Role of ataxia telangiectasia mutated in insulin signalling of muscle-derived cell lines and mouse soleus.

AIM: Ataxia telangiectasia mutated (ATM) reportedly plays a role in insulin-stimulated activation of Akt in some cell types but not in others. The role of ATM in insulin signalling has not been firmly resolved for skeletal muscle cells, for which Akt phosphorylation is a pivotal step in stimulation of glucose transport. Accordingly, our aim was to determine the role of ATM in insulin effects for cell lines derived from skeletal muscle and for skeletal muscle. METHODS: We examined insulin effects in L6 myotubes, mouse soleus, C2C12 myotubes and differentiated rhabdomyosarcoma (RD) cells in the presence and absence of a low concentration (1 microm) of the ATM inhibitor KU55933. We also compared insulin signalling in C2C12 cells expressing shRNA against ATM and control cell lines (empty vector; cells expressing non-targeting shRNA). RESULTS: In L6 myotubes and mouse soleus muscle, KU55933 inhibited insulin-stimulated phosphorylation of the 160 kDa substrate of Akt (AS160) despite no effect on Akt. In contrast, KU55933 prevented insulin-stimulated Akt phosphorylation in C2C12 myotubes. Furthermore, C2C12 myotubes expressing shRNA against ATM displayed reduced insulin-stimulated Akt phosphorylation compared to controls. KU55933 also decreased insulin-stimulated Akt phosphorylation in differentiated RD cells. CONCLUSION: These model-dependent differences in the role of ATM in insulin action demonstrate a role of ATM in insulin-stimulated phosphorylation of Akt (in C2C12 and RD cells) but also allow the elucidation of a novel, Akt-independent role of ATM (in L6 myotubes and mouse soleus, at the level of AS160) in insulin signalling.

The relative sensitivity of sperm, eggs and embryos to copper in the blue mussel (Mytilus trossulus).

Copper, an essential element, is toxic at elevated concentrations, and as a result of anthropogenic activities is becoming increasingly prevalent in marine environments. In this study, we examined the effects of copper on early life stages of the blue mussel, Mytilus trossulus. We assessed the impacts of increasing copper concentrations on embryo development, egg viability, sperm fertilization capacity and, in particular, on sperm swimming speed using computer-assisted sperm analysis. Sensitivity to copper followed the pattern: embryos > sperm > eggs. A dramatic increase in abnormal embryo development was observed following exposure to copper concentrations exceeding 10 microg/L. Sperm swimming speeds decreased significantly when exposed to 100 microg/L of copper, but lower doses did not influence sperm swimming speed. Copper exposure (at any tested concentration) did not affect sperm flagellum length, or alter egg viability. Based on our results, we suggest that exposure of sperm to copper may interfere with mitochondrial activity, which reduces sperm swimming speed during the extended duration of sperm motility in blue mussel.

Branchial cadmium and copper binding and intestinal cadmium uptake in wild yellow perch (Perca flavescens) from clean and metal-contaminated lakes.

Branchial binding kinetics and gastro-intestinal uptake of copper and cadmium where examined in yellow perch (Perca flavescens) from a metal-contaminated lake (Hannah Lake, Sudbury, Ontario, Canada) and an uncontaminated lake (James Lake, North Bay, Ontario, Canada). An in vivo approach was taken for gill binding comparisons while an in vitro gut binding assay was employed for gastro-intestinal tract (GIT) uptake analysis. By investigating metal uptake at the gill and the gut we cover the two main routes of metal entry into fish. Comparisons of water and sediment chemistries, metal burdens in benthic invertebrate, and metal burdens in the livers of perch from the two study lakes clearly show that yellow perch from Hannah L. are chronically exposed to a highly metal-contaminated environment compared to a reference lake. We found that metal-contaminated yellow perch showed no significant difference in gill Cd binding compared to reference fish, but they did show significant decreases in new Cd binding and absorption in their GITs. The results show that gill Cd binding may involve low-capacity, high-affinity binding sites, while gastro-intestinal Cd uptake involves binding sites that are high-capacity, low-affinity. From this we infer that Cd may be more critically controlled at the gut rather than gills. Significant differences in branchial Cu binding (increased binding) were observed in metal-contaminated yellow perch. We suggest that chronic waterborne exposure to Cu (and/or other metals) may be the dominant influence in gill Cu binding rather than chronic exposure to high Cu diets. We give supporting evidence that Cd is taken up in the GIT, at least in part, by a similar pathway as Ca(2+), principally that elevated dietary Ca(2+) reduces Cd binding and uptake. Overall our study reveals that metal pre-exposure via water and diet can alter uptake kinetics of Cu and Cd at the gill and/or the gut.