Elevated serum leptin in patients with coronary artery ...

International Journal of Obesity (2000) 24, 369±375 ß 2000 Macmillan Publishers Ltd All rights reserved 0307±0565/00 $15.00 www.nature.com/ijo

Elevated serum leptin in patients with coronary artery disease: no association with the Trp64Arg polymorphism of the b3-adrenergic receptor K Stangl1*, I Cascorbi2, M Laule1, V Stangl1, M Vogt1, S Ziemer3, I Roots2, K Wernecke4, G Baumann1 and H Hauner5 1

Medizinische Klinik und Poliklinik I, Berlin, Germany; 2Institut fuÈr Klinische Pharmakologie, Berlin, Germany; 3Institut fuÈr Pathologische und Klinische Biochemie, Berlin, Germany; 4Institut fuÈr Biometrie der ChariteÂ, Berlin, Germany; and 5DiabetesForschungsinstitut an der Heinrich-Heine-UniversitaÈt DuÈsseldorf, DuÈsseldorf, Germany

BACKGROUND: Serum leptin is associated with the occurrence of cardiovascular risk factors but it is unknown whether leptin is also associated with cardiovascular disease. Another open question is whether the Trp64Arg polymorphism of the b 3-adrenergic receptor (b3-AR) is a determinant of circulating leptin. OBJECTIVES: We measured serum leptin concentrations in a large group of patients with angiographically assessed coronary artery disease (CAD) and investigated the relationship between the Trp64Arg polymorphism of the b3adrenergic receptor (AR) and serum leptin. PATIENTS AND METHODS: Leptin was measured in the fasting state in 1000 consecutive patients with angiographically con®rmed CAD by radioimmunoassay. The codon 64 T=C polymorphism of the b 3-AR gene was analysed by the polymerase chain reaction=restriction fragment length polymorphism (PCR=RFLP) technique. Controls were 1000 age-, gender- and weight-matched subjects without clinical signs of CAD. RESULTS: Serum leptin concentrations were signi®cantly higher in patients with CAD than in those without CAD (median: 6.8 vs 6.1 ng=ml, P < 0.001). In a multiple regression analysis leptin was found to be a determinant of CAD (P 0.005) along with established risk factors. No differences in serum leptin were observed between wild-type and heterozygous carriers of the Trp64Arg mutation of the b3-AR gene, whereas the small group of homozygous carriers had higher leptin due to their higher BMI. In a multiple linear regression analysis, body mass index, gender and fasting insulin were the main signi®cant determinants of serum leptin, whereas the b3-AR polymorphism had no effect. CONCLUSIONS: Patients with coronary artery disease exhibit higher serum leptin concentrations than age- and gender-matched controls of comparable BMI, indicating that leptin could contribute to the development of cardiovascular disease, possibly via activation of the sympathetic nervous system. The Trp64Arg variant of the b3adrenoceptor did not in¯uence serum leptin. International Journal of Obesity (2000) 24, 369±375 Keywords: b3-adrenergic receptor gene polymorphism; coronary artery disease; leptin; insulin; obesity

Introduction Leptin is a 16 kDa peptide which is almost exclusively produced by adipocytes and plays an important role in the regulation of energy balance.1,2 Serum leptin concentrations re¯ect adipose tissue mass,3 but may also be in¯uenced by pathophysiological conditions such as insulin resistance.4 It is now well known that leptin is subject to short-term regulation by a variety of hormones and metabolites.2 Recent data indicate positive associations between serum leptin and metabolic risk factors for cardiovascular disease,5 but no information is currently available on serum leptin *Correspondence: K. Stangl, Medizinische Klinik und Poliklinik, Kardiologie, ChariteÂ, Campus Mike, Schumannstr 20=21, 10117 Berlin. Received 22 March 1999; revised 16 August 1999; accepted 27 October 1999

concentrations in patients with coronary artery disease (CAD). Increasing evidence suggests that leptin has broader actions than originally believed, also including effects on sympathetic nerve and cardiovascular functions.6 In rodents, systemic administration of leptin increased sympathetic nerve activity in some organs, but did not acutely elevate arterial blood pressure and heart rate,7 whereas intracerebroventricular infusion of leptin increased both sympathetic nerve activity and blood pressure.8 In fasted mice, intraperitoneal infusion of leptin was followed by increases of circulating glucose, insulin and glucagon via sympathetic nerves.9 These ®ndings may be of interest for humans as it is known from clinical trials that a high sympathetic nerve activity is associated with an increased risk of cardiovascular disease.10 However, studies on rodent adipocytes and in humans also demonstrated that catecholamines exert

Elevated serum leptin in CAD K Stangl et al

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a suppressive effect on leptin production.11 ± 16 The b3adrenoceptor (b3-AR) appears to be critically involved in this down-regulation, at least in rodent adipose tissue. It has been reported that functional b3-adrenergic receptors are present in human adipose tissue, particularly in the intra-abdominal fat depots.17 ± 19 One of these studies postulated that the b3-AR plays a pathogenic role for the development of metabolic disturbances in abdominal obesity.19 Recent studies demonstrated that a mutation of the human b3-AR gene, characterized by substitution of arginine for tryptophan at position 64 (Trp64Arg), is associated with increased capacity for body weight gain, features of insulin resistance, and early onset of type-2 diabetes mellitus,20 ± 22 all considered to be cardiovascular risk factors. However, subsequent studies reported con¯icting results on the functional role of the b3-AR polymorphism with respect to body weight gain and metabolic disorders.23 ± 30 Irrespective of this controversy, it is tempting to speculate that the Trp64Arg polymorphism may be associated with a dysregulation of leptin production and may contribute to elevated serum leptin. It was consequently the objective of this crosssectional study ®rst to investigate the role of serum leptin as a possible determinant of CAD in humans and, in this context, to examine the contribution of the b3-AR polymorphism to serum leptin concentrations in patients with and without CAD.

based on both reference of patient case notes using WHO criteria,31 and angiographical ®ndings; patients with equivocal results were excluded. Body mass index (BMI) was calculated as weight (kg) divided by height (m) squared (kg=m2). Waist-tohip (WHR) was calculated as waist circumference divided by hip circumference (cm=cm). Subjects were de®ned as smokers if they were current smokers or had smoked within the last 10 y. All others were classi®ed as non-smokers. Patients were de®ned as having hypertension if they presented with a mean blood pressure 140=90 mmHg on repeated measurements under standardized conditions and=or were under antihypertensive drug treatment. Laboratory determinations

Blood was collected in the morning after an overnight fast of at least 10 h. EDTA-containing tubes served for analysis of plasma lipids. Total cholesterol, HDLcholesterol and triglycerides were measured by enzymatic methods (Boehringer, Mannheim, Germany). LDL-cholesterol was calculated using the Friedewald formula; apo-A1 and apo-B were determined by immunoturbidimetric assays (Tina-quant, Boehringer Mannheim). Measurement of serum leptin (Linco, St Charles, MO) and insulin (Pharmacia, Freiburg, Germany) were performed by radioimmunoassay according to the instructions of the manufacturers. Polymerase chain reaction=restriction fragment length polymorphism

Patients and methods Between October 1995 and January 1997, 1000 consecutive Caucasian patients from the Berlin area, who had been admitted for angiography for suspected CAD and=or for elective or emergency interventions, were recruited at the ChariteÂ University Medical Center. In a case ± control design, an additional 1000 patients matched for age, gender and race served as controls. The patients of the control group had no evidence of CAD or of other cardiovascular diseases on the basis of data from patient histories, physical examination, ECG and echocardiography. We excluded individuals with severe systemic diseases which could have potentially in¯uenced variables of the cardiovascular risk factor pro®le. All cases and controls gave written consent according to the study protocol which was approved by the Ethical Committee of the ChariteÂ Hospital. Cases were classi®ed by angiographic criteria. Angiograms were reviewed by experienced cardiologists who were blind to patient identity and outcome. CAD was de®ned as stenosis 50% in at least one major coronary artery or major branch. The severity of CAD was classi®ed according to the number of affected arteries, ie as one-, two- or three-vessel disease. Diagnosis of myocardial infarction was International Journal of Obesity

A 10 ml venous blood sample, drawn with EDTA as anticoagulant, was obtained from each subject and stored at 720 C. After thawing, erythrocytes were disrupted for 30 min in a hypoosmolaric buffer containing 155 mmol=l NH4Cl, 10 mmol=l KH2CO3 and 0.1 mmol=l EDTA, at pH 8.0. Leukocytes were isolated by centrifugation at 1000 g at 4 C for 30 min, resuspended in 20 mmol=l Tris ± HCl, 2 mmol=l EDTA and 30 mmol=l NaCl, at pH 7.5, and stored at 720 C. DNA was isolated manually by a standard three-step phenol=chloroform extraction32 after digestion with proteinase K (Boehringer, Mannheim), or with a 341A Applied Biosystems DNA extractor (Biosystems, Weiterstadt, Germany). DNA was dissolved overnight at 55 C in 10 mmol=l Tris and 1 mmol=l EDTA buffer (pH 8.0), and stored at 4 C until further analysis. The T=C point mutation leading to a Trp=Arg exchange in codon 64 of the b3-AR gene was evaluated by polymerase chain reaction=restriction fragment length polymorphism (PCR=RFLP). Primers were designed from the sequence published by Van Spronsen et al.33 A 192-bp fragment taken from exon 1 of the b3-AR gene was ampli®ed using 0.5 units of AmpliTaq Gold1 (Perkin Elmer, Weiterstadt, Germany), 0.2 mmol=l deoxynucleotides, 0.2 mmol=l downstream-primer b3-1 50 GGC AAC CTG CTG GTC ATC, 0.2 mmol=l upstream-primer b3-2 50 -GTC


CAC CGA GGT CCA CAG and 1.0 mmol=l MgCl2 in a ®nal volume of 25 ml. PCR conditions were 45 cycles for 30 s at 94 C, 30 s at 57 C, and 30 s at 72 C using a Perkin-Elmer GeneAmp1 9600 or 9700 thermocycler. PCR ef®ciency was checked on a 2% agarose gel, for 20 min at 120 V. A 15 ml aliquot of the ampli®ed product was incubated with 90 units of MspI (New England Biolabs, Schwalbach, Germany), in a ®nal volume of 25 ml, at 37 C overnight. Fragments were separated on 3.5% NuSieve 3:1 gel (Biozym, Hessisch Oldendorf, Germany) for 75 min at 100 V. The wild-type presented a constitutive restriction site resulting in a 118- and a 74-bp fragment, whereas mutated alleles presented three fragments of 88, 74, and 30 bp.

Statistical analysis

In this case ± control study each group originally comprised 1000 subjects. Samples were matched for age, gender and race but did not include matched pairs. Values are presented as median and 25th and 75th percentiles. Comparisons with respect to continuous variables were performed using the Mann ± Whitney U-test, as most continuous variables were not normally distributed, even after log transformation. Differences in frequencies were tested by w2-test. We employed multiple logistic regression analysis with CAD as dependent variable including age, BMI, gender, leptin, fasting insulin, diabetes, smoking, hypertension and hypercholesterolaemia, to examine the relationship between leptin and CAD. Determinants of serum leptin levels were further analysed by linear regression analysis with BMI, gender, b3-AR genotypes and other possible determinants as covariates.

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Results Table 1 presents selected characteristics of the case and the control group including risk factors for CAD. The groups were comparable for age, gender distribution, BMI and waist=hip ratio, but differed signi®cantly with respect to the prevalence of diabetes mellitus, hypercholesterolaemia, hypertension, and smoking Ð as well as for serum concentrations of triglycerides, total cholesterol, LDL-cholesterol, apoA1 and apo-B. The case group demonstrated varying degrees of severity of CAD; 66.7% suffered from acute myocardial infarction or had a history of previous myocardial infarction. Serum leptin was found to be signi®cantly higher in the subjects with angiographically assessed CAD compared to the control subjects (6.8 vs 6.1 ng=ml, P < 0.001; Mann ± Whitney U-test; Table 1). This was also true when males and females were analysed separately (males: 5.8 vs 5.0 ng=ml; females, 15.9 vs 12.8 ng=ml, each P < 0.01; Mann ± Whitney U-test). In addition, fasting insulin was elevated in the patients with CAD as compared to controls without evidence of CAD (13.3 vs 11.5 mU=ml, P < 0.001; Mann ± Whitney U-test). Presence of diabetes, hypertension and hypercholesterolaemia were associated with higher plasma leptin concentrations, whereas smokers had lower leptin concentrations than non-smokers, both in the cases and controls (Table 2). In a multiple logistic regression analysis with CAD as dependent variable, serum leptin was found to be an independent determinant of angiographically assessed coronary heart disease in addition to other established cardiovascular risk factors (Table 3). When both groups were subdivided according to the b3-AR genotype, the respective difference in leptin

Table 1 Basal characteristics of the case and control group. Values for continuous variables are presented as median and 25th and 75th percentiles

Age (y) Female (%) History (%): diabetes mellitus hypertension hypercholesterolaemia smoking myocardial infarction Body mass index (kg=m2) Waist=hip ratio Triglycerides (mmol=l) Cholesterol (mmol=l) HDL-cholesterol (mmol=l) LDL-cholesterol (mmol=l) Apo-A1 (mg=ml) Apo-B (mg=ml) Leptin (ng=ml) Fasting insulin (mU=ml)

Cases (n 1000)

Controls (n 1000)

60.6 (55.1 ± 67.1) 24.1

60.5 (54.5 ± 66.5) 24.1

22.8 55.2 52.7 44.0 66.7 (24.2 ± 28.6) (0.9 ± 1.0) (1.3 ± 2.4) (5.1 ± 6.6) (0.9 ± 1.4) (3.1 ± 4.5) (1.2 ± 1.6) (1.0 ± 1.4) (4.3 ± 11.2) (9.1 ± 21.2)

11.4 35.9 30.3 35.2 0 (24.0 ± 28.7) (0.9 ± 1.0) (1.1 ± 2.0) (4.7 ± 6.3) (0.9 ± 1.4) (2.9 ± 4.2) (1.2 ± 1.6) (0.9 ± 1.3) (3.5 ± 10.8) (8.5 ± 16.6)

26.3 1.0 1.7 5.8 1.1 3.8 1.4 1.2 6.8 13.3

26.0 1.0 1.5 5.5 1.1 3.6 1.4 1.1 6.1 11.5

P-value 0.477 0.001* 0.001* 0.001* 0.001* 0.182 0.659 0.001 0.001 0.727 0.001 0.01 0.001 0.001 0.001

Differences between groups were tested using the Mann ± Whitney U-test for continuous variables and w2-test (*), respectively. International Journal of Obesity


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Table 2 Serum leptin concentrations (median, 25th and 75th percentiles) in subjects with CAD and controls according to risk factorsa CAD Diabetes present Diabetes absent P-value Hypertension present Hypertension absent P-value

Controls

7.5 (5.1 ± 12.4) 6.6 (4.1 ± 11.1) 0.013

7.0 (3.9 ± 11.3) 6.0 (3.4 ± 10.7) 0.189

7.7 (4.9 ± 13.6) 6.0 (3.8 ± 9.6) < 0.001

7.5 (4.4 ± 13.6) 5.4 (3.1 ± 9.5) < 0.001

7.0 (4.6 ± 11.8)

7.2 (4.1 ± 12.2)

6.6 (4.0 ± 10.9)

5.8 (3.3 ± 9.9)

Hypercholesterolaemia present Hypercholesterolaemia absent P-value

0.037

< 0.001

Smokers Non-smokers P-value

6.4 (3.9 ± 10.5) 7.1 (4.5 ± 11.9) 0.008

5.2 (2.7 ± 9.0) 6.8 (4.1 ± 11.8) < 0.001

a

Mann ± Whitney U-test.

concentrations between the patients with CAD and the control subjects remained unchanged, but was statistically signi®cant only in the wild-type group and the group of homozygote carriers of the mutation (Table 4). A striking ®nding in the case group was that leptin concentrations were almost twice as high among the nine homozygous carriers than among both the wildtype and heterozygous carriers (14.1 vs 6.8 and 6.7 ng=ml, respectively; P < 0.01 each). In the control group, serum leptin also tended to be higher in the homozygous carriers than in the two other subgroups (Table 4). In Figure 1, leptin was plotted against BMI for the wild-type and the heterozygote variant stratiTable 3 Multiple logistic regression analysis with CAD as dependent variable Variable

b coefficient

s.e.

P-value

7 0.003 7 0.348 7 0.470 0.049 0.014 0.024 0.739 0.494 0.099

0.006 0.150 0.016 0.012 0.004 0.009 0.102 0.106 0.100

0.955 0.020 0.026 0.000 0.002 0.005 0.000 0.000 0.000

Age (y) Gender BMI (kg=m2) Diabetes Fasting insulin (mU=ml) Leptin (ng=ml) Hypertension Smoking Hypercholesterolaemia

Table 4 Serum leptin in cases and controls according to the b3-AR genotype. Data are presented as median (25th and 75th percentiles)a Leptin (ng=ml) b3-AR genotype Trp=Trp Trp=Arg Arg=Arg

a

Cases

Controls

P-value

6.8 (4.2 ± 11.1) (n 826) 6.7 (4.3 ± 11.1) (n 157) 14.1 (6.6 ± 25.1) (n 9)

6.2 (3.6 ± 10.6) (n 813) 6.7 (3.3 ± 10.8) (n 171) 9.2 (2.8 ± 15.3) (n 4)

< 0.002

Mann ± Whitney U-test.

International Journal of Obesity

n.s. < 0.01

®ed for gender and group. Only in the females with the Trp=Arg genotype did the regression lines differ between cases and controls, indicating that female heterozygote carriers of the Trp64Arg mutation in the CAD group had higher leptin concentrations at a given BMI than the female heterozygote carriers of the control group. The carriers with the Arg=Arg genotype were excluded from this analysis due to the small number of subjects. We ®nally investigated which factors contributed to serum leptin concentrations. A multiple linear regression analysis was performed for cases and controls separately. This calculation revealed that in the case group gender, BMI, fasting insulin, age and presence of diabetes were signi®cant determinants of serum leptin, whereas in the control group gender, BMI and fasting insulin independently contributed to serum leptin (Table 5). In contrast, the other variables in the model including the b3-AR genotype had no signi®cant effect on serum leptin concentrations.

Discussion The recent identi®cation and characterization of leptin introduced a new candidate which may contribute to some of the metabolic disturbances associated with obesity2,4 and, subsequently, also with its consequences, particularly cardiovascular disease. In clinical studies, plasma concentrations of leptin were found to be positively associated with insulin resistance and metabolic risk factors for cardiovascular disease.5 However, to date no information is available on the relationship between circulating leptin levels and coronary artery disease. On the basis of a large sample of patients, we now report higher serum leptin concentrations in subjects with angiographically assessed CAD as compared to controls without clinical evidence of CAD, although both groups were comparable with regard to age and gender distribution and did not differ in BMI. The statistical analysis using a multiple regression model showed that serum leptin was a signi®cant determinant of CAD in addition to other established cardiovascular risk factors. However, it must be kept in mind that the design of the study does not allow the conclusion that leptin is a causal risk factor for coronary artery disease. To study a possible cause ± effect relationship a prospective study would be necessary. How can the association between leptin and CAD be explained? It is well known that leptin increases the activity of the sympathetic nervous system.2,6 This has been demonstrated in animal studies after administration of leptin into the cerebroventricular ¯uid or at peripheral sites.7 ± 9 In a recent study in healthy adult subjects by Snitker et al, there was a positive correlation between basal muscle sympathetic nerve activity


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Figure 1 Relationship between BMI and serum leptin according to gender and b3-AR genotype (wildtype and heterozygote variant Table 5 Determinants of serum leptin, separated according to case and control group in a multiple linear regression analysis with serum leptin as dependent variablea

Age (y) Gender BMI (kg=m2) Insulin (mU=ml) Diabetes

b coefficient, cases=controls

s.e., cases= controls

P-value, cases=controls

0.079=0.023 11.690=9.265 0.947=0.850 0.047=0.041 7 0.084=0.042

0.024=0.018 0.497=0.403 0.059=0.045 0.011=0.012 0.038=0.043

0.001=0.201 0.000=0.000 0.000=0.000 0.000=0.001 0.027=0.329

a

b3-AR polymorphism genotypes, hypertension, hypercholesterolaemia and smoking did not reach statistical signi®cance in either group.

(MSNA), a direct measure of sympathetic out¯ow, and plasma leptin concentrations of a magnitude similar to that between MSNA and percentage body fat. Therefore, leptin could be the peripheral signal that explains the correlation between MSNA and percentage body fat.34 However, the situation is complicated by the fact that body fat is associated with both leptin and MSNA, by the poorly characterized phenomenon of leptin resistance,3 by the possible stimulatory effect of insulin on MSNA36 and by the observation that an increased sympathetic out¯ow in turn may decrease leptin production in adipose tissue.11 ± 16 Therefore, further detailed studies are required to obtain a better understanding of the role of leptin in the regulation of the sympathetic nervous system in humans. In a further analysis gender, BMI and fasting insulin proved to be signi®cant determinants of serum leptin. Separate effects were also observed for

age and the presence of diabetes in the patients with CAD. In contrast, the b3-AR genotype and the other parameters used in the model including hypercholesterolaemia, hypertension and smoking had no signi®cant in¯uence on serum leptin. As gender distribution and BMI were similar between the two groups, although the latter gives only limited information on percentage body fat, the role of fasting insulin is an interesting ®nding. The moderately elevated leptin concentrations in patients with CAD, but also in patients with cardiovascular risk factors such as hypertension, could be at least partially due to the higher insulin levels compared with the control group. It is well established that insulin is able to increase leptin production in cultured human fat cells.37 Furthermore, clinical studies using the euglycaemic ± hyperinsulinaemic clamp technique indicate that prolonged hyperinsulinaemia increases serum leptin levels in a dose-dependent fashion.38 There is some evidence that the b3-AR may be involved in the pathogenesis of at least some obesitylinked metabolic disturbances.19 This adrenergic receptor subtype was recently found to be expressed in human adipose tissue, particularly in the omental fat depot,17 and may contribute to the regulation of catecholamine-induced lipolysis.18 To date, there is no information available on the role of the b3-AR in leptin production in humans, whereas in animals b3AR agonists were found to be potent suppressors of leptin production.11 ± 14 Clinical studies reported an association between a polymorphism of the b3-adrenoceptor at codon 64 and International Journal of Obesity


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insulin resistance, weight gain and other metabolic disorders suggesting that this polymorphism could be of clinical signi®cance for humans,20 ± 22 although subsequent studies did not uniformly con®rm these ®ndings.23 ± 30 Irrespective of this controversy, we tested the hypothesis that a possible dysfunction of the b3-AR due to a mutation at codon 64 could affect leptin production in adipose tissue and may result in elevations of serum leptin concentrations. However, the results presented here clearly indicate that at least the heterozygous form of the mutation has no physiologically signi®cant effect on serum leptin. An interesting ®nding was that serum leptin concentrations were elevated among the homozygous carriers of the b3-AR polymorphism. It appears justi®ed to assume that this observation is the result of the enlarged body-fat mass in this group rather than of diminished suppression of leptin production due to the mutation. The mean BMI in the small group of homozygote carriers of the mutation was signi®cantly higher than in the carriers of the wild-type and the heterozygous mutation (median BMI: 28.0 vs 26.1 vs 26.0 kg=m2). In conclusion, the results of this study reveal: (1) higher serum concentrations of leptin in patients with angiographically assessed CAD as compared to ageand gender-matched controls with similar BMI which may support the hypothesis that leptin is a determinant of cardiovascular disease possibly via its stimulatory action on the sympathetic nervous system; and (2) the lack of an association between the Trp64Arg polymorphism of the b3-AR gene and serum leptin arguing against a dysregulation of leptin production by this mutation. The elevated leptin concentrations in patients with coronary artery disease may partially be the consequence of elevated serum insulin concentrations which are also known to activate sympathetic nerve activity. Further prospective studies are required to assess the role of leptin in the pathogenesis of coronary artery disease. Acknowledgements

We wish to thank Dr R Michael, Department of Nuclear Medicine, ChariteÂ, Berlin for providing expertise and technical assistance in leptin and insulin determinations. Parts of this work were supported by the German Federal Ministry of Education, Science, Research and Technology (grant no 01EC9408=0). References

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