Leptin, a 16-kilodalton protein, is involved in the. Gender-Dependent Alterations in Serum Leptin in Alcoholic Cirrhosis. Materials and Methods

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1 GASTROENTEROLOGY 1998;115: Gender-Dependent Alterations in Serum Leptin in Alcoholic Cirrhosis ARTHUR J. MCCULLOUGH,* ELISABETTA BUGIANESI, GIULIO MARCHESINI, and SATISH C. KALHAN* *Departments of Medicine and Pediatrics, Case Western Reserve University, Cleveland, Ohio; and Departments of Medicine and Pediatrics, University of Bologna, Bologna, Italy Background & Aims: Leptin is a peptide that decreases food intake and increases energy expenditure. It is produced in fat cells, is stimulated by cytokines, and its levels in serum are higher in females. Because anorexia, hypermetabolism, and elevated cytokine levels are frequently observed in cirrhosis, we hypothesized that the serum leptin level would be elevated in cirrhosis. The aim of this study was to investigate the relationship of serum leptin to gender, body composition, and tumor necrosis factor (TNF). Methods: Male (n 18) and female (n 10) abstinent alcoholic cirrhotic patients were studied and compared with control subjects (15 male and 8 female). Fat mass, fat-free body mass, and body cell mass were calculated by using H 2 [ 18 O] and bromide dilution methodology. Serum leptin and TNF concentrations were measured by immunoassays. Results: Fat mass was decreased only in male cirrhotics (P 0.05), whereas body cell mass was decreased in both male and female cirrhotics (P 0.01). Leptin levels were elevated in female (P 0.001) but not male cirrhotics compared with controls. When expressed per kilogram of fat mass, leptin was elevated in both male (P 0.01) and female (P 0.01) cirrhotics. Women in both cirrhotic and control groups had higher leptin levels than men. TNF was elevated in both male and female cirrhotics and did not correlate with leptin levels. Conclusions: Cirrhotics have elevated serum leptin levels, which are related to both gender- and gender-dependent alterations in body composition. Leptin, a 16-kilodalton protein, is involved in the regulation of food intake and body composition 1 through a central feedback mechanism 2,3 and has been proposed to be a pathophysiological factor in human obesity. 1 Its level is higher in females 4,5 and correlates with fat mass. 5 In ob/ob mice, which have a genetic defect in leptin production, administration of recombinant leptin diminishes food intake, decreases body weight, and increases energy expenditure. 6 Leptin is expressed and secreted by adipocytes, 1 the major secretogogues being cytokines (particularly tumor necrosis factor [TNF]- and interleukin [IL]-1), corticosteroids, and insulin. 1,7 9 Because the levels of all three of these secretogogues are increased in cirrhosis, 10,11 a disease frequently associated with hypermetabolism 12 and altered body composition, 13 we hypothesized that leptin levels may be increased in cirrhosis and be involved in the malnutrition so prevalent in this disease. However, inconsistent with this hypothesis is the normal or diminished fat mass observed in cirrhosis, because, with the exception of patients receiving renal dialysis, 14 leptin levels have correlated directly with fat mass in all other patient groups studied to date. 1,4,5,15,16 In addition, it is unclear what effect the gender-related alterations in sex steroids and body composition associated with cirrhosis 13,17 might have on leptin. We therefore analyzed serum leptin in relationship to TNF and body composition in both female and male alcoholic cirrhotics and controls. Materials and Methods Twenty-eight patients with biopsy-proven alcoholic cirrhosis (18 men and 10 women) and 23 normal controls (15 men and 8 women) were studied. At the time of the study, ascites was present in 10 cirrhotic patients (6 men and 4 women) but no other active complications of liver disease (encephalopathy, gastrointestinal bleeding, or alcoholic hepatitis) were present. Abstinence from alcohol for at least 1 month before the study was confirmed by a family member. Controls were considered normal on the basis of history, physical examination, and screening blood chemistries. Informed written consent was obtained from all subjects before the study, which was approved by the Institutional Committee on Human Investigation. Study Protocol Both cirrhotic and control subjects were instructed to consume a daily diet containing 1 g/kg body wt of protein and 30 kcal/kg body wt of nonprotein calories for 1 week before the study. After the patients fasted overnight, an accurately weighed Abbreviations used in this paper: BCM, body cell mass; BMI, body mass index; FFBM, fat-free body mass; IL, interleukin; TNF, tumor necrosis factor by the American Gastroenterological Association /98/$3.00

2 948 MCCULLOUGH ET AL. GASTROENTEROLOGY Vol. 115, No. 4 amount (approximately 2 g) of H 2 [ 18 O], 97.4 atom % access 18 O (Merck & Co. Rahway, NJ), was given orally at 8 AM for the measurement of total body water. 13 In addition, a 4-mmol oral dose of NaBr (approximately 320 mg of bromide) was also given for the measurement of extracellular water content. 13 Arterialized venous blood samples from a heated hand vein and breath samples were obtained at 30-minute intervals during the 6-hour study. Blood samples were centrifuged immediately, and the plasma samples were stored at 70 C until analysis. Breath samples for 18 O enrichment of expired CO 2 were collected by having the subject breathe normally through a mouthpiece and one-way valve to fill a 5-L anesthesia bag. An aliquot of the sample was then placed in an evacuated tube as described previously. 13,18 Immunoassays Serum leptin concentrations were measured by using a commercially available radioimmunoassay based on double antibody methodology 19 specific for human leptin (Linco, St. Charles, MO). The assay is linear to 100 ng/ml leptin and sensitive to 0.5 ng/ml. Serum TNF- concentrations were measured by a commercially available high-sensitivity enzymelinked immunosorbent assay specific for human TNF- (R&D Systems, Minneapolis, MN). The assay is linear to 32 pg/ml and sensitive to 0.18 pg/ml. For both leptin and TNF-, the intra-assay and interassay coefficients of variation were 6% and 8%, respectively. Analysis of Breath Samples The [ 18 O] enrichment of the CO 2 in expired air was measured by using an isotope-ratio mass spectrometer after separation of the CO 2 by cryogenic distillation in vacuum, as described previously. 13,18 Bromide Analysis Before analysis, 1.0 ml of serum was deproteinized by centrifuging through a PLGC Millipore ultrafilter (14-mm disc size; Waters Chromatography Division, Millipore Corp., Bedford, MA) using an Amicon micro-partition device (Division of W.R. Grace & Co., Danvers, MA). The protein-free serum ultrafiltrate obtained from the procedure was used for the subsequent bromide analysis. Chromatographic analysis of bromide was performed as reported previously with certain modifications by using a Waters high-performance liquid chromatography system. 13 Calculations Total body water (TBW) was calculated by measuring the dilution of H 2 [ 18 O] tracer. Because [ 18 O] equilibrates rapidly with water and CO 2 in the body in the presence of carbonic anhydrase, [ 18 O] enrichment was measured in the expired CO 2 as follows: TBW (kg) Dose of H 2[ 18 O] Administered (g) APE f, (E t E O ) 1000 where APE is [ 18 O] enrichment of H 2 [ 18 O] administered; f is fractionation factor for the equilibration of H 2 O and CO at 37 C; and E t and E o are [ 18 O] enrichments of expired CO 2 at plateau and at time 0. Extracellular water was calculated by the dilution of bromide in the calculation of the bromide space. 13 The corrected bromide space (CBS) is calculated from the serum bromide concentration according to the following formula: Br Dose Administered (mmol/l) CBS (1) , [Br F ] [Br B ] (mmol/l) where Br F is the final serum Br concentration, Br B is the basal serum Br concentration, 0.90 is the correction factor for Br distribution in nonextracellular sites, and 0.95 is the correction for Donnan s equilibrium. Correction for the water concentration in serum is not required in this calculation because Br concentrations were determined in a protein-free ultrafiltrate of serum. Fat-free body mass (FFBM) and body cell mass (BCM) were calculated from the measurements of total body and intracellular water content, respectively. It was assumed that water constitutes 73.25% of the FFBM 20 and 70% of the intracellular compartment. 21,22 The hydration of the intracellular compartment in cirrhotic subjects is the same or increased compared with controls. 21,22 Fat mass was calculated from the body weight and total body water gradient. The body mass index (BMI) was calculated from Weight (kg)/height (m 2 ). Nutritional status was estimated by a single observer (A.J.M.) as no, mild, or severe malnutrition from a global assessment based on a history of weight loss, muscle wasting, diminished fat stores, and evidence of vitamin deficiency (cheilosis or glossitis). Statistics All data are expressed as mean SEM. Comparisons between subject groups were performed using the Student t test for unpaired data. The linear correlation between two mutually dependent variables was determined and expressed as the correlation coefficient (r). Results Patient Profiles and Body Composition There was no significant difference in the distribution of Child Pugh class between men and women. The mean score was 7.8 in male and 7.4 in female cirrhotics; the number of Child Pugh class A, B, and C was 6, 8, and 4 in male and 4, 4, and 2 in female cirrhotics, respectively. Nutritional status based on subjective global assessment showed no, mild, or severe malnutrition to be present in 8, 8, and 2 male cirrhotics and 7, 2, and 1 female cirrhotics. This assessment of malnutrition correlated with Child Pugh score (r 0.52; P 0.05) but not with serum leptin levels or the measured body

3 October 1998 LEPTIN IN ALCOHOLIC CIRRHOSIS 949 compartments. All blood chemistries were within normal range for all control subjects (data not shown). As shown in Table 1, no significant differences were found in age, height, weight, or BMI (an estimate of percentage of body fat) between men and women in the control and cirrhotic groups. The body surface area was less in male cirrhotics (P 0.05) but did not differ between female controls and cirrhotics. Figure 1A and B shows the fat mass and BCM (expressed as percentage of body weight) in male (Figure 1A) and female (Figure 1B) subjects, as calculated from tracer dilution. The BMI is also provided as a comparison to the fat mass calculation derived by tracer dilution. Although there was no significant difference in the BMI between male cirrhotics and controls ( vs ), both the fat mass expressed as percentage of body weight ( vs ; P 0.05) and body cell mass expressed as percentage of FFBM ( vs ; P 0.001) were decreased in the male cirrhotics. In female cirrhotics, the BCM was also decreased compared with controls ( vs FFBM; P 0.002). However, in contrast to the males, neither the fat mass (20.5% 2.7% vs. 23.5% 1.2% body wt) nor the BMI ( vs ) was decreased in female cirrhotics. The fat mass (expressed as percentage of body weight) was significantly less in male than in female cirrhotics ( vs ; P 0.05), in male controls compared with female ( vs ; P 0.05), and in cirrhotics with ascites compared with those without ascites ( vs ; P 0.03). The BCM (expressed as percentage of FFBM) did not differ between female and male cirrhotics ( vs ), but was significantly less in female controls than in male ( vs ; P 0.003) and in ascitic compared with nonascitic cirrhotics ( vs ; P 0.001). Figure 2 shows the correlation between the two estimates of fat mass as a percentage of body weight; the BMI and the fat mass measured by tracer dilution. There was a significant correlation between these two measures of percentage of body fat in controls (r 0.52; P 0.02) but not in cirrhotics (r 0.05). Leptin Levels Figure 3 shows serum leptin levels expressed in both absolute terms and corrected per kilogram of fat mass. In absolute terms, leptin was increased in female ( vs ng/ml; P 0.02) but not in male ( vs ng/ml) cirrhotics. Leptin level was increased in both female cirrhotics ( vs ng/ml; P 0.001) and controls ( vs ng/ml; P.04) compared with their male counterparts. However, when expressed per kilogram of fat mass, leptin was increased in both male ( vs ; P 0.02) and female ( vs ; P 0.02) cirrhotics. Female cirrhotics had higher leptin levels than male cirrhotics ( vs ng/ml), but this did not quite reach significance (P 0.07). Female controls had higher leptin per kilogram of fat mass than male controls ( vs ; P 0.05). No significant difference was found between cirrhotics with and without ascites. TNF Levels As shown in Figure 4, serum TNF levels were increased above controls in both male ( vs pg/ml; P 0.004) and female ( vs pg/ml; P 0.003) cirrhotics. There were no significant differences between male and female subjects in either the cirrhotic or control groups. Cirrhotic patients with ascites had higher TNF levels than patients without ascites ( vs pg/ml; P 0.005). Correlations Figure 5 shows the correlations between leptin and fat mass as percentage of body weight. As shown, Table 1. Demographics and Calculated Body Composition in Male and Female Cirrhotic and Control Subjects Subjects Age (yr ) Weight (kg) Height (cm) Surface area (m 2 ) BMI Men Control (n 15) (NS) (NS) (NS) (P 0.05) (NS) Cirrhotic (n 18) Women Control (n 8) (NS) (NS) (NS) (NS) (NS) Cirrhotic (n 10) NOTE. Values in parentheses represent the level of significance for differences between men and women in the control and cirrhotic groups. NS, no significant difference.

4 950 MCCULLOUGH ET AL. GASTROENTEROLOGY Vol. 115, No. 4 Figure 1. Body composition in (A) male and (B) female subjects. Male cirrhotics had decreased fat mass and body cell mass, but only the body cell mass was decreased in female cirrhotics. *P 0.04; P vs. controls. leptin correlates with fat mass in both cirrhotics (r 0.62; P 0.001) and controls (r 0.61; P 0.006). There was a negative correlation between leptin and BCM in the total study population (r 0.49; P 0.005) and the cirrhotic group (r 0.42; P 0.04). No correlation between leptin and the BCM was observed in the control group (r 0.39). No significant correlation between leptin and TNF was found in either controls or cirrhotics. Figure 2. Correlation between BMI and fat mass/body weight (%). There was a significant correlation in (A) controls (r 0.52; P 0.02) but not in (B) cirrhotics. Discussion Leptin, a recently recognized protein, has been proposed to physiologically regulate body weight by suppressing appetite and increasing energy expenditure. 1,4,23 Because cirrhosis is associated with anorexia and increased energy expenditure, 12 the elevated serum leptin levels in cirrhosis, as reported in the present paper, may be involved in the high prevalence of malnutrition in cirrhotic patients. However, the biological significance of leptin is being scrutinized. Initially, low leptin levels (as observed in the ob/ob mouse model of obesity 6 ) had been proposed as a pathophysiological mechanism involved in human obesity. However, high leptin levels were observed in obese humans, 5 and these high leptin levels correlated directly with the BMI. 1,5 Other studies 4,24 have confirmed this direct correlation between leptin and BMI (i.e., the more Figure 3. Serum leptin levels. Absolute leptin levels were increased only in female cirrhotics (P 0.02) but were increased in both male (P 0.02) and female (P 0.02) cirrhotics when adjusted for fat mass. In all groupings, women had higher leptin levels than men. *P 0.02 vs. controls, same gender. P at least 0.05 vs. different gender in the same group.

5 October 1998 LEPTIN IN ALCOHOLIC CIRRHOSIS 951 Figure 4. Serum TNF levels. TNF was elevated to a similar degree in both male and female cirrhotics. *P fat, the higher the leptin level). These data suggest that resistance to the biological effects of leptin exists not only in obesity 1,3,5 but also in diabetes mellitus 4,25 and endstage renal disease. 14 Therefore, not only is it important to measure leptin levels, but these levels also must be related to body composition, particularly the fat mass. In addition, leptin levels are sex dependent, higher in women than in men. 4,5 These concepts are especially Figure 5. Correlation between leptin and fat mass. Serum leptin correlated with fat mass in both (A) cirrhotics (P 0.005) and (B) controls (P 0.005). important in cirrhosis because cirrhotics have genderdependent alterations in body composition 13 and sex steroids. 17 Using precise analytical measures of body composition in the present study, the fat mass was determined to be decreased in male cirrhotics but normal in female cirrhotics. The BCM, which is the central energy exchanging compartment, was decreased in both male and female cirrhotics and worsened with deterioration in liver function. The calculation used to estimate BCM from intracellular and extracellular water compartments (as used in the present study) is based on the fact that 73.25% of the fat-free mass is water and 70% of the cell is water. The percentage for the FFBM was derived from normal subjects and has not been measured in cirrhotic subjects. However, for our observation of a decreased BCM in cirrhosis to be incorrect, the intracellular water concentration in cirrhosis would have to be lower than normal. Although the concentration of water within the cell was not measured in the current study, previous measurements in muscle cells and white blood cells from cirrhotics have found intracellular water concentrations to be normal or increased. 21,22 Therefore, any inaccuracy in the measurement of BCM mass using the current figures would result in underestimating the decrease in BCM in cirrhosis (i.e., the BCM mass in cirrhosis may actually be lower than we have estimated). The importance of determining accurate body composition measurements while investigating physiological processes in cirrhosis is now widely accepted. 13 The present study emphasizes the importance of this issue, especially because leptin is derived solely from fat cells. In this study, only female cirrhotics had increased leptin levels based on absolute levels. However, when adjusted for fat mass, the leptin level was increased in both male and female cirrhotics. This observation would not have been made if only BMI was used to estimate fat mass. The excess in extracellular fluid that occurs at all stages of liver disease 12,26 causes weight-to-height parameters (such as the BMI) to be inaccurate in cirrhosis. This concept is confirmed in the present study, because the BMI correlated significantly with the measured fat mass in control subjects, but not in cirrhotics. Consistent with previous data, leptin levels were gender dependent in this study. Both female controls and female cirrhotics had higher serum leptin levels than their male counterparts. This observation of higher serum leptin levels in women could not be related simply to the higher fat mass in the female groups because leptin levels remained higher in the women even after adjusting for fat mass. The cause of this gender dependence in leptin levels

6 952 MCCULLOUGH ET AL. GASTROENTEROLOGY Vol. 115, No. 4 is not clear, but it may be related to the sensitizing effect of estrogen on cytokine release. 27 The cause of increased leptin levels in cirrhosis remains to be determined. Because the degradative pathways of leptin have not been completely defined, it is possible that elevated levels result from the accumulation of leptin caused by impaired hepatic clearance. However, this seems unlikely for a number of reasons. First, a study of leptin receptor tissue distribution has shown that the highest levels of expression for the receptor are found in the kidney and lung. 3 Second, the molecular weight and calculated plasma half-life for leptin 28 are similar to those of other peptide hormones that are degraded by the proximal renal tubule. 14 Lastly, in the present study, the serum leptin levels did not correlate with the severity of liver disease as characterized by either the Child Pugh class, its point score, or the presence or absence of ascites. A more likely cause is the stimulation of leptin secretion and/or production by one or more of leptin s known secretogogues. Of particular interest are the cytokines. TNF- and IL-1 stimulate leptin secretion. 7 A TNF- polymorphism has been described that exacerbates the alterations in leptin levels normally found among insulin-resistant subjects. 29 In addition, the leptin receptor is now known to be a single membrane spanning receptor most closely related to a class of cytokine receptors including IL-6. 3 TNF-, IL-1, and IL-6 concentrations are all elevated in alcoholic cirrhosis. 11 However, TNF- did not correlate with serum leptin levels in the present study; other possible mechanisms may be involved. Other known or possible leptin secretogogues that are also elevated in cirrhosis include insulin, 9 corticosteroids, 8 and nitric oxide. 30 Which, if any, of these factors might be involved in the leptin alterations in cirrhosis will require further study. Because most previous studies have investigated diseases or conditions that are associated with excess fat, the biological significance of elevated leptin levels in the face of apparent leptin resistance has been questioned. 1,3 In addition, whether hypermetabolism or appetite suppression 4 is the major mechanism of leptin-mediated weight loss is still unclear. However, in the present study, despite a normal or decreased amount of fat mass, increased leptin levels were found in cirrhosis (a disease associated with both hypermetabolism and anorexia), especially when adjusted for fat mass. Therefore, in contrast to previous studies on other diseases, the diminished BCM and fat mass in cirrhosis observed in the present study are at least consistent with the possibility that leptin may be exerting its biological effects of hypermetabolism and anorexia in this disease. In summary, this study raises the possibility that leptin may be involved in the malnutrition of cirrhosis. 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