Leucocytes are a major source of circulating nicotinamide phosphoribosyltransferase (NAMPT)/pre-B cell colony (PBEF)/visfatin linking obesity and inflammation in humans

Abstract

Aims/hypothesis: Nicotinamide phosphoribosyltransferase (NAMPT) is a multifunctional protein potentially involved in obesity and glucose metabolism. We systematically studied the association between circulating NAMPT, obesity, interventions and glucose metabolism and investigated potential underlying inflammatory mechanisms.

Methods: Fasting morning NAMPT serum levels were measured in cohorts of lean vs obese children, cohorts of intervention by lifestyle, exercise and bariatric surgery, and during an OGTT. In addition, mRNA expression, protein production and enzymatic activity of NAMPT were assessed from isolated leucocytes and subpopulations.

Results: Circulating NAMPT was significantly elevated in obese compared with lean children and declined after obesity interventions concomitantly with the decline in BMI, high-sensitivity C-reactive protein (hsCrP) and leucocyte counts. Circulating NAMPT significantly correlated with glucose metabolism and cardiovascular variables in univariate analyses, but only the association with glucose response during an OGTT was independent from BMI. We therefore assessed the NAMPT dynamic following an oral glucose load and found a significant decline of NAMPT levels to 77.0 ± 0.1% as a function of time, and insulin-to-glucose ratio during an OGTT in obese insulin-resistant adolescents. Circulating NAMPT was, however, most strongly associated with leucocyte counts (r = 0.46, p < 0.001). The leucocyte count itself determined significantly and independently from BMI insulin resistance in multiple regression analyses. We systematically evaluated NAMPT expression among several tissues and found that NAMPT was predominantly expressed in leucocytes. In subsequent analyses of leucocyte subpopulations, we identified higher NAMPT protein concentrations in lysates of granulocytes and monocytes compared with lymphocytes, whereas granulocytes secreted highest amounts of NAMPT protein into cell culture supernatant fractions. We confirmed nicotinamide mononucleotide enzymatic activity of NAMPT in all lysates and supernatant fractions. In monocytes, NAMPT release was significantly stimulated by lipopolysaccharide (LPS) exposure.

Conclusions: Leucocytes are a major source of enzymatically active NAMPT, which may serve as a biomarker or even mediator linking obesity, inflammation and insulin resistance.

Figures

Fig. 1

NAMPT association with obesity. a Obese children (n = 86) had significantly higher NAMPT levels (p = 0.031) compared with lean children (n = 70). Data are mean ± SEM. b NAMPT serum levels correlated with BMI (r = 0.35, p < 0.001). c Changes of NAMPT levels and BMI after 1-year lifestyle intervention in obese children and adolescents (n = 36), 6 months following bariatric surgery in severely obese adults and adolescents (n = 14), and 6 month exercise programme in normal weight adults (n = 15). Data for BMI (open bars) and NAMPT levels (black bars) are given relative to the basal situation (hatched bar), which was set to 1.0. Data are mean ± SEM. Statistical significance was assessed by paired t test. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 2

NAMPT association with glucose/insulin metabolism. Correlation of NAMPT serum levels with AUC BG during OGTT (a) (r = 0.25, p = 0.001) and Matsuda ISI (b) (r = −0.29, p < 0.001). White circles = lean children, black circles = obese children. c Children with impaired insulin sensitivity according to Matsuda ISI < 4 (n = 44) had higher NAMPT levels compared with normal insulin sensitivity (n = 105, p = 0.003). Data are given as mean ± SEM and were analysed by t test of log-transformed NAMPT. d Correlation of NAMPT decline (given as mean ratio of NAMPT between 60–120 min to basal NAMPT) and Matsuda ISI (r = 0.48, p = 0.017). Course of blood glucose (e), insulin (f) and NAMPT (g) serum levels during OGTT in insulin-sensitive (n = 11) and insulin-resistant (n = 13) obese children. For NAMPT, data are given as ratio of NAMPT levels at single time points compared with basal NAMPT at t = 0 (pAnova = 0.003). White circles, normal insulin; black circles, hyperinsulinemia. h Three-dimensional plot of NAMPT as a function of insulin-to-glucose ratio and time during OGTT

Fig. 3

NAMPT association with leucocytes in the Leipzig Atherobesity Childhood cohort. Correlation of NAMPT serum levels with EPC count (a) (r = −0.29, p < 0.001) and hsCrP (b) (r = 0.34, p < 0.001). Correlations of WBC count with metabolic variables AUC insulin (c) (r = 0.35, p < 0.001) and Matsuda ISI (d) (r = −0.33, p < 0.001). The strongest correlation was achieved between NAMPT and WBC count (e) (r = 0.46, p < 0.001). White circles, lean children; black circles, obese children. Correlation coefficients were determined by Pearson correlation analyses of log-transformed variables

Fig. 4

NAMPT expression pattern, production and secretion by leucocyte subpopulations. a The expression of NAMPT mRNA was significantly higher in PBL than in all other tissues, including adipose tissue and liver (pAnova < 0.0001). Significance was calculated by one-way ANOVA with Dunnett’s post test (compared with PBL). b The mRNA expression of NAMPT was more than fivefold higher in granulocytes and monocytes compared with lymphocytes. c Higher amounts of NAMPT protein were detected in cell lysates of granulocytes and monocytes compared with lymphocytes. d Granulocytes secreted more than 22-fold higher amounts of NAMPT protein into cell culture supernatant fractions (n = 12) when normalised to total protein. Serum concentrations of NAMPT were highly correlated to leucocyte count in particular to neutrophil granulocyte (e) (r = 0.92, p = 0.009) and monocyte (f) (r = 0.94, p = 0.005) count but not to lymphocyte count (g) (n = 6, p = 0.41). h NAMPT enzymatic activity was present in cell lysates and supernatant fractions of all leucocyte subpopulations (n = 5). iSIRT1 mRNA expression was significantly higher in granulocytes compared with lymphocytes and monocytes (n = 12). j The release of NAMPT was significantly increased from monocytes and granulocytes after stimulation with 1 μg/ml LPS for 24 h in n = 3 independent experiments. Data are mean ± SEM. Statistical significance was assessed by Student’s t test and Pearson correlation analysis: *p < 0.05, **p < 0.01, ***p < 0.0001. G, granulocytes; L, lymphocytes; M, monocytes; PBL, peripheral blood leucocytes