Therefore, the precise relationship between elevated eNAMPT and type 2 diabetes remains unresolved. Nicotinamide phosphoribosyltransferase exists in intracellular (iNAMPT) and extracellular (eNAMPT) forms. diabetes; (1) a mouse model of diabetes fed a high-fat diet (HFD) for 10?weeks received i.p. injections with an anti-monomeric-eNAMPT antibody; and (2) lean non-diabetic mice received i.p. injections with recombinant monomeric eNAMPT daily for 14?days. Results Serum monomeric eNAMPT levels were elevated in HFD-fed mouse models of diabetes, whilst eNAMPT-dimer levels were unchanged. eNAMPT-monomer neutralisation in HFD-fed mice resulted in lower blood glucose levels, amelioration of impaired glucose tolerance (IGT) and whole-body insulin resistance, improved pancreatic islet function, and reduced inflammation. These effects were maintained for at least 3?weeks post-treatment. eNAMPT-monomer administration induced a diabetic phenotype in mice, characterised by elevated blood glucose, IGT, impaired pancreatic insulin secretion 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 this 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 usually 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 in type 2 diabetes patients [2], whilst raised eNAMPT levels strongly correlate with declining beta cell function [3]. Therefore, a pathophysiological role is usually 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 usually widely expressed and is a well-characterised NAD biosynthetic enzyme [8]. In contrast, the function of eNAMPT is usually 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 usually reportedly essential for the biosynthetic functions of NAMPT [5, 15]. The eNAMPT-monomer has potential NAD-independent proinflammatory effects. However, the precise structureCfunction associations have not yet been fully investigated, particularly within the context of type 2 diabetes pathophysiology. Given the crucial role of chronic inflammation in type 2 diabetes pathophysiology, we hypothesised that eNAMPT-monomer levels will Carnosol be selectively elevated in type 2 diabetes and by potentially acting in a proinflammatory manner, may play a key role in type 2 diabetes pathophysiology. Methods Animal studies For immunoneutralisation experiments, 8-week-old male C57Bl/6 mice (Charles River, Margate, UK) were Mouse monoclonal antibody to hnRNP U. This gene belongs to the subfamily of ubiquitously expressed heterogeneous nuclearribonucleoproteins (hnRNPs). The hnRNPs are RNA binding proteins and they form complexeswith heterogeneous nuclear RNA (hnRNA). These proteins are associated with pre-mRNAs inthe nucleus and appear to influence pre-mRNA processing and other aspects of mRNAmetabolism and transport. While all of the hnRNPs are present in the nucleus, some seem toshuttle between the nucleus and the cytoplasm. The hnRNP proteins have distinct nucleic acidbinding properties. The protein encoded by this gene contains a RNA binding domain andscaffold-associated region (SAR)-specific bipartite DNA-binding domain. This protein is alsothought to be involved in the packaging of hnRNA into large ribonucleoprotein complexes.During apoptosis, this protein is cleaved in a caspase-dependent way. Cleavage occurs at theSALD site, resulting in a loss of DNA-binding activity and a concomitant detachment of thisprotein from nuclear structural sites. But this cleavage does not affect the function of theencoded protein in RNA metabolism. At least two alternatively spliced transcript variants havebeen identified for this gene. [provided by RefSeq, Jul 2008] fed a high-fat diet (HFD; 60% wt/wt excess fat; 58Y1; Test Diets, St. Louis, MO, USA) or a control diet (CON) for 10 or 13?weeks, and then injected i.p. with a rabbit polyclonal mouse anti-eNAMPT antibody (2.5?g/ml; LS-“type”:”entrez-nucleotide”,”attrs”:”text”:”C48964″,”term_id”:”2386217″,”term_text”:”C48964″C48964; LifespanBio, WA, USA) or a non-immune IgG comparative (four separate doses during weeks 9C10). Antibodies were validated via immunoprecipitation and Carnosol immunoblotting. Mice were then killed either directly post-treatment (at 10?weeks) or 3?weeks later (at 13?weeks; ribosomal RNA levels (Applied Biosystems, Warrington, UK). Changes in gene expression are normalised to control. For details of primers (Eurogentec, Southampton, UK), see ESM Table 1 Mouse islet isolation and insulin secretion Mouse pancreases were digested in 2?ml Hanks Buffered Salt Answer (HBSS) containing 1?mg/ml collagenase P and 0.15?mg/ml DNAse I (both Roche Diagnostics). Islets were hand-picked and transferred into RPMI 1640 medium for RNA extraction or insulin secretion assays. For islet insulin secretion assays, batches of eight size-matched islets were pre-incubated for 1?h at 37C in HBSS containing 3?mmol/l glucose, 10?mmol/l HEPES (pH 7.4) and 0.2% BSA (wt/vol.). For glucose-stimulated insulin secretion (GSIS) analysis, islets were incubated for 1?h at 37C in HBSS containing 10?mmol/l HEPES (pH 7.4) and 0.2% BSA, supplemented with 3?mmol/l or 17?mmol/l glucose. After 1?h, the medium was collected for determination of insulin levels by ELISA (see ESM Methods islet isolation and insulin secretion ex vivo for further Carnosol details). Immunofluorescence analysis of mouse pancreatic sections Immunostaining was conducted as previously described [7]. Briefly, the whole pancreas was fixed in buffered formalin, embedded in paraffin, cut into sections and stained with guinea.