International Journal of Obesity (1998) 22, 728±733 ß 1998 Stockton Press All rights reserved 0307±0565/98 $12.00 http://www.stockton-press.co.uk/ijo Short term response of circulating leptin to feeding and fasting in man: in uence of circadian cycle J Dallongeville 1, B Hecquet 2, P Lebel 1, J-L Edme 3, C Le Fur 4, J-C Fruchart 1, J Auwerx 1 and M Romon 4 1 INSERM U-325 and DeÂpartement d'atheâroscleârose, Institut Pasteur de Lille; 2 Centre Oscar Lambret; 3 CERESTE, CHRU de Lille and 4 Service de Nutrition, CHRU de Lille, Lille, France OBJECTIVE: To investigate whether acute feeding induces changes in circulating leptin levels in humans and whether these changes vary according to nycthemeral cycle. METHODS: First experiment. Eighteen male subjects were given a fatty meal at 08.00 h. Blood sampling was performed for 10 h following this meal. Second experiment. Thirteen male subjects were given either a mixed meal or remained fasting either at night (starting at 01.00 h) or during the day (starting at 13.00 h). Blood samples were drawn every hour for a period of 8 h. RESULTS: First experiment. Serum leptin levels increased progressively from a mean (s.d.) baseline of 3.8 2.2 ng=ml to a value of 4.5 2.7 ng=ml (P < 0.01) 8 h after the fatty meal. Second experiment. During the day, serum leptin levels increased progressively from 2.65 1.7 to 3.34 2.2 ng=ml (P < 0.001) 6 h after the test-meal and decreased from 2.68 1.5 to 1.9 1.1 ng=ml (P < 0.001) 8 h after the beginning of the fasting experiment. Similar results were obtained at night. No statistically signi cant differences in leptin levels were observed between day and night sessions in response to feeding (mean area under the curve: 3.0 4.1 vs 4.1 4.1 ng=ml) and fasting (72.9 2.2 vs 71.5 2.2 ng=ml). CONCLUSION: In two independent experiments, human serum leptin levels increase following food intake. This response is not in uenced by nycthemeral cycle. Keywords: obesity; food intake; insulin; leptin; circadian rhythm Introduction Obesity is an important public health problem that contributes to cardiovascular morbidity and mortality. 1 The discovery of a hormone secreted by adipose tissue has raised the hope of a better understanding of the regulation of body weight. In fact, leptin appears to play a major role in body weight regulation. In animals, this protein acts through appetite and energy expenditure, 2,3 while in man, leptin is in uenced by fat mass, with higher levels observed in obesity. 4,5 The role of feeding=fasting cycles on circulating leptin levels is still a matter of debate. Initial experiments in humans have failed to demonstrate any acute effects of food intake on serum leptin levels. 4±9 Recent studies, however, have challenged this concept. First, fasting is associated with a clear decrease in serum leptin levels, 10 ± 12 which returns to the baseline value upon refeeding. Second, short term massive overfeeding in humans (120 kcal kg 71 over 12 h) is accompanied by a 40% increase in serum leptin concentration over baseline value. 13 Third, adipose Correspondence: Dr Jean Dallongeville, INSERM U-325-SERLIA, Institut Pasteur de Lille, 1 rue du Professeur Calmette, 59019 Lille, Cedex, France. Received January 1997; revised October 1997; accepted March 1998 tissue leptin production is increased 5 h after a high carbohydrate meal. 14 In these studies, the changes in circulating leptin levels were independent of changes in body weight, suggesting short term energy balance has an impact on circulating leptin levels. A number of studies have demonstrated a circadian 6,15 ± 17 cycle of circulating leptin levels in humans. Generally these experiments have shown higher values of circulating leptin late at night and lower values in the morning. Since previous studies have focused on meals taken during the day and that the nycthemeral cycle appears to control leptin levels, we assess whether food intake has a short term in uence on circulating leptin and whether the time of feeding might affect the response to a meal. In order to assess the circadian variation of leptin response to a meal, the subjects were submitted to a 12 h shift in meal timing and were also studied during fasting in the same time schedule. Methods Experiment 1 Subjects. Eighteen healthy male subjects (mean s.d.: 23.5 1.5 y) participated in the study. Their mean body mass index (BMI) was 22.5 2.1 kg=m 2 ranging from 22±26 kg=m 2. None of
these subjects had diabetes, liver, renal or thyroid disease. All participants were on their habitual diet at the time of the experiment. The subjects were admitted to hospital on the morning of the study and remained hospitalized for 10 h. All participants signed an informed written consent. The protocol was reviewed by an INSERM ethical board and approved by the local ethics committee. 729 Test meal and study protocol. After an overnight fast (12 h), subjects were given a fatty breakfast (65% of calories as fat, 20% as carbohydrates and 15% as protein) containing 1 g of fat=kg body weight. The mean caloric intake was 2.32 0.22 MJ. This breakfast was ingested in 15 min, beginning between 07.45 and 08.00 h. After the meal, the subjects remained fasting for 10 h, but were allowed to drink as much water as desired. Blood samples were drawn every 2 h for leptin and triglyceride measurements. Experiment 2 Subjects. Thirteen healthy male subjects were recruited from the University of Lille II for the study. Obesity (BMI > 27 kg=m 2 ), unstable weight (delta weight > 2 kg in the last three months), smoking, drug intake, working at night or in shifts, and travel across time zones within the preceding six months were exclusion criteria. The mean (s.d.) age and BMI of the participants were 25.2 5.5 y and 22.3 1.7 kg=m 2, respectively. Body fat was assessed by anthropometry. Skin-fold thickness was measured at four sites ± biceps, triceps, subscapular and supra iliac. Body density was calculated using the formula of Durnin and Womersley 18 and percent body fat was derived by the equation of Siri. 19 Mean (s.d.) body fat mass was 9.73 2.9 kg. All potential volunteers had a fasting blood sample analyzed to exclude diabetes, hyperlipidaemia and impaired thyroid, hepatic or renal function. Furthermore, they had to answer a questionnaire concerning their daily routine as well as their sleep and meal schedule. Subjects with irregular habits were excluded. The protocol was approved by the hospital (CHRU de Lille) ethics committee according to the current regulations in France. Study protocol (Figure 1). The study was performed in the Centre d'investigation Clinique of the Lille University Hospital. There were four subsets of experiments performed in a randomized order: two feeding sessions (at 13.00 h and 01.00 h) and two fasting sessions (at 13.00 h and 01.00 h). Between each session, there was a minimum time span of ve days. All subjects completed the four sessions in < 5 weeks. During the 24 h preceding each session, the subjects were instructed to refrain from alcoholic beverages and strenuous exercise, and not to change their dietary habits. Food intake of the preceding day was assessed by a 24 h recall that did not show any Figure 1 Schematic representation of Experiment 2. statistical signi cant difference before the four sessions. At each session, the subjects arrived six hours before the test-meal: 07.00 h (day session) and 19.00 h (night session) and a blood sample was collected. Then the participants were given a light meal (`pre test-meal') and stayed on the ward. At 12.50 h for the day session or 00.50 h for the night session, a blood sample was collected and the subjects were given either a test-meal (feeding experiment) or 300 ml of water alone (fasting control). The test-meal was consumed in <15 min. Blood samples were taken every 20 min during the 8 h following the ingestion of the test-meal or water. During the sessions, the subjects were allowed to watch entertainment movies, but were not allowed to sleep. Test meal composition. The test-meal was a mixed meal, composed of bread, butter, ham, apple, marmalade and cottage cheese, and was consumed with 300 ml water. The composition of each meal during the day and night sessions was similar. Its energy content was 40% of the estimated energy needs (4.6 0.2 MJ). The percentages of total energy intake derived from protein, fat and carbohydrates were 15%, 40% and 45%, respectively. The pre testmeal which was given six hours before the beginning of the fasting or feeding experiment, covered 20% (2.3 0.1 MJ) of the daily energy needs. The relative protein, fat and carbohydrate content was identical to that of the experimental meal. Biochemical measurements Blood was collected in dry tubes and allowed to clot for one hour. Serum was separated by centrifugation (2500 rpm) for 20 min at 4 C. Glucose (Glucose, hexokinase method, Randox Laboratories Ltd, Roissy, France) and triglycerides (Triglycerides GPO-PAP, Boehringer Mannheim, Mannheim, Germany) were determined enzymatically. Hormone levels were determined using commercially available immunological assays. Insulin was measured by radio-immunoassay (Bi-Insulin RIA, ERIA, Diagnostics Pasteur, Maine la Coquette, France). C-peptide levels were determined by radio-immunoassay (RIA-coat C-Peptid, ByK- Sangtek Diagnostica GMBH & Co. KG, Bad Homburg,
730 Germany). Cortisol concentration was measured by a direct immunoassay (Enzymuntest-Cortisol, Boehringer-Mannheim, Mannheim, Germany). Leptin levels were measured in duplicate by radio-immunoassay (Human leptin RIA kit, Wak-Chemie, Medical GMBH, Bad Homburg, Germany). The lower limit of sensitivity of the assay is 0.5 ng=ml. The intra- and inter-assay-variability are 3.7% and 12.6%, respectively, for values between 1.3±1.9 ng=ml. Statistical analysis To test whether food intake has an impact on circulating biological variables (leptin and others) we used one-way analysis of variance with repeated measures followed by the Dunnet test for post-hoc analysis. The response to the test meal (area under the curve, AUC) was assessed as follows: response (AUC) ˆ S[Var Tx 7 Var T0 ], where Var is the biological variable of interest, Tx represents time after the meal (x range from 0±10 h in experiment 1 or 0±8 h in experiment 2) and T0 is the baseline value of the variable of interest. Correlation analysis was performed using the Pearson correlation analysis. In experiment 2, comparison of day and night values was performed using a Paired t-test. Figure 2 Experiment 1: Mean (s.e.m.) concentration of triglycerides (upper panel) and leptin (lower panel) before and after food intake. ANOVA with repeated measures, P < 0.0001 for triglycerides and P < 0.0001 for leptin; *P< 0.05, **P< 0.01, *** P < 0.001 relative to T0 (Dunnet test). Results Experiment 1 ANOVA with repeated measures indicates a statistically signi cant effect of fatty breakfast intake on plasma triglycerides (P < 0.0001) and leptin (P < 0.0001). Mean plasma triglycerides (TG) levels increased progressively to reach the highest value 4 h postprandially (post-hoc Dunnet test: P < 0.0001) and returned to baseline value 7 h after the ingestion of the fat-rich meal (Figure 2). Mean leptin levels decreased between baseline and 2 h postprandially, and then increased to reach the highest value 8 h postprandially (P < 0.01) (Figure 2). Mean leptin was still signi cantly higher than baseline (P < 0.05) 10 h after the test-meal. The postprandial peak of leptin occurred three hours after the TG peak. There was no statistically signi cant correlation between the postprandial TGs and leptin responses (AUC). Experiment 2 Baseline levels. Baseline leptin was measured twice at 13.00 h and twice at 01.00 h, 6 h after a standard pre test-meal, containing 20% of the family energy needs. In these conditions, serum leptin measurements were highly comparable within subjects at 13.00 h (r ˆ 0.95, P < 0.0001) and at 01.00 h (r ˆ 0.96, P < 0.0001) and also between night (0.1.00 h) and day (13.00 h) (r ˆ 0.97, P < 0.0001). Serum leptin was correlated with body fat (r ˆ 0.74, P < 0.01). There was, however, a statistically signi cant difference between day and night (13.00 h vs 01.00 h) values for baseline glucose (P < 0.03) and cortisol (P < 0.0001) (Table 1). Both variables were higher during the day than at night, indicating a circadian in uence for these variables. In contrast, there was no statistically signi cant difference between day and night concentrations of leptin, insulin and C-peptide values (Table 1). Feeding±fasting manipulations. Glucose levels peaked 60 min postprandially and returned to the basal levels 6 h after the meal. Similarly, the changes in plasma insulin (Figure 3) and C-peptide levels were in agreement with expected postprandial changes. Oneway ANOVA with repeated measures shows a statistically signi cant effect of food intake on circulating Table 1 Baseline values (Mean s.d.) of the 13 study subjects. Blood samples were taken before the test meal or before the fasting experiment. Subjects had had the same light meal 6 h (at 7.00 h or 19.00 h) before experiment. Values are mean (s.d.) of two samples taken at the same hour Baseline 13.00 h 01.00 h P a Glucose (mmol/l) 5.02 0.26 4.79 0.22 <0.03 Leptin (ng/ml) 2.66 1.6 2.47 1.4 NS Insulin (pmol/l) 53.4 30.2 50.1 16 NS C-peptide (nmol/l) 0.49 0.19 0.43 0.15 NS Cortisol (nmol/l) 195.5 55 56.5 45 <0.0001 a Paired t-test was used to compare night and day values. NS ˆ not statistically signi cant.
731 Figure 3 Experiment 2: Mean ( s.e.m.) concentration of the insulin (upper panels) and leptin (lower panels), according to time after the postprandial or fasting experiment. Circles (squares) represent the values obtained after the feeding (fasting) experiments. Open (dark) signs are the results during the day (night). ANOVA with repeated measures: P < 0.0001 for postprandial insulin (both at night and during the day), not statistically signi cant (NS) for insulin during the fasting experiment; P < 0.0001 for postprandial leptin (both at night and during the day), and P < 0.005 for leptin during the fasting experiment (both at night and during the day); * P < 0.05, ** P < 0.01, ***P< 0.001 relative to T0 (Dunnet test). leptin (P < 0.0001, both at night and during the day), and of fasting (both at night P < 0.005 and during the day P < 0.0001). During the day, mean serum leptin increased progressively from 2.65 1.7±3.34 2.2 ng=ml (P < 0.001 with post-hoc Dunnet test) 6 h after the test-meal (Figure 3). Fasting was associated with a progressive decrease in circulating leptin levels from 2.68 1.5±1.92 1.1 ng=ml (P < 0.001 with post-hoc Dunnet test) 8 h after the beginning of the fasting experiment (Figure 3). Similar results were obtained at night. Circulating levels of leptin increased from 2.66 1.7±3.65 2.5 ng=ml (P < 0.001 with post-hoc Dunnet test) 8 h post-prandially and decreased from 2.29 1.2±1.70 0.9 ng=ml (P < 0.05 with post-hoc Dunnet test) after an 8 h fast (Figure 3). Day and night comparison. Table 2 summarizes the results of the postprandial or fasting responses during the day and at night. The postprandial glucose response was signi cantly higher (P < 0.01) at night than during the day. Similarly, the glucose response to fasting was signi cantly higher (P < 0.05) at night than during the day. This indicates a circadian cycle for glucose response. In contrast, the postprandial or fasting responses of leptin, insulin, C-peptide and cortisol were not statistically different between night and day. Table 2 Response (Mean s.d.) to postprandial or fasting experiments of the 13 study subjects, according to the time of the meal. The response is assessed as the area under the postprandial curve as described in methods Test meal P a Fasting P a Day Night Day Night Glucose (mmol/l) b 25.5 12.1 37.7 11.7 <0.01 0.83 3.9 4.55 3.5 <0.05 Leptin (ng/ml) c 3.0 4.1 4.1 4.1 NS 72.9 2.2 71.5 2.2 NS Insulin (pmol/l) b 4860 2220 5340 2100 NS 252 642 174 222 NS C-peptide (nmol/l) b 36.4 10 38 11 NS 72.5 5.3 70.06 2.3 NS Cortisol (nmol/l) d 375 358 488 524 NS 7121 358 727.6 422 NS a Paired t-test was used to compare night and day values. b Response calculated on blood samples taken every 20 min for 6 h. c Response calculated on blood samples taken every hour for 8 h. d Response calculated on blood samples taken every 20 min for 2 h. NS ˆ not statistically signi cant.
732 Discussion The design of the present study allowed the investigation of the acute role of food intake on circulating leptin levels and whether the time of feeding affects this response to a meal. Food intake was associated with an increase, whereas fasting was associated with a decrease, in circulatory leptin levels. The postprandial increase in leptin was not affected by circadian cycle. These results indicate that leptin levels are modulated by food intake in humans. In two independent studies with different test-meals (a fatty breakfast and a mixed meal), food intake resulted in a postprandial rise in circulating leptin levels. As expected, glucose levels increased abruptly 60 min after the mixed meal and returned to baseline value 6 h later. C-peptide and insulin levels also increased rapidly after the mixed meal. These rapid indications were in marked contrast with the slower response of serum leptin to the test meal, which only began to reach statistical signi cance 4 h postprandially. This nding is in agreement with the other observations, 10,20 which showed a delayed increase in circulating leptin levels after infusion of insulin. This time course supports the concept that postprandial leptin response in humans could be the consequence of the trophic effect of insulin on adipocytes, as suggested by others. 3 In the present studies, blood sampling was performed in the 10 h and 8 h after food intake. This duration was too short to allow serum leptin to return completely to baseline and suggested that acute food ingestion has a delayed and prolonged effect on circulating leptin levels. Previous feeding experiments did not show any signi cant changes in postprandial leptin levels in humans. 4±9 The reasons for the apparent discrepancies with the present study, could be the lack of standardization of the test-meals, differences in sampling intervals and duration of the experiments. In the report of Maffei et al, 4 leptin measurements were performed one hour after each meal. Sinha et al 6 analyzed circulating leptin before and 0.5, 1 and 2 h after food intake. Dagogo-Jack et al 7 determined plasma leptin 3 h after eating a usual lunch. Weigle et al 8 performed measurements on pooled samples including early postprandial values. Finally, Clapham et al 9 measured leptin, 1, 2 and 3 h following a mixed meal. In agreement with these observations, we did not observe any statistically signi cant change in leptin levels up to 4 h postprandially. Another possible source of disagreement is the inclusion of obese subjects in the postprandial experiment, such as in the report of Considine et al. 5 Recent studies in rodents have demonstrated that leptin response to intra-peritoneal injection of glucose is blunted in obese mice as compared to lean controls. 21 If such observation is true in humans, the combined analysis of obese and lean subjects might have masked a potential effect of food intake on leptin response. Other observations, however, support our data: Kolaczynksi et al 13 reported that massive overfeeding results in an increase in leptin level which peaks between 5 and 10 h after starting the overfeeding program; Coppack et al 14 found that leptin production from adipose tissue is increased ve hours after a high carbohydrate meal; and Schoeller et al 22 found that the diurnal rhythm of plasma leptin in young males is entrained to meal timing. Our work extends these observations to a normo-caloric mixed meal and circulating leptin levels in humans. In contrast to the glucose postprandial response, which was higher at night than during the day, the leptin response was not different between night and day. This suggests that, under carefully controlled conditions of food intake, leptin levels are less affected by circadian rhythm than by food intake. This observation is in apparent contradiction with previous published studies which concluded that leptin was in uenced by a circadian rhythm. 6,15 ± 17 A number of reasons may explain this difference. In 6,15 ± 17 previous experiments, the diurnal cycle in uences were assessed in subjects during a usual sleep=wake cycle, that is, eating during the day and sleeping during the night. Such experimental design does not allow the sorting out of the effect of food intake and=or sleep from the true effect of circadian rhythm. Therefore, because of the in uence of food intake on circulating leptin levels, the reported 6,15 ± 17 increase in serum leptin levels at night time could be the consequence of the relatively slow and cumulative effect of diurnal meals. In the present study, the subjects were exactly in the same nutritional state at night or during the day. They did not modify their food intake the day preceding the experiment, as assessed by dietary recall. Furthermore, six hours before baseline determination, they had the same light meal containing 20% of theoretic energy need. Finally, in the present study the subjects were not allowed to sleep, which permitted the assessment of the effect of the diurnal cycle and food intake, independently of sleep related metabolic changes 23 ± some of which may affect leptin levels. 24 In the present study, the circadian cycle was demonstrated by cortisol changes, and the lack of sleep-related hormonal disturbances by the absence of sleep-related growth hormone peak (data not shown). Accordingly, we observed the expected circadian modulation of glucose response to a meal 25 but no in uence on the leptin response. Conclusion Leptin levels increase after food intake and decrease during fasting, indicating that leptin levels are under feed-back control of food intake. In addition, the leptin response to food intake is not affected by the
circadian cycle. This suggests that leptin is submitted to short term regulation by food intake in lean men. Acknowledgements The authors would like to thank the staff of the C.I.C. du CHRU de Lille for their excellent clinical intervention. This study was supported by the grant 931501 from INSERM, by grant from MinisteÁre de la Sante et Solidarite Nationale and from GIP Cereste. References 1 Meisler JG, Jeor SS. Summary and recommendations from the American Health Foundation's Expert Panel on healthy weight. Am J Clin Nutr 1996; 3S: 474S±476S. 2 Trayhurn P. Leptin the `new' player in energy balance and obesity. BNF Nutr Bull 1997; 22: 7±14. 3 Caro JF, Sinha MK, Kolacynski JW, Zhang PL, Considine RV. Leptin: the tale of an obesity gene. Diabetes 1996; 45: 1455± 1462. 4 Maffei M, Halaas J, Ravussin E, Pratley RE, Lee GH, Zhang Y, Fei H, Kim S, Lallone R, Ranganathan S, Kern PA, Friedman JM. 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