24In striated muscle, EPA and DHA have differential effects on the metabolism of glucose and 25 differential effects on the metabolism of protein. We have shown that, despite similar incorporation, 26 treatment of C 2 C 12 myotubes (CM) with EPA but not DHA improves glucose uptake and protein 27 accretion. We hypothesized that these differential effects of EPA and DHA may be due to divergent 28 shifts in lipidomic profiles leading to altered proteomic profiles. We therefore carried out an 29 assessment on the impact of treating CM with EPA and DHA on lipidomic and proteomic profiles. 30 FAME analysis revealed that both EPA and DHA led to similar but substantial changes in fatty acid 31 profiles with the exception of arachidonic acid, which was decreased only by DHA, and DPA, which 32 was increased only by EPA treatment. Global lipidomic analysis showed that EPA and DHA induced 33 large alterations in the cellular lipid profiles and in particular, the phospholipid classes. Subsequent 34 targeted analysis confirmed that the most differentially regulated species were phosphatidylcholines 35 and phosphatidylethanolamines containing long chain fatty acids with 5 (EPA treatment) or 6 (DHA 36 treatment) double bonds. As these are typically membrane associated lipid species we hypothesized 37 that these treatments differentially altered the membrane-associated proteome. SILAC based 38 proteomics of the membrane fraction revealed significant divergence in the effects of EPA and DHA 39 on the membrane associated proteome. We conclude that the EPA specific increase in polyunsaturated 40 long chain fatty acids in the phospholipid fraction is associated with an altered membrane associated 41 proteome and these may be critical events in the metabolic remodelling induced by EPA treatment. 42

Aims/hypothesisThe aim of this study was to determine whether oral dosing with N-acetylcysteine (NAC) increases intraplatelet levels of the antioxidant, glutathione (GSH), and reduces platelet–monocyte conjugation in blood from patients with type 2 diabetes.MethodsIn this placebo-controlled randomised crossover study, the effect of oral NAC dosing on platelet–monocyte conjugation and intraplatelet GSH was investigated in patients with type 2 diabetes (eligibility criteria: men or post-menopausal women with well-controlled diabetes (HbA1c < 10%), not on aspirin or statins). Patients (n = 14; age range 43–79 years, HbA1c = 6.9 ± 0.9% [52.3 ± 10.3 mmol/mol]) visited the Highland Clinical Research Facility, Inverness, UK on day 0 and day 7 for each arm of the study. Blood was sampled before and 2 h after oral administration of placebo or NAC (1,200 mg) on day 0 and day 7. Patients received placebo or NAC capsules for once-daily dosing on the intervening days. The order of administration of NAC and placebo was allocated by a central office and all patients and research staff involved in the study were blinded to the allocation until after the study was complete and the data fully analysed. The primary outcome for the study was platelet–monocyte conjugation.ResultsOral NAC reduced platelet–monocyte conjugation (from 53.1 ± 4.5% to 42.5 ± 3.9%) at 2 h after administration and the effect was maintained after 7 days of dosing. Intraplatelet GSH was raised in individuals with depleted GSH and there was a negative correlation between baseline intraplatelet GSH and platelet–monocyte conjugation. There were no adverse events.Conclusions/interpretationThe NAC-induced normalisation of intraplatelet GSH, coupled with a reduction in platelet–monocyte conjugation, suggests that NAC might help to reduce atherothrombotic risk in type 2 diabetes.Funding:Chief Scientist Office (CZB/4/622), Scottish Funding Council, Highlands & Islands Enterprise and European Regional Development Fund.Trial registration:isrctn.org ISRCTN89304265Electronic supplementary materialThe online version of this article (doi:10.1007/s00125-012-2685-z) contains peer-reviewed but unedited supplementary material, which is available to authorised users.

BackgroundContrast-induced nephropathy is a common complication of contrast administration in patients with chronic kidney disease and diabetes. Its pathophysiology is not well understood; similarly the role of intravenous or oral acetylcysteine is unclear. Randomized controlled trials to date have been conducted without detailed knowledge of the effect of acetylcysteine on renal function. We are conducting a detailed mechanistic study of acetylcysteine on normal and impaired kidneys, both with and without contrast. This information would guide the choice of dose, route, and appropriate outcome measure for future clinical trials in patients with chronic kidney disease.Methods/DesignWe designed a 4-part study. We have set up randomised controlled cross-over studies to assess the effect of intravenous (50 mg/kg/hr for 2 hrs before contrast exposure, then 20 mg/kg/hr for 5 hrs) or oral acetylcysteine (1200 mg twice daily for 2 days, starting the day before contrast exposure) on renal function in normal and diseased kidneys, and normal kidneys exposed to contrast. We have also set up a parallel-group randomized controlled trial to assess the effect of intravenous or oral acetylcysteine on patients with chronic kidney disease stage III undergoing elective coronary angiography. The primary outcome is change in renal blood flow; secondary outcomes include change in glomerular filtration rate, tubular function, urinary proteins, and oxidative balance.DiscussionContrast-induced nephropathy represents a significant source of hospital morbidity and mortality. Over the last ten years, acetylcysteine has been administered prior to contrast to reduce the risk of contrast-induced nephropathy. Randomized controlled trials, however, have not reliably demonstrated renoprotection; a recent large randomized controlled trial assessing a dose of oral acetylcysteine selected without mechanistic insight did not reduce the incidence of contrast-induced nephropathy. Our study should reveal the mechanism of effect of acetylcysteine on renal function and identify an appropriate route for future dose response studies and in time randomized controlled trials.Trial registrationClinical Trials.gov: NCT00558142; EudraCT: 2006-003509-18.