Consistent with a beneficial effect of the eNAMPT-dimer, serum and white AT eNAMPT-dimer levels were unchanged or decreased in HFD-fed mice, whilst 14-day NMN administration lowered blood glucose and inflammation in non-diabetic mice. and whole-body insulin resistance, improved pancreatic AMG319 islet function, and reduced inflammation. These effects were maintained for at least 3?weeks post-treatment. eNAMPT-monomer administration induced AMG319 a diabetic phenotype in mice, characterised by elevated blood glucose, IGT, impaired pancreatic insulin secretion AMG319 and the presence of systemic and tissue inflammation, without changes in NAD levels. Conclusions/interpretation We demonstrate that elevation of monomeric-eNAMPT plays an important role in the pathogenesis of diet-induced diabetes via proinflammatory mechanisms. These data provide proof-of-concept evidence that the eNAMPT-monomer represents a potential therapeutic target for type 2 diabetes. Electronic supplementary material The online version of this article (doi:10.1007/s00125-016-4076-3) contains peer-reviewed but unedited supplementary material, which is available to authorised users. Keywords: Beta cell, Extracellular nicotinamide phosphoribosyltransferase, eNAMPT, Inflammation, Islet, Type 2 diabetes Introduction Type 2 diabetes is characterised by the presence of peripheral insulin resistance and pancreatic beta cell dysfunction [1]. Determining the precise pathophysiological mechanisms responsible for these processes is essential for the development of novel therapeutics. Serum concentrations of extracellular nicotinamide phosphoribosyltransferase (eNAMPT; also referred to as visfatin/pre-B cell colony-enhancing factor [PBEF]) are commonly elevated KIAA0564 in type 2 diabetes patients [2], whilst raised eNAMPT levels strongly correlate with declining beta cell function [3]. Therefore, a pathophysiological role is implied for eNAMPT in type 2 diabetes. However, other studies have reported both insulin sensitising and beta cell protective effects of eNAMPT [4C7]. Therefore, the precise relationship between elevated eNAMPT and type 2 diabetes remains unresolved. Nicotinamide phosphoribosyltransferase exists in intracellular (iNAMPT) and extracellular (eNAMPT) forms. iNAMPT is widely expressed and is a well-characterised NAD biosynthetic enzyme [8]. In contrast, the function of eNAMPT is unclear, although putative proinflammatory [9, 10], insulin-mimetic [11] and NAD biosynthetic functions [5, 12, 13] have been described. These disparate putative functions are controversial and have been challenged [11, 14], but may be explained by the presence of structurally and functionally distinct monomeric (50?kDa) and dimeric (100?kDa) forms of eNAMPT. Dimerisation is reportedly essential for the biosynthetic functions of NAMPT [5, 15]. The eNAMPT-monomer has potential NAD-independent proinflammatory effects. However, the precise structureCfunction romantic relationships never have however been looked into completely, inside the context of type 2 diabetes AMG319 pathophysiology particularly. Given the key function of chronic irritation in type 2 diabetes pathophysiology, we hypothesised that eNAMPT-monomer amounts will end up being selectively raised in type 2 diabetes and by possibly acting within a proinflammatory way, may play an integral function in type 2 diabetes pathophysiology. Strategies Animal research For immunoneutralisation tests, 8-week-old male C57Bl/6 mice (Charles River, Margate, UK) had been given a high-fat diet plan (HFD; 60% wt/wt unwanted fat; 58Y1; Test Diet plans, St. Louis, MO, USA) or a control diet plan (CON) for 10 or 13?weeks, and injected i then.p. using a rabbit polyclonal mouse anti-eNAMPT antibody (2.5?g/ml; LS-C48964; LifespanBio, WA, USA) or a nonimmune IgG similar (four separate dosages during weeks 9C10). Antibodies were validated via immunoblotting and immunoprecipitation. Mice were after that killed either straight post-treatment (at 10?weeks) or 3?weeks later (in 13?weeks; ribosomal RNA amounts (Applied Biosystems, Warrington, UK). Adjustments in gene appearance are normalised to regulate. For information on primers (Eurogentec, Southampton, UK), see ESM Desk 1 Mouse islet insulin and isolation secretion Mouse pancreases had been digested in 2?ml Hanks Buffered Sodium Alternative (HBSS) containing 1?mg/ml collagenase P and 0.15?mg/ml DNAse We (both Roche Diagnostics). Islets were hand-picked and transferred into RPMI 1640 moderate for RNA insulin or removal secretion assays. For islet insulin secretion assays, batches of eight size-matched islets had been pre-incubated for 1?h in 37C in AMG319 HBSS containing 3?mmol/l blood sugar, 10?mmol/l HEPES (pH 7.4) and 0.2% BSA (wt/vol.). For glucose-stimulated insulin secretion (GSIS) evaluation, islets had been incubated for 1?h in 37C in HBSS containing 10?mmol/l HEPES (pH 7.4) and 0.2% BSA,.