Molecular Characteristics of Serum Visfatin and Differential Detection by Immunoassays

Institut für Laboratoriumsmedizin, Klinische Chemie und Molekulare Diagnostik, University of Leipzig, Leipzig, Saxony, Germany
Journal of Clinical Endocrinology & Metabolism (Impact Factor: 6.21). 01/2008; 92(12):4783-91. DOI: 10.1210/jc.2007-1304
Source: PubMed


There are controversial results concerning the association of visfatin with obesity and diabetes. We aimed to characterize molecular features of visfatin, to assess visfatin detection by different immunoassays, and evaluate the association with human obesity and glucose metabolism.
Distinct preparations of human visfatin (recombinant, endogenously expressed from human adipocytes, and overexpressed in COS-7 cells) were readily identified by three currently available immunoassays. However, direct comparison of native human serum samples did reveal great discrepancies between these assays and complete lack of correlation. To specify putative molecular isoforms of visfatin, we fractionated iodine-125-labeled recombinant visfatin spiked into human serum and supernatants of visfatin-overexpressing COS-7 cells by size exclusion chromatography. We obtained a distinct peak at approximately 100 kDa that was confirmed by subsequent Western blotting of the fractions and is equivalent to the molecular mass of the dimer. Only one of the immunoassays detected a similar peak in native human size exclusion chromatography serum fractions, whereas the others detected a peak at more than 500 kDa or did not show any distinct peak. We did not observe any differences in visfatin serum levels between lean or obese patients. In addition, there was no correlation between visfatin serum levels with visfatin mRNA expression in sc or visceral fat and with parameters of glucose metabolism.
Differences in the qualitative and quantitative detection of visfatin by immunoassays need to be considered in clinical association studies and may explain the conflicting observations with respect to a putative relation of circulating visfatin to human obesity or insulin resistance.

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    • "Following final washing, peroxidase activity is quantified using TMB (3,3 ,5,5 -tetramethylbenzidine). This immunoassay has been demonstrated to determine visfatin values with high accuracy (Körner et al., 2007). Detection limit of the essay was 0.01 ng/ml. "
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    ABSTRACT: Visfatin is a recently described protein that is thought to regulate the process of adipocyte differentiation. Findings suggest that visfatin may be actively involved in the control of weight regulatory networks. However, to what extent and which role it plays in eating disorders is still poorly understood, as mixed results have been reported. The aim of the current study was to investigate serum visfatin concentrations on a cross sectional sample between acute anorexia nervosa patients (n=44), weight recovered patients (n=13) and healthy controls (n=46) and a longitudinal sample of acute patients (n=57) during weight recovery at three different time-points. Results did not show significant differences in visfatin between the three groups; however, acute patients showed a higher visfatin/BMI-SDS ratio than controls and recovered patients. Longitudinal results revealed an increase of visfatin levels during therapy. Our results suggest that high ratios of visfatin/BMI-SDS could be a state marker in acute anorexia nervosa, displaying a compensatory mechanism of the individual to maintain normal visfatin levels under malnourished conditions. Copyright © 2015 Elsevier Ltd. All rights reserved.
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    • "Use of nondenaturing conditions revealed that visfatin/Nampt stored and released by OA human tissues is naturally dimerized, because we detected the 120 kDa form. This natural dimeric form has been detected in human serum and is also constitutively released by human hepatocytes [40,41]. "
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    ABSTRACT: Visfatin is an adipokine that may be involved in intertissular joint communication in osteoarthritis (OA). With a homodimeric conformation, it exerts nicotinamide phosphoribosyltransferase (Nampt) enzymatic activity, essential for nicotinamide adenine dinucleotide biosynthesis. We examined the tissular origin and conformation of visfatin/Nampt in human OA joints and investigated the role of visfatin/Nampt in OA chondrocytes and osteoblasts by studying Nampt enzymatic activity. Synovium, cartilage and subchondral bone from human OA joints were used for protein extraction or incubated for 24 hours in serum-free media (conditioned media), and synovial fluid was obtained from OA patients. Visfatin/Nampt expression in tissular extracts and conditioned media was evaluated by western blot and enzyme-linked immunosorbent assay (ELISA), respectively. Nampt activity was assessed in OA synovium by colorimetric assay. Primary cultures of murine chondrocytes and osteoblasts were stimulated with visfatin/Nampt and pretreated or not with APO866, a pharmacologic inhibitor of Nampt activity. The effect on cytokines, chemokines, growth factors and hypertrophic markers expression was examined by quantitative reverse transcriptase polymerase chain reaction and/or ELISA. In tissular explants, conditioned media and synovial fluid, visfatin/Nampt was found as a homodimer, corresponding to the enzymatically active conformation. All human OA joint tissues released visfatin/Nampt (synovium: 628 +/- 106 ng/g tissue; subchondral bone: 195 +/- 26 ng/g tissue; cartilage: 152 +/- 46 ng/g tissue), with significantly higher level for synovium (P <0.0005). Nampt activity was identified ex vivo in synovium. In vitro, visfatin/Nampt significantly induced the expression of interleukin 6, keratinocyte chemoattractant and monocyte chemoattractant protein 1 in chondrocytes and osteoblasts. APO866 decreased the mRNA and protein levels of these pro-inflammatory cytokines in the two cell types (up to 94% and 63% inhibition, respectively). Levels of growth factors (vascular endothelial growth factor, transforming growth factor beta) and hypertrophic genes were unchanged with treatment. Visfatin/Nampt is released by all human OA tissues in a dimeric enzymatically active conformation and mostly by the synovium, which displays Nampt activity. The Nampt activity of visfatin is involved in chondrocyte and osteoblast activation, so targeting this enzymatic activity to disrupt joint tissue interactions may be novel in OA therapy.
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    • "However, others report opposite findings with decreasing or no changes in plasma/serum Nampt levels associated with diabetes/obesity [11]–[16]. The conflicting results may be due, in part, to the types of populations studied, small sample size, and/or variability in the types of assays used to measure serum/plasma eNampt [17]. The role of eNampt in maintaining normal metabolic responses has been demonstrated using rodent models. "
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    ABSTRACT: Nicotinamide phosphoribosyltransferase (Nampt) is a rate-limiting enzyme in the mammalian NAD+ biosynthesis of a salvage pathway and exists in 2 known forms, intracellular Nampt (iNampt) and a secreted form, extracellular Nampt (eNampt). eNampt can generate an intermediate product, nicotinamide mononucleotide (NMN), which has been reported to support insulin secretion in pancreatic islets. Nampt has been reported to be expressed in the pancreas but islet specific expression has not been adequately defined. The aim of this study was to characterize Nampt expression, secretion and regulation by glucose in human islets. Gene and protein expression of Nampt was assessed in human pancreatic tissue and isolated islets by qRT-PCR and immunofluorescence/confocal imaging respectively. Variable amounts of Nampt mRNA were detected in pancreatic tissue and isolated islets. Immunofluorescence staining for Nampt was found in the exocrine and endocrine tissue of fetal pancreas. However, in adulthood, Nampt expression was localized predominantly in beta cells. Isolated human islets secreted increasing amounts of eNampt in response to high glucose (20 mM) in a static glucose-stimulated insulin secretion assay (GSIS). In addition to an increase in eNampt secretion, exposure to 20 mM glucose also increased Nampt mRNA levels but not protein content. The secretion of eNampt was attenuated by the addition of membrane depolarization inhibitors, diazoxide and nifedipine. Islet-secreted eNampt showed enzymatic activity in a reaction with increasing production of NAD+/NADH over time. In summary, we show that Nampt is expressed in both exocrine and endocrine tissue early in life but in adulthood expression is localized to endocrine tissue. Enzymatically active eNampt is secreted by human islets, is regulated by glucose and requires membrane depolarization.
    Full-text · Article · Mar 2013 · PLoS ONE
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