Relationship of the white blood cell count to body fat: role of leptin

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1 British Journal of Haematology, 1997, 99, Relationship of the white blood cell count to body fat: role of leptin CHARLTON A. WILSON, 1,2 GENENE BEKELE, 1,3 MARGERY NICOLSON, 4 ERIC RAVUSSIN 1 AND RICHARD E. PRATLEY 1 1 Clinical Diabetes and Nutrition Section, Phoenix Epidemiology and Clinical Research Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Phoenix, Arizona, 2 Department of Internal Medicine, Phoenix Indian Medical Center, Indian Health Service, Phoenix, Arizona, 3 Carl T. Hayden Veterans Affairs Medical Center, Phoenix Arizona, and 4 Amgen Inc., Thousand Oaks, California, U.S.A. Received 17 April 1997; accepted for publication 30 July 1997 Summary. The white blood cell (WBC) count is correlated to the amount of body fat in humans, but the mechanism for this association is unknown. Leptin, a 16 kd protein produced in adipocytes, circulates in humans in direct proportion to the amount and percentage of body fat. Recent evidence suggests that leptin and the leptin receptor are part of a novel pathway which stimulates haemopoiesis. This study was designed to test whether fasting plasma leptin concentrations contribute to the relationship between the WBC count and body fat. 117 Pima Indians with a wide range in body composition were studied. The WBC count was positively correlated with percentage of body fat (r ¼ 0. 44, P ¼ ) and fasting plasma leptin concentration (r ¼ 0. 38, P ¼ ). In multiple regression analyses, age, gender and percent body fat were significant independent determinants of the WBC count. After controlling for age and gender, percent body fat accounted for 23% of the variance in the WBC count (partial r ¼ 0. 48, P ¼ ). In similar models which also included plasma leptin concentration, percent body fat remained significantly related to the WBC count, but only accounted for 7% of its variance (partial r ¼ 0. 27, P ¼ ). Based on these results, and the demonstration that leptin directly effects the stimulation of proliferation of haemopoietic stem cells in vitro, we hypothesize that the relation of the WBC count to percent body fat may be mediated, in part, through the effect of leptin. Keywords: haemopoiesis, leptin, body fat, humans, white blood cell count. We, and others, have shown that the white blood cell (WBC) count is correlated to the amount of body fat in humans (Nanji & Freeman, 1985; Hansen et al, 1990; Freidman et al, 1990; Nieto et al, 1992; Pratley et al, 1995) independent of age, gender and ethnicity. Although there are several plausible explanations for this, the mechanism by which body fat is related to the WBC count remains speculative. Leptin, a recently discovered 16 kd protein produced by adipocytes, is present in the circulation in direct proportion to the amount of body fat in humans and in most animal models of obesity (Maffei et al, 1995; Caro et al, 1996). In the ob/ob mouse a genetic deficiency of leptin results in massive obesity and diabetes, whereas administration of leptin decreases food intake, increases energy expenditure and sympathetic nerve outflow, and decreases body weight (Caro Correspondence: Dr Richard E. Pratley, Clinical Diabetes and Nutrition Section, NIDDK/NIH, 4212 North 16th Street, Phoenix, AZ 85016, U.S.A Blackwell Science Ltd et al, 1996). A specific receptor for leptin has been cloned and is expressed in the hypothalamus in regions involved in food intake and energy expenditure (Caro et al, 1996). Based on these findings, it has been hypothesized that leptin acts as a lipostat signalling peripheral energy stores to central hypothalamic regulatory areas in a negative feedback loop which serves to regulate energy balance (Caro et al, 1996). However, leptin receptors are also found on many non-neural tissue cells, suggesting a broader role for this hormone (Caro et al, 1996; Cioffi et al, 1996; Bennet et al, 1996). A novel cytokine receptor expressed in human CD34 þ cells has recently been identified based on its homology to gp130 (Cioffi et al, 1996). This receptor is identical to the receptor for leptin (Cioffi et al, 1996; Bennet et al, 1996), and appears to be part of a unique pathway which stimulates haemopoiesis at the stem/progenitor cell level (Cioffi et al, 1996; Bennet et al, 1996). In culture, the administration of leptin stimulates proliferation of haemopoietic stem cells (Bennet et al, 1996). 447

2 448 Charlton A. Wilson et al Table I. Subject characteristics. Variable Men Women P value N Age (years) 29 1 (19 46) 29 1 (18 44) Body fat (%) 29 1 (13 42) 39 1 (20 48) Leptin (ng/ml) ( ) ( ) WBC ( 10 9 /l) ( ) ( ) RBC ( /l) ( ) ( ) HCT (%) ( ) ( ) PLT ( 10 9 /l) ( ) ( ) Values are mean SEM (range). When considered together, the observations that leptin is directly related to the amount of body fat and stimulates haemopoietic stem cells suggest that leptin may be the mechanism linking obesity and higher WBC counts. To test this, we measured the WBC count, fasting plasma leptin concentrations and body composition in 117 healthy Pima Indians who participated in an ongoing study of risk factors for non-insulin-dependent diabetes mellitus between 1988 and 1994 (Lillioja et al, 1993). METHODS Subjects. All subjects provided written informed consent prior to participation and upon admission all underwent a medical history and physical examination and a fasting laboratory profile, including a complete blood count (CBC). Individuals with significant abnormalities on screening, including symptoms or signs of infection or diabetes, were excluded from analysis. None of the subjects were taking medications which could affect the WBC count. Complete blood count determination. From 1988 to 1991, CBCs were performed using an Ortho ELT-8 (Ortho, Braintree, Mass.) automated analyser. Subsequently, a Coulter STKS (Coulter Electronics, Hialeah, Fla.) automated analyser was used. We have previously reported that WBC counts determined using the two systems were highly correlated and it is not necessary to correct for this change in methodology (Pratley et al, 1995). Leptin determination. Fasting plasma leptin concentrations were measured in duplicate with a solid-phase sandwich enzyme immunoassay using an affinity-purified polyvalent antibody immobilized in microtitre wells. Bound leptin was detected with affinity purified antibody conjugated to horseradish peroxidase, and quantified with a chromogenic substrate (TMB/peroxide). The concentration of leptin in plasma was calculated from a standard curve generated in each assay with recombinant human leptin. This assay is sensitive to leptin concentrations of 50 pg/ml. The intra- and the inter-assay coefficients of variation were 6. 5% and 8. 45%, respectively. Body composition. Body density was determined by hydrodensitometry corrected for residual lung volume (Goldman & Buskirk, 1961) which was simultaneously measured using the helium dilution technique (Warren E. Collins Inc., Braintree, Mass.). Percent body fat was calculated from body density measurements (Keys & Brozek, 1953). Statistics. All data were analysed using SAS (Cary, N.C.). Plasma leptin concentrations were log transformed to achieve a normal distribution prior to parametric analyses. Differences between the genders were tested with unpaired t-tests. Simple and multiple linear regression models with calculation of partial correlation coefficients were used to analyse the relationships among selected variables. P values < were considered significant. All data are presented as means SEM. RESULTS Subjects Thirty-five percent of the subjects were women, all of whom were premenopausal. Although the group as a whole was obese, there was a wide range in body composition among both men and women (Table I). Percent body fat, fasting plasma leptin concentrations, and platelets were higher in women than in men. In contrast, the red blood cell (RBC) count, haematocrit (HCT) and haemoglobin levels were significantly lower in women than in men. Relations among percent fat, leptin and haematological parameters In the group as a whole, fasting plasma leptin concentrations were highly correlated to percent body fat Table II. Relation of haematological values to percent body fat and fasting plasma leptin concentration. Percent body fat Log 10 [fasting leptin] WBC ( ) ( ) RBC ¹0. 27 (0. 003) ¹0. 28 (0. 002) HCT ¹0. 38 ( ) ¹0. 49 ( ) PLT (0. 007) (0. 002) Values are Pearson product-moment correlation coefficients (P value).

3 Leptin and WBC 449 Fig 1. Relation between log 10 [fasting plasma leptin] and percent body fat measured by hydrodensitometry (r ¼ 0. 84, P ¼ ). (r ¼ 0. 84, P ¼ , Fig 1). The WBC and the RBC counts were negatively correlated with age (r ¼¹0. 20 and ¹0. 25 respectively, P values < 0. 05). The WBC count was positively correlated with percent body fat (r ¼ 0. 44, P ¼ , Fig 2) and leptin concentrations (r ¼ 0. 38, P ¼ , Fig 3). Similarly, the platelet (PLT) count was positively correlated to percent body fat and leptin (Table II). In contrast, the RBC count, haematocrit, and haemoglobin Fig 2. Relation between the WBC and percent body fat (r ¼ 0. 44, P ¼ ).

4 450 Charlton A. Wilson et al Fig 3. Relation between the WBC and log 10 [fasting plasma leptin] (r ¼ 0. 38, P ¼ ). levels were negatively correlated with percent body fat and leptin (Table II). In multiple regression analyses age, gender and percent body fat were all significantly related to the WBC count, confirming our earlier observations (Pratley et al, 1995). After controlling for age and gender, percent body fat accounted for 23% of the variance in the WBC count (partial r ¼ 0. 48, P ¼ ). In models which also controlled for fasting plasma leptin concentration, percent body fat remained significantly related to the WBC count (partial r ¼ 0. 27, P ¼ ), but the variance explained by percent body fat dropped to 7%. Percent body fat and leptin were not significantly related to the RBC count, haematocrit or platelet count after controlling for the effects of age and gender. DISCUSSION The results of this study confirmed that the WBC count is related to percent body fat. Furthermore, they have indicated that most of the variance in the WBC count attributable to percent body fat could be accounted for by fasting plasma leptin concentrations. This suggested that the correlation between the WBC count and the amount of body fat previously observed (Pratley et al, 1995; Nanji & Freeman, 1985; Hansen et al, 1990; Freidman et al, 1990; Nieto et al, 1992), could be mediated, in part, through leptin. The observation that leptin has direct effects to stimulate haemopoiesis in vitro supports a causal relation between leptin and the WBC count. Cioffi et al (1996) identified the presence of an isoform of the leptin receptor on murine and human haemopoietic stem cells, suggesting a role for leptin in haemopoietic stem cell biology. Bennet et al (1996) also identified variants of the leptin receptor in human and murine haemopoietic stem cells and further observed that leptin increased the proliferation of stem cell populations at the level of a multilineage progenitor as shown by increased myelopoiesis, erythropoiesis and lymphopoiesis. In the same report, analysis of db/db mice, in which the leptin receptor is truncated and non-functional, revealed that steady-state levels of peripheral B cells and CD4-expressing T cells were dramatically reduced. The db/db mice also demonstrated decreased lymphopoietic progenitors in marrow and poor recovery of marrow following irradiation. Also, despite normal peripheral blood erythrocyte counts, decreased spleen erythrocyte production was noted in these mice. Two subsequent studies (Gainsford et al, 1996; Mikhail et al, 1997) have confirmed the observation that leptin plays an important role in haemopoietic stem cell proliferation. Human obesity is characterized by increased circulating concentrations of leptin, suggesting a functional resistance to the central effects of leptin (Maffei et al, 1995; Considine et al, 1996; Caro et al, 1996). The cause of this resistance is not known but may relate to reduced transport of leptin across the blood brain barrier (Caro et al, 1996). Obesity may therefore expose bone marrow to higher concentrations of leptin and the result may be increased stem cell proliferation leading to higher WBC counts. The RBC count, haematocrit or platelets were not related to obesity or fasting plasma leptin concentration when gender was also included in multivariate analyses. In the db/db mouse model (Bennet et al, 1996) peripheral erythrocyte levels were unaffected by

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