Association of NAMPT with normal development in healthy lean children
We identified no differences in circulating NAMPT levels with progression through puberty in normal healthy lean boys or girls (ESM Fig. 1
a). Also, there was no correlation between NAMPT levels and height SDS, circulating IGF-I and markers of pubertal development and adrenarche, including oestradiol, testosterone or dehydroepiandrosterone-sulfate (DHEA-S). There were no differences between boys and girls in the entire cohort (ESM Fig. 1
b) or if analysis was restricted to adolescent children.
Association of NAMPT with obesity and obesity interventions
We applied the Leipzig Atherobesity Childhood cohort (ESM Table 2
) to evaluate differences in NAMPT serum concentrations between lean and obese children. NAMPT levels were significantly higher in the obese children compared with lean controls (Fig. ). NAMPT levels correlated with BMI (Fig. ) and other variables of obesity and body fat, including BMI SDS as a normalised index of the degree of overweight, leptin as a biomarker of fat mass, and skinfold thickness as an index of subcutaneous fat (Table ).
We further assessed NAMPT serum concentrations in the context of three distinct intervention studies: (1) a 12 month lifestyle intervention programme in children and adolescents; (2) bariatric surgery by sleeve gastrectomy in severely obese adolescents and adults; and (3) a 6 month exercise intervention in normal weight young healthy adults (ESM Table 3
). The strategies differed in the extent of weight loss and improvement of HOMA-IR achieved (Fig. , ESM Table 3
After both the bariatric surgery and the exercise interventions we found significantly lower NAMPT serum concentrations, whereas we observed no significant change after lifestyle intervention (Fig. ). The decrease in NAMPT correlated significantly with the decrease in BMI in the surgery group only (ESM Fig. 2
In addition to BMI, the bariatric surgery group showed the strongest decrease in white blood cell count (WBC) and high-sensitivity C-reactive protein (hsCrP; ESM Fig. 2
a–c) and there was a significant correlation between decrease in hsCrP and NAMPT in this group (ESM Fig. 2
f). In multiple regression analyses, decrease in hsCrP (β
0.033) and decrease in BMI (β
0.029) were significant predictors for the NAMPT decrease, whereas decrease in WBC and basal WBC, hsCrP and BMI did not contribute (R2
0.035 for the model).
Association of NAMPT with glucose metabolism
NAMPT significantly correlated with all variables of glucose and insulin metabolism (Table , Fig. ). Children with impaired insulin sensitivity had higher NAMPT levels compared with normal insulin-sensitive children (Fig. ). When we adjusted for BMI SDS in partial correlation analyses, only mean blood glucose (BG) and AUC BG as dynamic variables during OGTT remained significant, but most other significances were lost (Table ). In multiple regression analyses, NAMPT was significantly associated with AUC BG (β
0.004), whereas BMI SDS, age, sex and pubertal stage did not contribute significantly.
To investigate this finding in more detail, we analysed the dynamic of NAMPT serum concentrations during an OGTT in 24 obese adolescents (age 11.7
4.1 years, BMI SDS 2.60
0.53). Compared with basal levels, NAMPT levels declined to 77
0.0029). This decline was stronger in the insulin-sensitive obese participants, as evidenced by a negative correlation of NAMPT with Matsuda ISI (Fig. ). We thus stratified groups into insulin-resistant and insulin-sensitive patients on the basis of peak insulin during OGTT >1,000 and <600 pmol/l, respectively (Fig. ). Accordingly, Matsuda ISI was significantly impaired in the hyperinsulinaemic group (8.02
0.70 vs 2.59
0.0001). There was no difference in the degree of obesity between the two groups (2.72
0.11 vs 2.48
0.20 BMI SDS, p
0.2). The decline of NAMPT, however, was significantly more pronounced in the insulin-sensitive group (Fig. ) and correlated with AUC-insulin-to-AUC-BG ratio (r
0.038). Likewise, individual NAMPT levels during OGTT correlated with the insulin-to-glucose ratio at each time point (r
0.034). Thus, NAMPT levels declined after an oral glucose load as a function of time and insulin-to-glucose ratio (Fig. ).
Association of NAMPT with cardiovascular parameters and leucocytes
We assessed the correlation of NAMPT levels with cardiovascular variables (random and 24 h blood pressure), functional variables of early vascular impairment (IMT, endothelial function), and regenerative capacity (EPC count and migratory function) in our Atherobesity Childhood cohort. We identified significant correlations of NAMPT with systolic blood pressure and endothelial function that did, however, not withstand adjustment for BMI SDS in partial correlation analyses (Table ). By contrast, the cellular variables EPC and WBC counts, and hsCrP, were significantly associated with NAMPT serum levels (Fig. , Table ). In multiple regression analyses, NAMPT was the strongest predictor for EPC as well as for WBC counts (Table ).
Multiple regression analyses for independent associations of NAMPT serum levels
Considering this predominant association of NAMPT with WBCs, we hypothesised that WBCs may influence the associations of NAMPT with metabolic variables and evaluated the correlation of WBCs themselves with metabolic and cardiovascular variables. We identified significant BMI-independent correlations with AUC insulin, Matsuda ISI (Fig. ), systolic blood pressure (r
0.024), but most strongly with NAMPT serum levels (Fig. ). Likewise, in multiple regression analyses, WBCs contributed significantly and independently of age, sex and BMI SDS to Matsuda ISI (Table ).
Hence, NAMPT is most strongly associated with WBC count and WBCs themselves are significantly associated with variables of insulin resistance.
Expression pattern of NAMPT in tissues and leucocytes
Considering the strong association of circulating NAMPT levels with leucocytes in the clinical study, we systematically evaluated the expression pattern of NAMPT among 14 metabolically, endocrine and immunologically active tissues. We found that NAMPT mRNA was predominantly expressed in peripheral blood leucocytes (Fig. ).
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 (more ...)
To determine which subpopulation(s) of leucocytes potentially contribute to NAMPT levels in peripheral blood, we assessed NAMPT mRNA and NAMPT protein expression in granulocytes, lymphocytes and monocytes isolated from peripheral blood of 12 children and adolescents. The mRNA expression was more than fivefold higher in granulocytes and monocytes compared with lymphocytes (Fig. ). Consistent with this, we detected a higher amount of NAMPT protein in cell lysates of granulocytes and monocytes compared with lymphocytes (Fig. ). Granulocytes secreted highest amounts of NAMPT protein into cell culture supernatant fractions (Fig. ). Consistently, serum concentrations of NAMPT were highly correlated to leucocyte count, particularly to neutrophil granulocyte and monocyte count but not to lymphocyte count (Fig. ). Hence, NAMPT is predominantly produced and secreted by leucocytes, in particular by granulocytes.
NAMPT function and interaction with SIRT1 and LPS in distinct leucocyte subpopulations
We evaluated the enzymatic activity of the NAMPT protein by quantification of nicotinamide mononucleotide (NMN) synthesis from nicotinamide and confirmed that NAMPT enzymatic activity was present in cell lysates and supernatant fractions of all leucocyte subpopulations (Fig. ).
To assess a downstream target of NAMPT activity, we examined SIRT1 mRNA expression in the leucocyte subpopulations. The expression of SIRT1 was significantly higher in granulocytes compared with lymphocytes and monocytes (Fig. ).
To finally evaluate whether NAMPT is stimulated by inflammatory agents, we analysed NAMPT release following stimulation with LPS. LPS significantly increased NAMPT release from monocytes and slightly enhanced NAMPT release from granulocytes (Fig. ).
Leucocyte counts and NAMPT release from leucocytes during OGTT
We assessed whether the decline of NAMPT during OGTT is attributable to reduced leucocyte counts or reduced NAMPT release from leucocytes in 11 additional patients. The leucocyte, lymphocyte and neutrophil counts did not change significantly after oral glucose intake; only monocytes were significantly decreased after 60 min (ESM Fig. 3
a). Ex vivo, we found a slight decrease in NAMPT release after 120 min (ESM Fig. 3
b). Finally, to assess whether glucose has a direct effect on NAMPT release, we incubated isolated leucocytes with glucose, but we did not find significant changes (ESM Fig. 3