NNMT (nicotinamide N-methyltransferase, E.C. 2.1.1.1) catalyses the N-methylation of nicotinamide to 1-methylnicotinamide. NNMT expression is significantly elevated in a number of cancers, and we have previously demonstrated that NNMT expression is significantly increased in the brains of patients who have died of Parkinson's disease. To investigate the cellular effects of NNMT overexpression, we overexpressed NNMT in the SH-SY5Y cell line, a tumour-derived human dopaminergic neuroblastoma cell line with no endogenous expression of NNMT. NNMT expression significantly decreased SH-SY5Y cell death, which correlated with increased intracellular ATP content, ATP/ADP ratio and Complex I activity, and a reduction in the degradation of the NDUFS3 [NADH dehydrogenase (ubiquinone) iron-sulfur protein 3] subunit of Complex I. These effects were replicated by incubation of SH-SY5Y cells with 1-methylnicotinamide, suggesting that 1-methylnicotinamide mediates the cellular effects of NNMT. Both NNMT expression and 1-methylnicotinamide protected SH-SY5Y cells from the toxicity of the Complex I inhibitors MPP+ (1-methyl-4-phenylpyridinium ion) and rotenone by reversing their effects upon ATP synthesis, the ATP/ADP ratio, Complex I activity and the NDUFS3 subunit. The results of the present study raise the possibility that the increase in NNMT expression that we observed in vivo may be a stress response of the cell to the underlying pathogenic process. Furthermore, the results of the present study also raise the possibility of using inhibitors of NNMT for the treatment of cancer.

Background Tranexamic acid reduces surgical bleeding and reduces death due to bleeding in patients with trauma. Meta-analyses of small trials show that tranexamic acid might decrease deaths from gastrointestinal bleeding. We aimed to assess the effects of tranexamic acid in patients with gastrointestinal bleeding. Methods We did an international, multicentre, randomised, placebo-controlled trial in 164 hospitals in 15 countries. Patients were enrolled if the responsible clinician was uncertain whether to use tranexamic acid, were aged above the minimum age considered an adult in their country (either aged 16 years and older or aged 18 years and older), and had significant (defined as at risk of bleeding to death) upper or lower gastrointestinal bleeding. Patients were randomly assigned by selection of a numbered treatment pack from a box containing eight packs that were identical apart from the pack number. Patients received either a loading dose of 1 g tranexamic acid, which was added to 100 mL infusion bag of 0•9% sodium chloride and infused by slow intravenous injection over 10 min, followed by a maintenance dose of 3 g tranexamic acid added to 1 L of any isotonic intravenous solution and infused at 125 mg/h for 24 h, or placebo (sodium chloride 0•9%). Patients, caregivers, and those assessing outcomes were masked to allocation. The primary outcome was death due to bleeding within 5 days of randomisation; analysis excluded patients who received neither dose of the allocated treatment and those for whom outcome data on death were unavailable. This trial was registered with Current Controlled Trials, ISRCTN11225767, and ClinicalTrials.gov, NCT01658124.

Nicotinamide N-methyltransferase (NNMT, E.C. 2.1.1.1) N-methylates nicotinamide to produce 1-methylnicotinamide (MeN). We have previously shown that NNMT expression protected against neurotoxin-mediated cell death by increasing Complex I (CxI) activity, resulting in increased ATP synthesis. This was mediated via protection of the NDUFS3 subunit of CxI from degradation by increased MeN production. In the present study, we have investigated the effects of NNMT expression on neurone morphology and differentiation. Expression of NNMT in SH-SY5Y human neuroblastoma and N27 rat mesencephalic dopaminergic neurones increased neurite branching, synaptophysin expression and dopamine accumulation and release. siRNA gene silencing of ephrin B2 (EFNB2), and inhibition of Akt phosphorylation using LY294002, demonstrated that their sequential activation was responsible for the increases observed. Incubation of SH-SY5Y with increasing concentrations of MeN also increased neurite branching, suggesting that the effects of NNMT may be mediated by MeN. NNMT had no significant effect on the expression of phenotypic and post-mitotic markers, suggesting that NNMT is not involved in determining phenotypic fate or differentiation status. These results demonstrate that NNMT expression regulates neurone morphology in vitro via the sequential activation of the EFNB2 and Akt cellular signalling pathways.

Nicotinamide N-methyltransferase (NNMT, E.C. 2.1.1.1) N-methylates nicotinamide to 1-methylnicotinamide. We have previously shown that NNMT is significantly overexpressed in the brains of patients who have died of Parkinson's disease, and others have shown that NNMT is significantly overexpressed in a variety of diseases ranging from cancer to hepatic cirrhosis. In vitro overexpression has revealed many cytoprotective effects of NNMT, in particular increased complex I activity and ATP synthesis. Although this appears to be mediated by an increase in 1-methylnicotinamide production, the molecular mechanisms involved remain unclear. In the present study, we have investigated the role that sirtuins 1, 2 and 3, class III DNA deacetylase enzymes known to regulate mitochondrial energy production and cell cycle, have in mediating the effects of NNMT upon complex I activity. Expression of NNMT in SH-SY5Y human neuroblastoma cells, which have no endogenous expression of NNMT, significantly increased the expression of all three sirtuins. siRNA-mediated silencing of sirtuin 3 expression decreased complex I activity in NNMT-expressing SH-SY5Y cells to that observed in wild-type SH-SY5Y, and significantly reduced cellular ATP content also. These results demonstrate that sirtuin 3 is a key mediator of NNMT-induced complex I activity and ATP synthesis. These results further reinforce a central role for NNMT in the regulation of energy homeostasis and provide further mechanistic insight into the consequences of enhanced NNMT expression.

Nicotinamide N-methyltransferase (NNMT) is responsible for the N-methylation of nicotinamide to 1-methylnicotinamide. Our recent studies have demonstrated that NNMT regulates cellular processes fundamental to the correct functioning and survival of the cell. It has been proposed that NNMT may possess β-carboline (BC) N-methyltransferase activity, endogenously and exogenously produced pyridine-containing compounds which, when N-methylated, are potent inhibitors of Complex I and have been proposed to have a role in the pathogenesis of Parkinson's disease. We have investigated the ability of recombinant NNMT to N-methylate norharman (NH) to 2-N-methylnorharman (MeNH). In addition, we have investigated the toxicity of the BC NH, its precursor 1,2,3,4-tetrahydronorharman (THNH) and its N-methylated metabolite MeNH, using our in vitro SH-SY5Y NNMT expression model. Recombinant NNMT demonstrated NH 2N-methyltransferase activity, with a Km of 90 ± 20 µM, a kcat of 3 × 10(-4) ± 2 × 10(-5) s(-1) and a specificity constant (kcat/Km) of 3 ± 1 s(-1) M(-1) THNH was the least toxic of all three compounds investigated, whereas NH demonstrated the greatest, with no difference observed in terms of cell viability and cell death between NNMT-expressing and non-expressing cells. In NNMT-expressing cells, MeNH increased cell viability and cellular ATP concentration in a dose-dependent manner after 72 and 120 h incubation, an effect that was not observed after 24 h incubation or in non-NNNT-expressing cells at any time point. Taken together, these results suggest that NNMT may be a detoxification pathway for BCs such as NH.