- Email: [email protected]
Serum leptin levels in patients with hyperlipidemias
APPLIED NUTRITIONAL INVESTIGATION
Serum Leptin Levels in Patients With Hyperlipidemias Martin Haluzı´k, MD, PhD, Jirı´ Fiedler, MD, Jara Nedvı´dkova´, PhD, and Richard Cˇesˇka, MD, PhD rd From the 3 Medical Department, 1st Faculty of Medicine, Charles University, Prague; and the Institute of Endocrinology, Prague, Czech Republic Leptin is a protein hormone produced by adipocytes that reflects the body fat content. The aim of our study was to compare serum leptin levels in randomly selected untreated males and females with hypercholesterolemia and combined hyperlipidemia and in healthy control subjects matched for age and body mass index and to study the relations between leptin and serum lipids and lipoproteins. No statistically significant differences in serum leptin levels were found between the male control group (5.26 ⫾ 2.81 ng/mL⫺1) and the male group with hypercholesterolemia (8.16 ⫾ 3.85 ng/mL⫺1) or combined hyperlipidemia (7.51 ⫾ 4.83 ng/mL⫺1) and between the female control group (13.0 ⫾ 8.12 ng/mL⫺1) and the female group with hypercholesterolemia (15.36 ⫾ 8.89 ng/mL⫺1) or combined hyperlipidemia (18.63 ⫾ 10.15 ng/mL⫺1). Leptin concentration in male group with hypercholesterolemia did not differ significantly from the female control group; in the other male groups, leptin levels were significantly lower than those of the other female groups. Serum leptin levels in all studied groups except for the male group with hypercholesterolemia positively correlated with body mass index. Serum leptin levels correlated negatively with high-density lipoprotein cholesterol in the female group with hypercholesterolemia (r ⫽ ⫺0.67, P ⬍ 0.01) and the male group with combined hyperlipidemia (r ⫽ ⫺0.56, P ⬍ 0.01). A positive correlation between serum leptin and high-density lipoprotein cholesterol (r ⫽ 0.67, P ⬍ 0.01) and between leptin and lipoprotein (a) (r ⫽ 0.71, P ⬍ 005) was found in female group with combined hyperlipidemia. No other significant relationships between leptin and serum lipids or lipoproteins were found. We conclude that serum leptin levels in patients with hyperlipidemias do not significantly differ from those healthy control subjects matched by age and body mass index. Nutrition 2000; 16:429 – 433. ©Elsevier Science Inc. 2000 Key words: leptin, body mass index, hyperlipidemia, cholesterol, triacylglycerols
INTRODUCTION Leptin is a protein hormone produced by adipocytes that was identified by cloning the mouse ob gene.1 This substance reflects in most cases the body fat content i.e., its serum concentrations are elevated in obese people in comparison with lean people, and a strong positive correlation has been found between leptin and body fat content and between leptin body mass index (BMI).2,3 Some studies have shown that the degree of positive correlation is higher between leptin and subcutaneous fat than between leptin and visceral fat.4 Serum leptin levels are significantly higher in females than in males of similar age, BMI, and body fat content.2 It is not clear whether this sex difference is estrogen or gestagen dependent. Studies concerning the influence of estrogens or progesterone on serum leptin levels have shown rather conflicting results.5– 8 Information concerning relations between serum leptin concentrations and the levels of serum lipids are limited. Leyva et al.9 studied the relation of plasma leptin and components of a meta-
This study was supported by grants IGA MHCR 4041–3 and 5455–3 and by grant CR 206017-01 from the Ministry of Education. Correspondence to: Martin Haluzı´k, MD, PhD, 3rd Medical Department, 1st Faculty of Medicine, U nemocnice 1, Praha 2, 128 08, Czech Republic. E-mail: [email protected] Date accepted: July 18, 1999. Nutrition 16:429 – 433, 2000 ©Elsevier Science Inc., 2000. Printed in the United States. All rights reserved.
bolic syndrome X in a group of middle-aged men. A positive correlation of leptin with BMI, systolic and diastolic blood pressure, fasting triacylglycerols, serum uric acid, fasting glucose and insulin, and negative correlation with insulin sensitivity were found in this study. No correlation between leptin and high-density lipoprotein (HDL) cholesterol concentrations was observed. The relation of serum leptin and a series of anthropometric and metabolic parameters in growth-hormone– deficient hypopituitary adults was investigated by Al-Shoumer et al.10 Serum leptin levels in hypopituitary subjects were significantly higher than those of healthy controls and correlated positively with BMI and body fat mass, whereas no significant relation with fasting insulin growth factor-I, glucose, insulin, total, HDL, low-density lipoprotein (LDL) cholesterol and triacylglycerols were found. Rainwater et al.11 tested the relation of serum leptin levels and nine different measures of HDL phenotype. Only two of these measures (HDL triacylglyerol concentrations and the proportion of apolipoprotein AI (apo A-I) on HDL particles larger than HDL) were independently correlated with leptin in this study. Couillard et al.12 compared leptin levels and metabolic profiles of men with and without ischemic heart disease. No differences in leptin levels between the two studied groups and no association between leptin and plasma lipid and lipoprotein concentrations, with the exception of a positive correlation between leptin with plasma triacylglycerols, were found. The relation of leptin to metabolic and anthropometric parameters in women with polycystic ovary syndrome was investigated by Chapman et al.13 No differences in serum leptin levels between the control group and the group with polycystic ovary syndrome were observed. Leptin levels in both groups correlated 0899-9007/00/$20.00 PII S0899-9007(00)00288-4
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Nutrition Volume 16, Number 6, 2000 TABLE I.
DATA FOR MALE AND FEMALE PATIENTS AND CONTROL SUBJECTS WITH REGARD TO HYPERLIPIDEMIA*
Age (y)
BMI (kg/[m2]⫺1)
Leptin (ng/ml⫺1)
Cholesterol (mmol/L⫺1)
Triacylglycerols (mmol/L⫺1)
HDL (mmol/L⫺1)
25.28 ⫾ 5.06
13.0 ⫾ 8.12 (2,6) 5.26 ⫾ 2.81 (1,3,4)(3,4,5,6) 15.36 ⫾ 8.89 (2,5,6) 18.63 ⫾ 10.15 (2,5,6) 8.16 ⫾ 3.85 (3,4) 7.51 ⫾ 4.83
4.98 ⫾ 0.62 (3,4,5,6) 5.16 ⫾ 0.64 (4,6) 7.34 ⫾ 1.31 (1,2) 7.8 ⫾ 1.09 (1,2) 7.4 ⫾ 0.85 (1,2) 7.0 ⫾ 1.14 (1,3,4)
1.3 ⫾ 0.50 (4,6) 1.35 ⫾ 0.57
1.64 ⫾ 0.52
Group
n
NC-F
14
45.86 ⫾ 7.6
NC-M
17
44.31 ⫾ 11.56 26.07 ⫾ 3.44
H-F
21
48.83 ⫾ 14.38
CH-F
12
48.61 ⫾ 13.65 26.21 ⫾ 4.72
H-M
12
45.26 ⫾ 12.11 26.35 ⫾ 2.05
CH-M
24
46.77 ⫾ 13.91 26.96 ⫾ 5.55
25.1 ⫾ 5.49
1.44 ⫾ 0.49 (4,6) 3.14 ⫾ 0.8 (1,2,3,5) 1.51 ⫾ 0.27 (4,6) 3.07 ⫾ 0.059 (1,2)
LDL (mmol/L⫺1)
2.74 ⫾ 0.44 (3,4,5,6) 1.42 ⫾ 0.53 3.11 ⫾ 0.70 (3,4,5,6) 1.89 ⫾ 0.51 4.9 ⫾ 1.03 (6) (1,2) (12) 1.88 ⫾ 1.05 4.81 ⫾ 1.13 (1,2) 1.43 ⫾ 0.29 4.99 ⫾ 1.43 (1,2) 1.15 ⫾ 0.32 4.31 ⫾ 0.95 (1,2,3,5) (3) (1,2)
lp(a)
apoB
—
—
—
—
0.59 ⫾ 0.49
1.37 ⫾ 0.33
0.27 ⫾ 0.33
1.16 ⫾ 0.6
0.31 ⫾ 0.13
1.31 ⫾ 0.37
0.33 ⫾ 0.24
1.29 ⫾ 0.24
* Data are expressed as mean ⫾ standard deviation. Numbers in parentheses denote significant intergroup differences (P ⬍ 0.05, Dunn’s test). apoB, apolipoprotein B; BMI, body mass index; CH-F, female group with combined hyperlipidemia; CH-M, male group with combined hyperlipidemia; HDL, high-density lipoprotein; H-F, female group with hypercholesterolemia; H-M, male group with hypercholesterolemia; LDL, low-density lipoprotein; lp(a), lipoprotein (a); NC-F, female control group; NC-M, male control group
positively only with BMI and HDL cholesterol. No significant relations were found between the other serum lipid parameters and leptin. Hirose et al.,14 who studied the relation of leptin levels with hematopoesis in adolescents, found a positive correlation of serum leptin levels and BMI, leukocyte count, and HDL cholesterol. The relation of serum leptin to lipids or lipoprotein levels is not definitely clear. In most of these studies, no relation of serum leptin to total and LDL cholesterol were found; in some studies, a positive correlation between leptin and triacylglycerols or between leptin and HDL cholesterol was reported. Moreover, to our best knowledge, no study investigating the relation of serum leptin levels to lipid concentrations in patients with hyperlipidemia has been published thus far. Leptin is a strong regulator of food intake and has a series of complex central and peripheral metabolic effects. Therefore, we hypothesized that the changes in serum leptin levels could contribute to lipid metabolism alterations in patients with hyperlipidemia. We compared serum leptin concentrations in patients of both sexes with different types of hyperlipidemia with healthy control subjects matched for age and BMI.
PATIENTS AND METHODS Sixty-nine patients (36 males and 33 females) were included in the study. The patients represent a randomly selected sample of subjects examined in the Lipid Clinic of the University Hospital, Prague because of hyperlipidemia. Subjects were matched by age and BMI. None of the patients had diabetes and none was administered hypolipidemic drugs or medication known to affect food intake and/or lipid metabolism. None of patients kept any special diet. The patients were grouped according to type of hyperlipidemia: 21 untreated females with hypercholesterolemia (total cholesterol concentration above 5.8 mmol/L⫺1, triacylglycerol concentration below 2.00 mmol/L⫺1), 12 untreated females with combined hyperlipidemia (total cholesterol concentration above 5.8 mmol/L⫺1, triacylglycerol concentration above 2.00 mmol/ L⫺1), 12 untreated males with hypercholesterolemia (total cholesterol concentration above 5.8 mmol/L⫺1, triacylglycerol concentration below 2.00 mmol/L⫺1), and 24 males with combined hyperlipidemia (total cholesterol concentration above 5.8 mmol/ L⫺1, triacylglycerol concentration above 2.00 mmol/L⫺1). Thirtyone subjects (14 females and 17 males) with total cholesterol
levels below 5.8 mmol/L⫺1 and triacylglycerols levels below 2.00 mmol/L⫺1 formed the control group. The study protocol was approved by the local ethical committee. All of the subjects included in the study were instructed about the purpose of the study and gave their consent to participate. Subjects were measured and weighed. The blood samples for leptin and serum lipid determination were taken after an overnight fast (14 h) at the same time (approximately at 8 AM). The serum leptin concentrations were determined with a commercial sandwich enzyme immunoassay (BioVendor, Czech Republic, Prague). This human leptin assay has been developed to measure human leptin in plasma or serum. It is a completely homologous assay because the antibody is raised against highly purified human leptin and standards are prepared with human leptin. The limit of sensitivity for the human leptin assay was 0.2 ng/mL⫺1. The results of this assay highly correlate with those of commercial RIA radioimmunossay (Linco, St. Charles, MO, USA). Serum lipid parameters were measured in the Department of Clinical Biochemistry of the University Hospital, Prague by standard laboratory methods. Serum LDL cholesterol levels were computed by using Friedwald’s formula. The lp(a) and apoB levels ˇ eska15). were measured as described previously (C Statistical analysis was performed by SigmaStat statistical software (Jandel Scientific, San Rafael, CA, USA). Means and standard deviations were computed, and the significance of differences between groups was evaluated with analysis of variance and Dunn’s test. The correlations between groups were determined by the Spearman correlation test. The distribution of apolipoprotein E isoforms in the study groups was compared using Fisher’s test. Significance level was set at P ⬍ 0.05.
RESULTS Groups were matched by age and BMI. Serum leptin levels in the male control group did not differ from the male groups with hypercholesterolemia or combined hyperlipidemia but were significantly lower than the levels in all female groups (Table I, Fig. 1). No difference was observed between leptin levels in males with hypercholesterolemia versus males with combined hyperlipidemia. Serum leptin levels between the female control group and the female group with hypercholesterolemia or combined hyperlipid-
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TABLE II. CORRELATION OF SERUM LEPTIN LEVELS WITH DIFFERENT VARIABLES ACROSS STUDY GROUPS*
FIG. 1. Serum leptin levels in female and male control groups (NC), female and male groups with hypercholesterolemia (H), and female and male groups with combined hyperlipidemia (CH) are expressed as means ⫾ SD. Statistically significant differences across groups (Dunn’s test, P ⬍ 0.05): 1, significantly different from the female control group; 2, significantly different from the female group with hypercholesterolemia; 3, significantly different from the female group with combined hyperlipidemia; 4, significantly different from the male control group; 5, significantly different from the male group with hypercholesterolemia; 6, significantly different from the male group with combined hyperlipidemia.
emia did not differ either (Table I, Fig. 1). Leptin levels in the male group with hypercholesterolemia did not differ from those of the female control group but were significantly lower than in the female groups with hypercholesterolemia and combined hyperlipidemia (Table I, Fig. 1). Serum leptin levels in the male group with combined hyperlipidemia were significantly lower than those in the female control group and in the female groups with hypercholesterolemia and combined hyperlipidemia (Table I, Fig. 1). Serum leptin levels correlated positively with BMI in all study groups, with the exception of the male group with hypercholesterolemia, where no significant relation between serum leptin and BMI was observed (Table II). The significant negative correlation between serum leptin levels and HDL cholesterol was found in the female group with hypercholesterolemia (Fig. 2) and in the male group with combined hyperlipidemia (Fig. 3). In the female group with combined hyperlipidemia, a positive correlation between leptin and HDL levels (Fig. 4) and between leptin and lipoprotein (a) was found (Table II). No additional significant relationship between leptin and the other lipid metabolism parameters was found in the other groups. Multivariate regression analysis was performed for groups in which significant associations between leptin and HDL were found. The partial correlation analysis confirmed significant correlations between serum leptin and HDL levels in the female groups with hypercholesterolemia and combined hyperlipidemia and in the male group with combined hyperlipidemia even after adjustment for BMI and age. The relative distribution of apolipoprotein E isoforms did not significantly differ in the four hyperlipidemia groups (results not shown). The patients were then assigned to two subgroups with higher and lower leptin levels (below and above 10 ng/mL⫺1) to analyze the relation of leptin levels and the apolipoprotein-E isoform distribution. The relative distribution of apolipoprotein-E isoforms did not differ significantly in these two subgroups.
Group
n
BMI
HDL
lp(a)
NC-F
14
NS
—
NC-M
17
NS
—
H-F
21 12
H-M CH-M
12 24
r ⫽ ⫺0.67 P ⬍ 0.01 r ⫽ 0.67 P ⬍ 0.01 NS r ⫽ ⫺0.56 P ⬍ 0.01
NS
CH-F
r ⫽ 0.71 P ⬍ 0.01 r ⫽ 0.62 P ⬍ 0.01 r ⫽ 0.74 P ⬍ 0.01 r ⫽ 0.64 P ⬍ 0.05 NS r ⫽ 0.57 P ⬍ 0.01
r ⫽ 0.71 P ⬍ 0.05 NS NS
* According to Spearman’s correlation test. BMI, body mass index; CH-F, female group with combined hyperlipidemia; CH-M, male group with combined hyperlipidemia; HDL, highdensity lipoprotein; H-F, female group with hypercholesterolemia; H-M, male group with hypercholesterolemia; lp(a), lipoprotein (a); NC-F, female control group; NC-M, male control group; NS, not significant
DISCUSSION The aim of this study was to compare the serum leptin levels in male and female patients patients with hypercholesterolemia and combined hyperlipidemia with healthy control subjects of both sexes matched by age and BMI. No significant differences in serum leptin levels were found between the control groups and the hyperlipidemia groups in either males or females. As expected, a strong positive correlation between leptin and BMI was found in all studied groups, with the exception of the male group with hypercholesterolemia. The suprising dissociation between leptin and BMI in this group was accompanied by higher serum leptin levels than expected from BMI values. However, the difference in leptin levels in the male group with hypercholesterolemia versus the male control group and the male group with combined hyperlipidemia did not reach statistical significance.
FIG. 2. The correlation of serum leptin levels with HDL cholesterol in females with hypercholesterolemia (r ⫽ ⫺0.67, P ⬍ 0.01, Pearson’s test). HDL, high-density lipoprotein.
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FIG. 3. The correlation of serum leptin levels with HDL cholesterol in males with combined hyperlipidemia (r ⫽ ⫺0.56, P ⬍ 0.01, Pearson’s test). HDL, high-density lipoprotein.
Moreover, the leptin concentration in the male group with isolated hypercholesterolamia did not differ from that in the female control group but was significantly lower than that in the female groups with hypercholesterolemia and combined hyperlipidemia. However, the male group with hypercholesterolemia was, from a statistical point of view, relatively small; therefore, this finding should be tested further in a group with more subjects. In the other groups, the sex differences, with a higher level of leptin in females than in males matched by age and BMI, were maintained. Leptin is a relatively recently discovered protein hormone that was identified in ob/ob mice by Zhang et al.1 The main sources of serum leptin are adipocytes, and additional sites of its production are Graafian folicles16 and the placenta.17 Plasma leptin concentrations are closely related to body fat content18 and have a circadian rhythm.19 One of the most important hormonal factors having an impact on serum leptin levels is insulin, which was reported to increase serum leptin levels in vivo and in vitro.20,21 The strongly pronounced sexual dimorphism in leptin levels is suggested to be dependent on sex hormones. However, reports
Nutrition Volume 16, Number 6, 2000 concerning a relation between leptin and estrogen, progesterone, or testosterone are contradictory.5–7,22 The relation between leptin and plasma lipids and between leptin and lipoproteins has been reported by series of studies in healthy individuals and in subjects with various diseases (e.g., growth-hormone deficiency and polycystic ovary syndrome), but to the best of our knowledge, no study has reported on leptin levels in patients with lipid metabolism disorders thus far. According to previously published studies, there is probably no clear, significant relation between leptin and serum lipids or between leptin and lipoproteins. Some studies have reported no correlation between leptin and serum lipid parameters.10 Other studies have observed a significant positive correlation between leptin and HDL cholesterol11,13,14 or between leptin and triacylglycerols.9,12 In our study, no clear relation between serum leptin and lipid or between leptin and lipoprotein levels was observed. However, in three of the four groups with hyperlipidemia, a significant relation between HDL cholesterol and leptin concentration was found, whereas no such relation was observed in either the male or the female control group. We do not have any clear explanation for this result, but we suggest that the relation between leptin and HDL cholesterol is opposite that reported by some investigators. In subjects with normal lipid and lipoprotein levels, this relationship is explainable by different lipid and lipoprotein concentrations in subjects with hyperlipidemia, even though their BMI and leptin concentration are not different from those of healthy subjects. To our knowledge, our study is the first study to analyze the relation of serum leptin levels and apolipoprotein-E isoform distribution. We did not find any statistically significant relationship between serum leptin levels and apolipoprotein-E isoform distribution after grouping hyperlipidemia patients into groups with higher and lower levels of leptin. We conclude that serum leptin levels in most types of hyperlipidemia simply reflect the degree of adiposity expressed by BMI and are not clearly related to serum lipid and/or lipoprotein concentrations. The only exception was the male group with hypercholesterolemia, in which the dissociation between serum leptin levels and BMI was found because of higher leptin levels than expected from BMI values. However, this result should be further supported by an additional study performed in more subjects.
ACKNOWLEDGMENTS The authors thank BioVendor company for kindly providing ELISA kits for leptin measurements.
REFERENCES
FIG. 4. The correlation of serum leptin levels with HDL cholesterol in females with combined hyperlipidemia (r ⫽ 0.67, P ⬍ 0.01, Pearson’s test). HDL, high-density lipoprotein.
1. Zhang Y, Proenca R, Maffei M, et al. Positional cloning of the mouse obese gene and its human homologue. Nature 1994;372:425 2. Maffei M, Halaas J, Ravussin E, et al. Leptin levels in human and rodent: measurement of plasma leptin and ob RNA in obese and weight-reduced subjects. Nat Med 1995;1:1155 3. Ostlund RE, Yang JW, Klein S, Gingerich R. Relation between plasma leptin concentration and body fat, gender, diet, age, and metabolic covariates. J Clin Endocrinol Metab 1996;81:3909 4. Dua A, Hennes MI, Hoffmann RG, et al. Leptin: a significant indicator of total body fat but not of visceral fat and insulin insensitivity in African-American women. Diabetes 1996;45:1635 5. Shimizu H, Shimomura Y, Nakanishi Y, et al. Estrogen increases in vivo leptin production in rats and human subjects. J Endocrinol 1997;154:285 6. Haffner SM, Mykka¨nen L, Stern MP. Leptin concentrations in women in the San Antonio Heart Study: effect of menopausal status and postmenopausal hormone replacement therapy. Am J Epidemiol 1997;146:581 7. Nedvı´dkova´ J, Haluzı´k M, Schreiber V. The decrease in serum leptin levels in estrogen treated male mice. Phys Res 1997;46:291 8. Hardie L, Trayhurn P, Abramovich D, Fowler P. Circulating leptin in women: a longitudinal study in the menstrual cycle and during pregnancy. Clin Endocrinol 1997;47:1011
Nutrition Volume 16, Number 6, 2000 9. Leyva F, Godsland IF, Ghatei M, et al. Hyperleptinemia as a component of a metabolic syndrome of cardiovascular risk. Arter Thromb Vasc Biol 1998;18:928 10. Al-Shoumer KAS, Anyaoku V, Richmond W, Johnston DG. Elevated leptin concentration in growth hormone-deficient hypopituitary adult. Clin Endocrinol 1997;47:153 11. Rainwater DL, Comuzzie AG, VandeBerg JL, Manahey MC, Blangero J. Serum leptin levels are independently correlated with two measures of HDL. Atherosclerosis 1997;132:237 12. Couillard C, Lamarche B, Mauriege P, et al. Leptinemia is not a risk factor for ischemic heart disease in men. Prospective results from the Quebec Cardiovascular Study. Diab Care 1998;21:782 13. Chapman IM, Wittert GA, Norman RJ. Circulating leptin concentrations in polycystic ovary syndrome: relation to anthropometric and metabolic parameters. Clin Endocrinol 1997;46:175 14. Hirose H, Saito I, Kawai T, et al. Serum leptin level: possible association with haematopoiesis in adolescents, independent of body mass index and serum insulin. Clin Sci 1998;94:633 15. Cˇesˇka R, Kvasilova´ M, Procha´zkova´ R, Sˇobra J. The determination of apo B by Sevac antiserum. Sbornı´k Le´k 1989;91:334
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16. Cioffi JA, Van Blerkom J, Antczak M, et al. The expression of leptin and its receptors in pre-ovulatory human follicles. Mol Hum Reprod 1997;3:467 17. Masuzaki H, Ogawa Y, Sagawa N, et al. Nonadipose tissue production of leptin: leptin as a novel placenta-derived hormone in humans. Nat Med 1997;3:1029 18. Considine RV, Sinha MK, Heiman ML, et al. Studies of circulating leptin by radioimmunoassay in human with and without obesity. N Engl J Med 1996;344: 293 19. Sinha MK, Ohannesian JP, Heimann ML, et al. Nocturnal rise of leptin in lean, obese and non-insulin dependent diabetes mellitus subjects. J Clin Invest 1996; 97:1344 20. Kolaczynski JW, Nyce MR, Considine RV, et al. Acute and chronic effect of insulin on leptin production in humans. Studies in vivo and in vitro. Diabetes 1996;45:699 21. Wabitz M, Jensen PB, Blum WF, et al. Insulin and cortisol promote leptin production in cultured human fat cells. Diabetes 1996;45:1435 22. Paolisso G, Ammendola S, Del Buono A, et al. Serum levels of insulin growth factor I and IGF-binding protein-3 in healthy centerarians: relationship with plasma leptin and lipid concentration, insulin action and cognitive function. J Clin Endocrinol Metab 1997;82:2204
Serum Leptin Levels in Patients With Hyperlipidemias Martin Haluzı´k, MD, PhD, Jirı´ Fiedler, MD, Jara Nedvı´dkova´, PhD, and Richard Cˇesˇka, MD, PhD rd From the 3 Medical Department, 1st Faculty of Medicine, Charles University, Prague; and the Institute of Endocrinology, Prague, Czech Republic Leptin is a protein hormone produced by adipocytes that reflects the body fat content. The aim of our study was to compare serum leptin levels in randomly selected untreated males and females with hypercholesterolemia and combined hyperlipidemia and in healthy control subjects matched for age and body mass index and to study the relations between leptin and serum lipids and lipoproteins. No statistically significant differences in serum leptin levels were found between the male control group (5.26 ⫾ 2.81 ng/mL⫺1) and the male group with hypercholesterolemia (8.16 ⫾ 3.85 ng/mL⫺1) or combined hyperlipidemia (7.51 ⫾ 4.83 ng/mL⫺1) and between the female control group (13.0 ⫾ 8.12 ng/mL⫺1) and the female group with hypercholesterolemia (15.36 ⫾ 8.89 ng/mL⫺1) or combined hyperlipidemia (18.63 ⫾ 10.15 ng/mL⫺1). Leptin concentration in male group with hypercholesterolemia did not differ significantly from the female control group; in the other male groups, leptin levels were significantly lower than those of the other female groups. Serum leptin levels in all studied groups except for the male group with hypercholesterolemia positively correlated with body mass index. Serum leptin levels correlated negatively with high-density lipoprotein cholesterol in the female group with hypercholesterolemia (r ⫽ ⫺0.67, P ⬍ 0.01) and the male group with combined hyperlipidemia (r ⫽ ⫺0.56, P ⬍ 0.01). A positive correlation between serum leptin and high-density lipoprotein cholesterol (r ⫽ 0.67, P ⬍ 0.01) and between leptin and lipoprotein (a) (r ⫽ 0.71, P ⬍ 005) was found in female group with combined hyperlipidemia. No other significant relationships between leptin and serum lipids or lipoproteins were found. We conclude that serum leptin levels in patients with hyperlipidemias do not significantly differ from those healthy control subjects matched by age and body mass index. Nutrition 2000; 16:429 – 433. ©Elsevier Science Inc. 2000 Key words: leptin, body mass index, hyperlipidemia, cholesterol, triacylglycerols
INTRODUCTION Leptin is a protein hormone produced by adipocytes that was identified by cloning the mouse ob gene.1 This substance reflects in most cases the body fat content i.e., its serum concentrations are elevated in obese people in comparison with lean people, and a strong positive correlation has been found between leptin and body fat content and between leptin body mass index (BMI).2,3 Some studies have shown that the degree of positive correlation is higher between leptin and subcutaneous fat than between leptin and visceral fat.4 Serum leptin levels are significantly higher in females than in males of similar age, BMI, and body fat content.2 It is not clear whether this sex difference is estrogen or gestagen dependent. Studies concerning the influence of estrogens or progesterone on serum leptin levels have shown rather conflicting results.5– 8 Information concerning relations between serum leptin concentrations and the levels of serum lipids are limited. Leyva et al.9 studied the relation of plasma leptin and components of a meta-
This study was supported by grants IGA MHCR 4041–3 and 5455–3 and by grant CR 206017-01 from the Ministry of Education. Correspondence to: Martin Haluzı´k, MD, PhD, 3rd Medical Department, 1st Faculty of Medicine, U nemocnice 1, Praha 2, 128 08, Czech Republic. E-mail: [email protected] Date accepted: July 18, 1999. Nutrition 16:429 – 433, 2000 ©Elsevier Science Inc., 2000. Printed in the United States. All rights reserved.
bolic syndrome X in a group of middle-aged men. A positive correlation of leptin with BMI, systolic and diastolic blood pressure, fasting triacylglycerols, serum uric acid, fasting glucose and insulin, and negative correlation with insulin sensitivity were found in this study. No correlation between leptin and high-density lipoprotein (HDL) cholesterol concentrations was observed. The relation of serum leptin and a series of anthropometric and metabolic parameters in growth-hormone– deficient hypopituitary adults was investigated by Al-Shoumer et al.10 Serum leptin levels in hypopituitary subjects were significantly higher than those of healthy controls and correlated positively with BMI and body fat mass, whereas no significant relation with fasting insulin growth factor-I, glucose, insulin, total, HDL, low-density lipoprotein (LDL) cholesterol and triacylglycerols were found. Rainwater et al.11 tested the relation of serum leptin levels and nine different measures of HDL phenotype. Only two of these measures (HDL triacylglyerol concentrations and the proportion of apolipoprotein AI (apo A-I) on HDL particles larger than HDL) were independently correlated with leptin in this study. Couillard et al.12 compared leptin levels and metabolic profiles of men with and without ischemic heart disease. No differences in leptin levels between the two studied groups and no association between leptin and plasma lipid and lipoprotein concentrations, with the exception of a positive correlation between leptin with plasma triacylglycerols, were found. The relation of leptin to metabolic and anthropometric parameters in women with polycystic ovary syndrome was investigated by Chapman et al.13 No differences in serum leptin levels between the control group and the group with polycystic ovary syndrome were observed. Leptin levels in both groups correlated 0899-9007/00/$20.00 PII S0899-9007(00)00288-4
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Nutrition Volume 16, Number 6, 2000 TABLE I.
DATA FOR MALE AND FEMALE PATIENTS AND CONTROL SUBJECTS WITH REGARD TO HYPERLIPIDEMIA*
Age (y)
BMI (kg/[m2]⫺1)
Leptin (ng/ml⫺1)
Cholesterol (mmol/L⫺1)
Triacylglycerols (mmol/L⫺1)
HDL (mmol/L⫺1)
25.28 ⫾ 5.06
13.0 ⫾ 8.12 (2,6) 5.26 ⫾ 2.81 (1,3,4)(3,4,5,6) 15.36 ⫾ 8.89 (2,5,6) 18.63 ⫾ 10.15 (2,5,6) 8.16 ⫾ 3.85 (3,4) 7.51 ⫾ 4.83
4.98 ⫾ 0.62 (3,4,5,6) 5.16 ⫾ 0.64 (4,6) 7.34 ⫾ 1.31 (1,2) 7.8 ⫾ 1.09 (1,2) 7.4 ⫾ 0.85 (1,2) 7.0 ⫾ 1.14 (1,3,4)
1.3 ⫾ 0.50 (4,6) 1.35 ⫾ 0.57
1.64 ⫾ 0.52
Group
n
NC-F
14
45.86 ⫾ 7.6
NC-M
17
44.31 ⫾ 11.56 26.07 ⫾ 3.44
H-F
21
48.83 ⫾ 14.38
CH-F
12
48.61 ⫾ 13.65 26.21 ⫾ 4.72
H-M
12
45.26 ⫾ 12.11 26.35 ⫾ 2.05
CH-M
24
46.77 ⫾ 13.91 26.96 ⫾ 5.55
25.1 ⫾ 5.49
1.44 ⫾ 0.49 (4,6) 3.14 ⫾ 0.8 (1,2,3,5) 1.51 ⫾ 0.27 (4,6) 3.07 ⫾ 0.059 (1,2)
LDL (mmol/L⫺1)
2.74 ⫾ 0.44 (3,4,5,6) 1.42 ⫾ 0.53 3.11 ⫾ 0.70 (3,4,5,6) 1.89 ⫾ 0.51 4.9 ⫾ 1.03 (6) (1,2) (12) 1.88 ⫾ 1.05 4.81 ⫾ 1.13 (1,2) 1.43 ⫾ 0.29 4.99 ⫾ 1.43 (1,2) 1.15 ⫾ 0.32 4.31 ⫾ 0.95 (1,2,3,5) (3) (1,2)
lp(a)
apoB
—
—
—
—
0.59 ⫾ 0.49
1.37 ⫾ 0.33
0.27 ⫾ 0.33
1.16 ⫾ 0.6
0.31 ⫾ 0.13
1.31 ⫾ 0.37
0.33 ⫾ 0.24
1.29 ⫾ 0.24
* Data are expressed as mean ⫾ standard deviation. Numbers in parentheses denote significant intergroup differences (P ⬍ 0.05, Dunn’s test). apoB, apolipoprotein B; BMI, body mass index; CH-F, female group with combined hyperlipidemia; CH-M, male group with combined hyperlipidemia; HDL, high-density lipoprotein; H-F, female group with hypercholesterolemia; H-M, male group with hypercholesterolemia; LDL, low-density lipoprotein; lp(a), lipoprotein (a); NC-F, female control group; NC-M, male control group
positively only with BMI and HDL cholesterol. No significant relations were found between the other serum lipid parameters and leptin. Hirose et al.,14 who studied the relation of leptin levels with hematopoesis in adolescents, found a positive correlation of serum leptin levels and BMI, leukocyte count, and HDL cholesterol. The relation of serum leptin to lipids or lipoprotein levels is not definitely clear. In most of these studies, no relation of serum leptin to total and LDL cholesterol were found; in some studies, a positive correlation between leptin and triacylglycerols or between leptin and HDL cholesterol was reported. Moreover, to our best knowledge, no study investigating the relation of serum leptin levels to lipid concentrations in patients with hyperlipidemia has been published thus far. Leptin is a strong regulator of food intake and has a series of complex central and peripheral metabolic effects. Therefore, we hypothesized that the changes in serum leptin levels could contribute to lipid metabolism alterations in patients with hyperlipidemia. We compared serum leptin concentrations in patients of both sexes with different types of hyperlipidemia with healthy control subjects matched for age and BMI.
PATIENTS AND METHODS Sixty-nine patients (36 males and 33 females) were included in the study. The patients represent a randomly selected sample of subjects examined in the Lipid Clinic of the University Hospital, Prague because of hyperlipidemia. Subjects were matched by age and BMI. None of the patients had diabetes and none was administered hypolipidemic drugs or medication known to affect food intake and/or lipid metabolism. None of patients kept any special diet. The patients were grouped according to type of hyperlipidemia: 21 untreated females with hypercholesterolemia (total cholesterol concentration above 5.8 mmol/L⫺1, triacylglycerol concentration below 2.00 mmol/L⫺1), 12 untreated females with combined hyperlipidemia (total cholesterol concentration above 5.8 mmol/L⫺1, triacylglycerol concentration above 2.00 mmol/ L⫺1), 12 untreated males with hypercholesterolemia (total cholesterol concentration above 5.8 mmol/L⫺1, triacylglycerol concentration below 2.00 mmol/L⫺1), and 24 males with combined hyperlipidemia (total cholesterol concentration above 5.8 mmol/ L⫺1, triacylglycerol concentration above 2.00 mmol/L⫺1). Thirtyone subjects (14 females and 17 males) with total cholesterol
levels below 5.8 mmol/L⫺1 and triacylglycerols levels below 2.00 mmol/L⫺1 formed the control group. The study protocol was approved by the local ethical committee. All of the subjects included in the study were instructed about the purpose of the study and gave their consent to participate. Subjects were measured and weighed. The blood samples for leptin and serum lipid determination were taken after an overnight fast (14 h) at the same time (approximately at 8 AM). The serum leptin concentrations were determined with a commercial sandwich enzyme immunoassay (BioVendor, Czech Republic, Prague). This human leptin assay has been developed to measure human leptin in plasma or serum. It is a completely homologous assay because the antibody is raised against highly purified human leptin and standards are prepared with human leptin. The limit of sensitivity for the human leptin assay was 0.2 ng/mL⫺1. The results of this assay highly correlate with those of commercial RIA radioimmunossay (Linco, St. Charles, MO, USA). Serum lipid parameters were measured in the Department of Clinical Biochemistry of the University Hospital, Prague by standard laboratory methods. Serum LDL cholesterol levels were computed by using Friedwald’s formula. The lp(a) and apoB levels ˇ eska15). were measured as described previously (C Statistical analysis was performed by SigmaStat statistical software (Jandel Scientific, San Rafael, CA, USA). Means and standard deviations were computed, and the significance of differences between groups was evaluated with analysis of variance and Dunn’s test. The correlations between groups were determined by the Spearman correlation test. The distribution of apolipoprotein E isoforms in the study groups was compared using Fisher’s test. Significance level was set at P ⬍ 0.05.
RESULTS Groups were matched by age and BMI. Serum leptin levels in the male control group did not differ from the male groups with hypercholesterolemia or combined hyperlipidemia but were significantly lower than the levels in all female groups (Table I, Fig. 1). No difference was observed between leptin levels in males with hypercholesterolemia versus males with combined hyperlipidemia. Serum leptin levels between the female control group and the female group with hypercholesterolemia or combined hyperlipid-
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TABLE II. CORRELATION OF SERUM LEPTIN LEVELS WITH DIFFERENT VARIABLES ACROSS STUDY GROUPS*
FIG. 1. Serum leptin levels in female and male control groups (NC), female and male groups with hypercholesterolemia (H), and female and male groups with combined hyperlipidemia (CH) are expressed as means ⫾ SD. Statistically significant differences across groups (Dunn’s test, P ⬍ 0.05): 1, significantly different from the female control group; 2, significantly different from the female group with hypercholesterolemia; 3, significantly different from the female group with combined hyperlipidemia; 4, significantly different from the male control group; 5, significantly different from the male group with hypercholesterolemia; 6, significantly different from the male group with combined hyperlipidemia.
emia did not differ either (Table I, Fig. 1). Leptin levels in the male group with hypercholesterolemia did not differ from those of the female control group but were significantly lower than in the female groups with hypercholesterolemia and combined hyperlipidemia (Table I, Fig. 1). Serum leptin levels in the male group with combined hyperlipidemia were significantly lower than those in the female control group and in the female groups with hypercholesterolemia and combined hyperlipidemia (Table I, Fig. 1). Serum leptin levels correlated positively with BMI in all study groups, with the exception of the male group with hypercholesterolemia, where no significant relation between serum leptin and BMI was observed (Table II). The significant negative correlation between serum leptin levels and HDL cholesterol was found in the female group with hypercholesterolemia (Fig. 2) and in the male group with combined hyperlipidemia (Fig. 3). In the female group with combined hyperlipidemia, a positive correlation between leptin and HDL levels (Fig. 4) and between leptin and lipoprotein (a) was found (Table II). No additional significant relationship between leptin and the other lipid metabolism parameters was found in the other groups. Multivariate regression analysis was performed for groups in which significant associations between leptin and HDL were found. The partial correlation analysis confirmed significant correlations between serum leptin and HDL levels in the female groups with hypercholesterolemia and combined hyperlipidemia and in the male group with combined hyperlipidemia even after adjustment for BMI and age. The relative distribution of apolipoprotein E isoforms did not significantly differ in the four hyperlipidemia groups (results not shown). The patients were then assigned to two subgroups with higher and lower leptin levels (below and above 10 ng/mL⫺1) to analyze the relation of leptin levels and the apolipoprotein-E isoform distribution. The relative distribution of apolipoprotein-E isoforms did not differ significantly in these two subgroups.
Group
n
BMI
HDL
lp(a)
NC-F
14
NS
—
NC-M
17
NS
—
H-F
21 12
H-M CH-M
12 24
r ⫽ ⫺0.67 P ⬍ 0.01 r ⫽ 0.67 P ⬍ 0.01 NS r ⫽ ⫺0.56 P ⬍ 0.01
NS
CH-F
r ⫽ 0.71 P ⬍ 0.01 r ⫽ 0.62 P ⬍ 0.01 r ⫽ 0.74 P ⬍ 0.01 r ⫽ 0.64 P ⬍ 0.05 NS r ⫽ 0.57 P ⬍ 0.01
r ⫽ 0.71 P ⬍ 0.05 NS NS
* According to Spearman’s correlation test. BMI, body mass index; CH-F, female group with combined hyperlipidemia; CH-M, male group with combined hyperlipidemia; HDL, highdensity lipoprotein; H-F, female group with hypercholesterolemia; H-M, male group with hypercholesterolemia; lp(a), lipoprotein (a); NC-F, female control group; NC-M, male control group; NS, not significant
DISCUSSION The aim of this study was to compare the serum leptin levels in male and female patients patients with hypercholesterolemia and combined hyperlipidemia with healthy control subjects of both sexes matched by age and BMI. No significant differences in serum leptin levels were found between the control groups and the hyperlipidemia groups in either males or females. As expected, a strong positive correlation between leptin and BMI was found in all studied groups, with the exception of the male group with hypercholesterolemia. The suprising dissociation between leptin and BMI in this group was accompanied by higher serum leptin levels than expected from BMI values. However, the difference in leptin levels in the male group with hypercholesterolemia versus the male control group and the male group with combined hyperlipidemia did not reach statistical significance.
FIG. 2. The correlation of serum leptin levels with HDL cholesterol in females with hypercholesterolemia (r ⫽ ⫺0.67, P ⬍ 0.01, Pearson’s test). HDL, high-density lipoprotein.
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Haluzı´k et al.
FIG. 3. The correlation of serum leptin levels with HDL cholesterol in males with combined hyperlipidemia (r ⫽ ⫺0.56, P ⬍ 0.01, Pearson’s test). HDL, high-density lipoprotein.
Moreover, the leptin concentration in the male group with isolated hypercholesterolamia did not differ from that in the female control group but was significantly lower than that in the female groups with hypercholesterolemia and combined hyperlipidemia. However, the male group with hypercholesterolemia was, from a statistical point of view, relatively small; therefore, this finding should be tested further in a group with more subjects. In the other groups, the sex differences, with a higher level of leptin in females than in males matched by age and BMI, were maintained. Leptin is a relatively recently discovered protein hormone that was identified in ob/ob mice by Zhang et al.1 The main sources of serum leptin are adipocytes, and additional sites of its production are Graafian folicles16 and the placenta.17 Plasma leptin concentrations are closely related to body fat content18 and have a circadian rhythm.19 One of the most important hormonal factors having an impact on serum leptin levels is insulin, which was reported to increase serum leptin levels in vivo and in vitro.20,21 The strongly pronounced sexual dimorphism in leptin levels is suggested to be dependent on sex hormones. However, reports
Nutrition Volume 16, Number 6, 2000 concerning a relation between leptin and estrogen, progesterone, or testosterone are contradictory.5–7,22 The relation between leptin and plasma lipids and between leptin and lipoproteins has been reported by series of studies in healthy individuals and in subjects with various diseases (e.g., growth-hormone deficiency and polycystic ovary syndrome), but to the best of our knowledge, no study has reported on leptin levels in patients with lipid metabolism disorders thus far. According to previously published studies, there is probably no clear, significant relation between leptin and serum lipids or between leptin and lipoproteins. Some studies have reported no correlation between leptin and serum lipid parameters.10 Other studies have observed a significant positive correlation between leptin and HDL cholesterol11,13,14 or between leptin and triacylglycerols.9,12 In our study, no clear relation between serum leptin and lipid or between leptin and lipoprotein levels was observed. However, in three of the four groups with hyperlipidemia, a significant relation between HDL cholesterol and leptin concentration was found, whereas no such relation was observed in either the male or the female control group. We do not have any clear explanation for this result, but we suggest that the relation between leptin and HDL cholesterol is opposite that reported by some investigators. In subjects with normal lipid and lipoprotein levels, this relationship is explainable by different lipid and lipoprotein concentrations in subjects with hyperlipidemia, even though their BMI and leptin concentration are not different from those of healthy subjects. To our knowledge, our study is the first study to analyze the relation of serum leptin levels and apolipoprotein-E isoform distribution. We did not find any statistically significant relationship between serum leptin levels and apolipoprotein-E isoform distribution after grouping hyperlipidemia patients into groups with higher and lower levels of leptin. We conclude that serum leptin levels in most types of hyperlipidemia simply reflect the degree of adiposity expressed by BMI and are not clearly related to serum lipid and/or lipoprotein concentrations. The only exception was the male group with hypercholesterolemia, in which the dissociation between serum leptin levels and BMI was found because of higher leptin levels than expected from BMI values. However, this result should be further supported by an additional study performed in more subjects.
ACKNOWLEDGMENTS The authors thank BioVendor company for kindly providing ELISA kits for leptin measurements.
REFERENCES
FIG. 4. The correlation of serum leptin levels with HDL cholesterol in females with combined hyperlipidemia (r ⫽ 0.67, P ⬍ 0.01, Pearson’s test). HDL, high-density lipoprotein.
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