 ml · kg⁻¹ · min ⁻¹) performed two 60 min bouts (Run 1 and Run 2) of downhill running (−13.5% gradient), separated by 14 days, at a speed eliciting 75% of their VO_2peak on a level grade. Saliva samples were collected before (baseline), immediately post exercise (IPE), and every hour for 12 h and every 24 h for 6 days after each run. Salivary sIgA concentration was measured and sIgA secretion rate was calculated. Results were analysed using repeated measures ANOVA (12 h period: 2x14; 24 h intervals: 2x7; p ≤ 0.05) with Tukey post-hoc tests where appropriate. Results are reported as means ± SE. There was a significant (p < 0.0001) interaction effect for sIgA secretion rate, IPE, with higher values after Run 2, as well as a significant (p < 0.01) time effect with elevated levels IPE and between 24 h and 144 h. There was a run effect (p < 0.0001), with the sIgA secretion rate significantly higher after Run 2. Repeated bouts of unaccustomed, eccentrically biased exercise induced alterations in the salivary sIgA secretion rate. This may serve as a protective mucosal adaptation to exercise-induced tissue damage.     

Dominant responses with conservation of T-cell receptor usage in the CD8+ T-cell recognition of a cancer testis antigen peptide presented through HLA-Cw7 in patients with multiple myeloma

Cancer testis antigens exhibit physiological expression within germ cells and are frequently expressed in malignant tissue. Interestingly, immunological tolerance to cancer testis proteins does not appear to be established, and the expression of CTAg proteins within malignant cells can therefore lead to induction of cellular and humoral immunity. A considerable body of evidence now indicates that CD8-specific immunity plays an important role in the control of cancer cell growth, and a number of vaccine studies are in progress to boost CTAg-specific cellular immune responses. We have previously identified CTAg-specific immune responses in patients with multiple myeloma and reported that recognition of the MAGE-A1_289–298 peptide, which is described as being restricted by HLA-B*0702, was the most frequent response seen with our peptide panel. Here, we studied seven CD8+ T-cell clones specific for this peptide which were isolated from three patients with myeloma at several time-points. The affinity of peptide recognition was high with 50% maximal interferon-γ production observed at a peptide concentration of 10⁻¹⁰ M and variation of only one order of magnitude between the affinities of the clones. Importantly, all the clones were able to recognise and kill multiple myeloma cell lines. Interestingly, one patient did not express HLA-B*0702, but three clones from this patient recognised the MAGE-A1_289–298 peptide on a lymphoblastoid cell line (LCLs) expressing HLA-Cw7, and we now show evidence that the MAGE-A1_289–298 peptide is expressed and recognised through Cw7. The T-cell receptor gene usage was determined in five clones and showed conserved features in both the α and the β chain genes indicating correlation between T-cell receptor usage and peptide specificity of cancer testis antigen-specific T-cell clones.