Leptin, an adipose tissue hormone, has circadian variations in its secretion. Aims of
this study were to show how circadian rhythm depends on fat tissue distribution in
obese and non-obese subjects. The research was carried out on 70 subjects (37 men and
33 women) with an average body mass index (BMI) of 25.22 kg/m2. Concentration of
leptin in blood was measured at 8.30 a.m., 12.30 p.m. and 6.30 p.m. Basal leptin level
correlated strongly with all isolated regions of subcutaneous fat tissue in women and
obese subjects. Circadian changes of blood leptin level in non-obese people are more significant
than these changes in obese people. Differences in circadian pattern of leptin secretion
between obese and non-obese subjects were probably caused by enlarged volume
of subcutaneous fat tissue in obese people. Lean subjects have subcutaneous fat in physiological
range which allows influence of some hormones (insulin or cortizol) or food intake
on leptin secretion

Ivan Karner

Mario Ćurković

Pašezada Kosović

Radivoje Radić

Robert Selthofer

Sven Kurbel

Vasilije Nikolić

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Circadian Rhythm of Blood Leptin Level in Obese and Non-Obese People

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Coll. Antropol. 27 (2003) 2: 555–561UDC 572.511:612.123Original scientific paperCircadian Rhythm of Blood LeptinLevel in Obese and Non-ObesePeopleRadivoje Radi}1, Vasilije Nikoli}1, Ivan Karner2, Pa{ezada Kosovi}2,Sven Kurbel3, Robert Selthofer1 and Mario ]urkovi}41 Department of Anatomy, School of Medicine, University »J. J. Strossmayer«, Osijek, Croatia2 Department of Patophysiology, School of Medicine, University »J. J. Strossmayer«, Osijek,Croatia3 Department of Physiology, School of Medicine, University »J. J. Strossmayer«, Osijek, Croatia4 Primary Health Care Office, Osijek, CroatiaA B S T R A C TLeptin, an adipose tissue hormone, has circadian variations in its secretion. Aims ofthis study were to show how circadian rhythm depends on fat tissue distribution inobese and non-obese subjects. The research was carried out on 70 subjects (37 men and33 women) with an average body mass index (BMI) of 25.22 kg/m2. Concentration ofleptin in blood was measured at 8.30 a.m., 12.30 p.m. and 6.30 p.m. Basal leptin levelcorrelated strongly with all isolated regions of subcutaneous fat tissue in women andobese subjects. Circadian changes of blood leptin level in non-obese people are more sig-nificant than these changes in obese people. Differences in circadian pattern of leptin se-cretion between obese and non-obese subjects were probably caused by enlarged volumeof subcutaneous fat tissue in obese people. Lean subjects have subcutaneous fat in physi-ological range which allows influence of some hormones (insulin or cortizol) or food in-take on leptin secretion.Key words: leptin, circadian rhythm, obese, non-obeseIntroductionLeptin is a peptide hormone producedin white adipose tissue that acts by acti-vating specific leptin receptors in hypo-thalamus. Leptin receptors are found inmany areas of the brain, including thehypothalamus, cerebellum, cortex, hippo-campus, thalamus, chorioid plexus, andbrain capillary endothelium1. Beside the555Received for publication June 8, 2003central nervous system, leptin receptorsare also found in liver, kidneys, pancreas,lungs and skeletal muscles2. Activation ofhypothalamic receptors results in decrea-sed food intake and increased energyconsumption3. In the arcuate nuclei of hy-pothalamus leptin inhibits production ofneuropeptide Y, thus reducing the appe-tite4,5. Basal leptin values are higher inwomen than in men, they are increasedduring pregnancy and they correlate withthe share of body fat and with the bodymass index (BMI)6,7. Different regions ofbody fat tissue do not seem to have thesame influence on the leptin blood level.The abdominal adipose tissue seems tohave the major effect8.Leptin concentration in blood showscircadian variations with the lowest val-ues at noon and the highest values aftermidnight9. Possible interpretations of thisdaily profile include insulin influence onchanging the blood leptin level10 and in-creased glomerular filtration in the mor-ning11 that might reduce the leptin bloodconcentration. It is still unclear how thesecircadian changes influence the leptinrole in weight control. Another interest-ing aspect is whether all parts of the adi-pose tissue have equal influence on theleptin circadian variations. In this studywe investigated the influence of obesitydegree on circadian rhythm of leptin se-cretion.Subjects and MethodsSubjectsThe research was carried out on 70healthy volunteers (37 men and 33 wo-men) who gave an informed consent toparticipate in this study. For the wholegroup of 70, the BMI was 24.88±3.87 kg/m2(in range between 17.57 and 33.33). Amongthem, 37 subjects had BMI lower than 25kg/m2 (22.06±2.08) while 33 subjects hadBMI above 25 kg/m2 (28.23±2.59).Anthropometric measurementsAll subjects gave consent to anthropo-metric and ultrasound measurements.Circumferences of the upper arm and fo-rearm, waist, hips, upper and lower legwere measured in the middle of each bodypart. The ranges between the front andthe back wall of thorax and abdomenwere also measured. The thickness ofsubcutaneous fat tissue was measured byultrasound with a linear probe made byShimadzu company at frequency of 7.5MHz at 16 points in body that were deter-mined in advance. List of these points, aswell as procedure of ultrasound measure-ment was fully described in previouspaper12. Subcutaneous fat tissue in theregions of the forearm, upper arm, upperleg and lower leg that are approximatelycircles in diameter was calculated as areadifference of two concentric circles. Oneach measuring point the thickness ofsubcutaneous fat tissue was measuredboth from the front and the back side andthe mean value was put in the formula.The trunk cross-section resembles ovalmore than circle, so we needed to changethe formula for calculating the area ofsubcutaneous fat tissue in that cross-sec-tion. Average thickness of thoracic subcu-taneous fat tissue was calculated as themean of values obtained on two measur-ing points (the middle of the line connect-ing the end of the large chest muscle andsternoclavicular joint). Average thicknessof abdominal subcutaneous fat tissue wascalculated as the mean of values obtainedon six measuring points. Since the thick-ness of subcutaneous fat tissue in regionsaround the joints is significantly differentit was decided to observe the length of 10cm on every limb to maintain the objec-tivity of research. In this way we man-aged to obtain the volume of subcutane-ous fat tissue that is comparable withother body regions13.556R. Radi} et al.: Circadian Rhythm of Blood Leptin, Coll. Antropol. 27 (2003) 2: 555–561Leptin level measurementsBlood samples were taken from allsubjects at 8.30 a.m., 12.30 p.m. and 6.30p.m., every time before the meal. Theblood leptin level was measured with po-lyclonal antibody radioimmunoassay byLinco Reasearch, Inc. (HL-81K). The as-say sensitivity was 0.5 ng/ml. Antibodieswere marked with 125I that has specificactivity of 135 Ci/g. Standard valuesfor non-obese people (BMI 18–25 kg/m2)were 2–5.6 ng/ml for men and 3.7–11.1ng/ml for women.StatisticsThe data were classified according toobesity degree as well as to blood takingtime. Student t-test for dependent sam-ples was used to test the significance ofdifference between morning, noon andevening leptin concentration in blood inall groups of data. For comparing the cor-relations among the examined indexesthe Pearson's method of linear regressionwas used. In tables it was presented asvalue (r) and probability (p) of linear cor-relation between two examined indexes.ResultsIn 33 obese subjects (BMI > 25 kg/m2)morning leptin concentration rose pro-portionally with the increase of BMI,whereas in 37 non-obese subjects (BMIup to 25 kg/m2) values of leptin level didnot correlate with BMI (Figure 1).Table 1 shows correlation (r) betweenthe volume of total subcutaneous fat tis-sue as well as the volume of isolated re-gions of subcutaneous fat tissue as inde-pendent variable and blood leptin level inthe morning (basal level) as dependentvariable in regard to sex and obesity de-gree. It is evident that basal leptin levelstrongly correlates with subcutaneous fatin all regions, except for the forearm andthe lower leg in men and the forearm innon-obese people.Table 2 shows differences of leptinconcentrations in the morning, at noonand in the evening. Using t-test for de-pendent samples the differences betweenmorning, noon and evening leptin levelswere checked. There were significant dif-ferences between morning and evening557R. Radi} et al.: Circadian Rhythm of Blood Leptin, Coll. Antropol. 27 (2003) 2: 555–561Fig. 1. Correlation between body mass index (BMI) and morning leptinconcentration in obese and non-obese subjects.and noon and evening leptin concentra-tions. The differences between morningand noon concentrations were not statis-tically significant (p<0.09). When non-obese subjects were observed separately(Table 3), there were significant differ-ences between morning and noon (p<0.01) and noon and evening values of theleptin concentration in blood (p=0.03). Inobese subjects, noon leptin levels were558R. Radi} et al.: Circadian Rhythm of Blood Leptin, Coll. Antropol. 27 (2003) 2: 555–561TABLE 1CORRELATION BETWEEN THE VOLUME OF SUBUCTANEOUS FAT TISSUE AS INDEPENDENTVARIABLE AND BLOOD LEPTIN LEVEL IN THE MORNING AS A DEPENDENT VARIABLE WITHREGARD TO SEX AND OBESITY DEGREESubcutaneousfat tissueBlood leptin level in the morningFemale MaleNon-obese(25 kg/m2)Obese(>25 kg/m2)Upper arm 0.74* 0.69* 0.58* 0.74*Forearm 0.77* 0.29 0.31 0.69*Upper leg 0.57* 0.55* 0.64* 0.58*Lower leg 0.43* 0.29 0.62* 0.46*Thorax 0.80* 0.75* 0.59* 0.72*Abdomen 0.84* 0.56* 0.50* 0.74*Whole body 0.82* 0.75* 0.71* 0.78** p < 0.05TABLE 2DIFFERENCES BETWEEN MORNING, NOON AND EVENING BLOOD LEPTIN CONCENTRATIONS(mg/L) IN ALL SUBJECTS (N=70) (STUDENT T-TEST FOR DEPENDENT SAMPLES)Leptin levels(X±SD)Noon (12.49±11.13) Evening (14.81±12.12)t p t pMorning (13.16±10.17) 1.68 0.09 3.2 <0.01Noon (12.49±11.13) 5.3 <0.01TABLE 3DIFFERENCES BETWEEN MORNING, NOON AND EVENING BLOOD LEPTIN CONCENTRATIONS(mg/L) IN MALE (N=37) AND FEMALE (N=33) SUBJECTS (STUDENT T-TEST FOR DEPENDENTSAMPLES)Leptin levels(X±SD)MalesNoon (8.17±5.74) Evening (9.41±6.42)t p t pMales Morning (8.74±6.21) 1.8 0.08 1.8 0.08Noon (8.17±5.74) 3.2 <0.01FemalesNoon (17.20±13.54) Evening (20.70±14.10)t p t pFemales Morning (17.97±11.47) 1.02 0.31 2.9 <0.01Noon (17.20±13.54) 4.5 <0.01not significantly lower than morning val-ues (p=0.78), whereas there was the dif-ference between noon and evening values(p=0.04). When subjects were divided ac-cording to sex (Table 4), it was evidentthat in female subjects there was no sig-nificant difference between morning andnoon values (p= 0.31).DiscussionLeptin in blood was obtained in freeand bound form. The share of free leptinin blood increases proportionally with theobesity degree14,15. We got similar resultsfor the whole group of 70 subjects. How-ever, when we divided them according toBMI into two subgroups of lean and obesesubjects, no significant correlation be-tween blood leptin levels and BMI wasfound in non-obese subjects. These datasuggest that the basal leptin value inblood is a relatively good index for thevolume of adipose tissue only in obesesubjects. It also indicates that blood lep-tin level does not correlate with bodymass index in eutrophic people. There aretwo explanations for this statement.First, level of free leptin in blood rises sig-nificantly when all leptin carriers are tiedand second, non-physiological excess offat tissue changes the pattern of leptinsecretion. We used three measurementsto get the average diurnal profile of bloodleptin concentration. The lowest valueswere obtained at 12.30 a.m., whereas theevening values were the highest, regard-less of the subject’s obesity degree. Thesedata are in accordance with studies ofSchoeller16 and Sinha17. Beside that, wenoticed a difference between non-obeseand obese subjects. In the subgroup ofnon-obese subjects morning values werehigher than the noon values, whileamong the obese subjects the noon valueswere higher than the morning values. Itseems that in obese subjects the nadir ofleptin concentration was shifted towardsmorning. Here are possible explanations.In this study it was pointed out that fe-male subjects had statistically significanthigher leptin levels, regardless of thetime of day. Both male and female sub-jects had higher leptin level in the eve-ning, while morning and noon levels werenot statistically different. In lean peoplemorning and noon leptin levels were sta-tistically different. They had the lowestpeak at noon, while the evening leptinlevel was increased. In fat people onlyevening leptin level was statistically in-creased. These findings suggest that559R. Radi} et al.: Circadian Rhythm of Blood Leptin, Coll. Antropol. 27 (2003) 2: 555–561TABLE 4DIFFERENCES BETWEEN MORNING, NOON AND EVENING BLOOD LEPTIN CONCENTRATIONS(mg/L) IN OBESE (N=33) AND NON-OBESE (N=37) SUBJECTS (STUDENT T-TEST FOR DEPENDENTSAMPLES)Leptin levels(X±SD)Obese subjectsNoon (17.55±13.73) Evening (18.97±14.21)t p t pObese subjects Morning (17.23±12.20) 0.4 0.67 2.1 0.04Noon (17.55±13.73) 2.6 0.02Non-obese subjectsNoon (8.12±5.42) Evening (11.20±8.65)t p t pNon-obese subjects Morning (9.63±6.28) 4.99 <0.01 2.40 0.02Noon (8.12±5.42) 4.80 <0.01there are no significant differences be-tween male and female subjects in leptincircadian rhythm. There is significantdifference only in lean people, no matterwhat sex they are. More complicated cir-cadian dynamics of leptin secretion inlean subjects is the result of the fact thatlean people have smaller amount of fattissue, so their leptin levels are in thenormal range. Thus the leptin level pres-ents dynamics of metabolism, which areconnected with metabolism-regulatedhormones and with intake of energy andfat in the body. Considering all these factsmakes circadian leptin dynamics in leanpeople more clear. Morning leptin levelsare relatively high because of night fast,after breakfast leptin is decreasing, sothe lowest value is at noon and the high-est values are obtained in the evening.The evening values are connected withnumerous factors like hormones, circa-dian rhythm and dynamics of food intakein the body. The fat subjects have largefat storage which causes higher morningleptin level and because of that there areno great changes of leptin level until eve-ning, when leptin level is statistically in-creased, possibly because of circadianrhythm of the whole body. For more pre-cise determining of leptin circadianrhythm, additional research should bedone which will include study of other cir-cadian hormones. So, the difference incircadian rhythm of the leptin secretionaccording to obesity degree is that boththe volume of subcutaneous fat and lep-tin secretion are in physiological range innon-obese people. This condition enablesinfluence of other physiological factors ondaily dynamics of the leptin level inblood, mostly the influence of insulin18-20or cortisol21. On the other hand, obesepeople have much more subcutaneous fattissue that produces proportionally moreleptin. Physiological influence on eleva-ted leptin concentration is of less impor-tance and the consequence is the absenceof regular decrease of blood leptin level atnoon. So, we can conclude that obese peo-ple have different diurnal rhythm of lep-tin secretion because of their leptin hy-perproduction.AcknowledgementsThis work was supported by the Cro-atian Ministry of Science and Technology(grant 0219011).R E F E R E N C E S1. MANTZOROS, C. S., Ann. Intern. Med, 130(1999) 671. — 2. MURAKAMI, T., T. YAMASHITA, M.IIDA, M. 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TASKINEN, S. L. KARONEN, H.YKI-JARVINEN, Diabetologia, 39 (1996) 993. — 21.MASUZAKI, H., Y. OGAWA, K. HOSODA, T. MIYA-WAKI, I. HANAOKA, J. HIRAOKA, J. Clin. Endocri-nol. Metab., 82 (1997) 2542.561R. Radi} et al.: Circadian Rhythm of Blood Leptin, Coll. Antropol. 27 (2003) 2: 555–561R. Radi}Department of Anatomy, School of Medicine, University »J. J. Strossmayer«,J. Huttlera 4, 31000 Osijek, CroatiaCIRKADIJADNI RITAM RAZINE LEPTINA U KRVI PRETILIH INEPRETILIH OSOBAS A @ E T A KLeptin, hormon kojeg izlu~uje masno tkivo, pokazuje cirkadijadne varijacije kon-centracije u krvi. Svrha ovog rada je prikazati utjecaj distribucije potko`nog masnogtkiva na dnevni profil koncentracije leptina u krvi u pretilih i ne-pretilih osoba. Istra-`ivanje je provedeno na 70 osoba (37 mu{karaca i 33 `ene) s prosje~nim indeksom tje-lesne mase (BMI) od 25.22 kg/m2. Koncentracija leptina u krvi je mjerena u 8.30, 12.30te 18.30 sati. Bazalne (jutarnje) vrijednosti leptina sna`no koreliraju s pojedina~nimvolumenima potko`nog masnog tkiva u `ena i pretilih osoba. Dnevne razlike u vrijed-nostima razine leptina u krvi su zna~ajnije u mr{avih u usporedbi s pretilim ispitani-cima. Primije}ene razlike u cirkadijadnom ritmu koncentracije leptina izme|u pretilihi ne-pretilih osoba su vjerojatno uvjetovane pove}anim volumenom masnog tkiva upretilih osoba budu}i da je volumen potko`nog masnog tkiva kod mr{avih ispitanika ufiziolo{kim granicama {to dozvoljava utjecaj nekih drugih hormona (inzulin, kortizol)ili unosa hrane na varijabilnost leptinske koncentracije.

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Circadian Rhythm of Blood Leptin Level in Obese and Non-Obese People

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