Leptin was assayed by RIA for human leptin (Phoenix. Pharmaceuticals, Inc., Phoenix, AZ). Intra- and .... different bowel kinetics, and/or modified absorption pat-.
0021-972X/99/$03.00/0 The Journal of Clinical Endocrinology & Metabolism Copyright © 1999 by The Endocrine Society
Vol. 84, No. 7 Printed in U.S.A.
Plasma Leptin Levels after Biliopancreatic Diversion: Dissociation with Body Mass Index L. DE MARINIS, A. MANCINI, D. VALLE, A. BIANCHI, D. MILARDI, A. PROTO, A. LANZONE, AND R. TACCHINO Departments of Endocrinology and Clinical Surgery (R.T.), Centro di Studio per la Fisiopatologia dello Shock-Centro Nazionale Ricerche (CNR), Catholic University School of Medicine, 00189 Rome, Italy; Eli Lilly & Co. (D.V.), 50121 Florence, Italy; and Oasi (A.P., A.L.), 94018 Troina, Italy ABSTRACT Human obesity is associated with increased leptin levels, related to body composition and fat mass (FM). Insulin has been suggested to be a regulator of in vivo leptin secretion. To further investigate the relationships between insulin and leptin levels in human obesity, we have studied 10 obese females, aged 26 –57 yr [body mass index (BMI), 42.9 6 6.3], successfully treated by biliopancreatic (BPD) diversion, in an early postoperative period (2 months after surgery, post-BPD I; BMI, 37.2 6 7.5) and a late postoperative period (16 –24 months after surgery; BMI, 27.6 6 3.96). Fourteen normal female subjects (18 –59 yr; BMI, 27.9 6 1.4 kg/m2) were studied as controls. In pre-BPD obese subjects, leptin levels were higher than those in controls (60.5 6 18.8 vs. 28.7 6 4.8 ng/mL; P , 0.001). BMI and insulin levels were also significantly greater (P , 0.0001 and P , 0.03, respectively). After surgery, the three parameters considered significantly decreased (P 5 0.0007 for BMI, P , 0.0001 for leptin, and P 5 0.038 for insulin, using Friedman’s test for repeated data). Concerning the correlation be-
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UMAN obesity is related to increased messenger ribonucleic acid (mRNA) and plasma leptin levels. A correlation has been clearly demonstrated with body composition and fat mass (FM) (1–3). Leptin is produced by adipose tissue in various mammals and humans (4 – 6); ob/ob mice, which show a nonsense mutation in the ob gene and lack circulating leptin, are characterized by abnormal body weight and adipose mass (7). In obese subjects, leptin is increased due to a high amount of fat mass and a higher production rate per U body fat with increased weight (8, 9); because the enhanced serum leptin concentrations did not prevent weight gain, leptin resistance is assumed (4, 10). Very recently, leptin deficiency due to mutation in the leptin gene has been reported to be associated with early-onset obesity (11); moreover, a homozygous mutation in the human leptin receptor gene that results in a truncated leptin receptor, lacking both the transmembrane and the intracellular domains, has been described (12). In addition to their early-onset morbid obesity, these patients have no pubertal development and exhibit reduced GH and TSH secretion (12). A relationship of human obesity with insulin levels has been hypothesized. Insulin has been suggested to be a Received April 16, 1998. Revision received June 3, 1998. Rerevision received March 12, 1999. Accepted March 23, 1999. Address all correspondence and requests for reprints to: Dr. Laura De Marinis, Catholic University School of Medicine, 901 Via Cassia, 00189 Rome, Italy.
tween leptin and FM in our patients, control subjects and pre-BPD subjects confirmed the correlation found in the general population (r 5 0.78; P , 0.01). On the contrary, post-BPD patients at 2 months lay outside the general correlation between FM and leptin; in fact, patients with low leptin levels still had a high FM. Moreover, in the post-BPD patients there was no longer a significant correlation between FM and leptin. Concerning the correlation between insulin and leptin levels, a significant correlation was present in control subjects and pre-BPD patients (r 5 0.46; P , 0.05). Using correlation analysis for repeated measures in surgically treated obese patients, a significant correlation within the subjects was present (r 5 0.91; P , 0.0001). After operation, BMI and leptin levels had a different pattern of decrease; leptin decreased rapidly, without correlation with BMI, indicating that body composition is not the only factor regulating leptin levels. The consistent correlation with insulin levels suggests an important interaction between these two hormones in post-BPD obese subjects. (J Clin Endocrinol Metab 84: 2386 –2389, 1999)
regulator of in vivo leptin secretion by adipose tissue in lean, but not in genetically obese (fa/fa), rats (13). In humans, a long term effect of insulin on leptin production has been demonstrated both in vivo and in vitro (14). In NIDDM patients, the concentration of plasma leptin is closely related to that of insulin, independent from insulin resistance (15). The effect of fasting, in both acute (16, 17) and chronic studies (1), has been investigated. Fasting is associated with a reduction of leptin levels, but the mechanism does not simply seem to be the result of the loss of adipose tissue. A short period of fasting induced a decrease in leptin levels, with reversion during refeeding, even if body weight did not vary (16). Leptin variation has also been reported in surgically induced weight loss (18), in a group of patients studied 24 –30 months after gastric by-pass operation. Biliopancreatic diversion is an important model of such weight loss, also associated with other important variations that can influence leptin levels. Only during the early period did biliopancreatic diversion (BPD) reduce insulin levels and restore insulin sensitivity (19). We have studied the pattern of variation in BMI and circulating levels of insulin and leptin in a group of 10 obese female subjects before and after BPD to investigate the timerelated changes in leptin levels after surgically induced weight loss, and we have further explored relationships between insulin and leptin levels in human obesity.
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LEPTIN LEVELS AFTER BILIOPANCREATIC DIVERSION Subjects and Methods This study protocol was approved by the ethical committee of our institution after informed consent was obtained from the subjects. We studied 10 morbidly obese patients who were scheduled to undergo therapeutic BPD (20) (pre-BPD subjects) because dietary therapy had failed. They were 10 females, aged 26 –57 yr, with a mean body mass index (BMI) of 42.9 6 6.3 kg/m2 (range, 32.5–54.6). They were studied at different intervals after successful BPD (post-BPD subjects): the early postoperative period (2 months after surgery, post-BPD I) and the late postoperative period (18 –24 months after surgery, post-BPD II). BPD consists of a partial gastrectomy with Roux-en-Y reconstruction. Gastric volumes range from 200 – 400 mL. The lengths of the alimentary tract and common tract are 200 and 50 cm, respectively. As a consequence, food is subverted from the normal action of biliary and pancreatic secretion, except for the common tract. The patients develop fat malabsorption (75% of ingested) and partial starch malabsorption, while maintaining a normal absorption of monodisaccharides (19% of ingested starch plus monodisaccharides) and a normal absorption of proteins (21). The demonstrated metabolic and hormone results (22–24) include 1) reversal of insulin resistance; 2) increase in dietinduced thermogenesis; and 3) modification of gut hormones, such as gastrin, enteroglucagon, neurotensin, and cholecystokinin. The control group consisted of 14 normal female subjects (aged 18 –59 yr; mean BMI, 27.9 6 1.4 kg/m2; range, 25.7–29.7 kg/m2). No patients of the other groups were taking medications. No patients suffered from diabetes mellitus, thyroid diseases, or chronic diseases. All groups were studied during their usual diet; mean energy intake was 2500 6 894 Cal/24 h (composed of 47 6 7% carbohydrates, 13 6 6% proteins, and 39 6 10% lipids) in pre-BPD subjects, 2940 6 1018 Cal/24 h (63 6 16% carbohydrates, 11 6 4% proteins, and 26 6 4% lipids) in post-BPD subjects, and 1460 6 230 Cal/24 h (55 6 5% carbohydrates, 13.6 6 2.8% proteins, and 30.6 6 4.1% lipids) in normal subjects. Basal samples of leptin and insulin levels were collected at 0800 h in the fasting state. Leptin was assayed by RIA for human leptin (Phoenix Pharmaceuticals, Inc., Phoenix, AZ). Intra- and interassay coefficients of variation were, respectively, 4.2% and 4.5%. The sensitivity of the method was 0.5 ng/mL. Insulin was assayed by RIA using kits from Abbott Diagnostics (Milan, Italy). Intra- and interassay coefficients of variation were, respectively, 4.5% and 5.6%. Normal basal plasma insulin levels ranged from 5–20 mU/mL. Total body mass, lean body mass, and FM in all subjects were determined by dual x-ray absorptiometry using a commercial scanner (Lunar DPX, Lunar Europe, Everberg, Belgium).
FIG. 1. Mean (6SEM) BMI and plasma leptin and insulin levels in 10 obese patients studied pre-BPD, 2 months after operation (post-BPD I), and 18 –24 months after operation (post-BPD II) and in 10 normal weight controls. *, P , 0.05 vs. control subjects (using MannWhitney U test; see text for details). #, P , 0.05 vs. pre-BPD patients (using Student-Newman-Keuls multiple comparison test; see text for details). E, P , 0.05 vs. post-BPD-I.
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Statistics The distribution of the data was tested with the Kolmogorov-Smirnov test and the Shapiro-Wilk test to verify whether the samples came from a specified distribution. As the data were not normally distributed, statistical analysis was performed using the Mann-Whitney U test when comparing controls vs. pre-BPD subjects and Friedman test for repeated data when evaluating longitudinal data in surgically treated obese subjects; then, Student-Newman-Keuls multiple comparisons test was used to determine significance among the three time points in obese patients. Spearman’s rank correlation and correlation analysis for repeated observations (25) were employed to evaluate the correlation between different parameters. For statistical evaluation we used the software package Primer of Biostatistics, version 4.02 (McGraw-Hill; for Windows95, New York, NY).
Results
The mean 6 sem levels of BMI, leptin, and insulin levels in our patients, tested before surgery (pre-BPD), in the early postoperative period (post-BPD I), and in the late postoperative period (post-BPD II), and in control subjects are shown in Fig. 1. In pre-BPD patients, BMI, leptin, and insulin levels were significantly greater than those in control subjects (P , 0.0001, P , 0.006, and P , 0.03, respectively). Friedman test for repeated data showed significant variations in the three parameters considered (P 5 0.0007 for BMI, P , 0.0001 for leptin, and P 5 0.038 for insulin). Student-Newman-Keuls multiple comparison test confirmed a significant difference between pre-BPD and post-BPD (both post-BPD I and BPDII, P , 0.05 for BMI, leptin, and insulin). No significant difference was observed in leptin levels between post-BPD I and post-BPD II; on the contrary, BMI showed a significant decrease between post-BPD-I and post-BPD-II (Fig. 1). Figure 2 shows the correlation between leptin and FM in our patients. Control subjects and pre-BPD subjects confirmed the correlation found in the general population (r 5 0.78; P , 0.01). The effect of surgically induced weight loss was to decrease both FM and leptin levels. Using correlation analysis for repeated evaluation, we found a significant correlation
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FIG. 2. Correlation between FM and leptin levels in our patients and normal controls. ƒ, Control subjects; ‚, obese subjects before surgery (pre-BPD); E, post-BPD I (2 months after surgery); L, post-BPD II (18 –24 months after surgery).
FIG. 3. Correlation between insulin and leptin levels in our patients and normal controls. ƒ, Control subjects; ‚, obese subjects before surgery (pre-BPD); E, post-BPD I (2 months after surgery); L, postBPD II (18 –24 months after surgery).
(r 5 0.79; P , 0.0001). However, post-BPD patients at 2 months lay outside the general correlation between FM and leptin; patients with low leptin levels still had a high FM. Moreover, in the post-BPD patients there was no longer a significant correlation between FM and leptin. Figure 3 shows the correlation between insulin and leptin levels in our patients. A significant correlation was present in control subjects and pre-BPD patients (r 5 0.46; P , 0.05). Using correlation analysis for repeated evaluation in surgically treated obese patients, a significant correlation within the subjects was present (r 5 0.91; P , 0.0001). Discussion
Our data show a dissociation in the decrease in leptin and insulin, on the one hand, and in body weight, on the other
hand, after surgically induced weight loss. Some data were available on leptin changes during diet-induced weight loss. Maffei et al. (1) showed decreased leptin levels after dieting in 14 obese females. Considine et al. (2) demonstrated a leptin reduction after 10% body weight loss. Our data are in agreement with those reported by Scholtz (26), who showed uncoupled decreases in leptin and body weight during a long term hypocaloric diet. He showed that more than 75% of the leptin changes occurred during the first 2 weeks. Also Sinha et al. (17) showed a 50% drop in free plasma leptin levels after short term fasting (24 h). Our data confirm the dissociation between leptin and BMI and were obtained in subjects who were not fasting, but were instead consuming a hypercaloric diet. Thus, they indicate a more complex regulation of leptin than previously hypothesized. Our study demonstrates a similar pattern of decrease in insulin levels after BPD. The role of insulin in the regulation of ob gene expression and leptin concentration has been investigated in rats and humans (27–29). In rats, insulin administration increases ob gene mRNA after 6 h (27). Two days of insulinization also increase ob gene expression in rats (28); 2-h exposure to insulin increases by 30% ob mRNA in cultured adipocytes (27) and stimulates dose-dependent leptin release from freshly isolated rat adipocytes (30). However, prolonged insulin infusion (8 h) is required in humans to increase circulating leptin (29). Very recently, it has been shown that 2-h hyperinsulinemia, obtained by euglycemic hyperinsulinemic clamp, increases circulating leptin levels in lean rats, whereas fa/fa rats, which exhibit an elevated basal leptin concentration, are not influenced by such a treatment (13). These data strongly support the role of insulin in regulating plasma leptin in animals. Our study is the first performed in subjects who were not receiving diet treatment, thus excluding the role of reduced caloric intake in the modulation of leptin levels. Instead, underlying the correlation between insulin and leptin, they can recognize other possible mechanisms: an altered release of gastroenteric hormones, due to the operation itself or to different bowel kinetics, and/or modified absorption patterns of metabolic fuels. A strong link with insulin is observed; the consistent correlation of leptin with insulin levels suggests an important interaction between these two hormones in post-BPD obese subjects. References 1. Maffei M, Halaas J, Ravussin E, et al. 1995 Leptin levels in human and rodent: measurement of plasma leptin and ob RNA in obese and weight-reduced subjects. Nat Med. 1:1155–1161. 2. Considine RV, Sinha MK, Heiman ML, et al. 1996 Serum immunoreactiveleptin concentrations in normal-weight and obese humans. N Engl J Med. 334:292–295. 3. Dagogo-Jack S, Fanelli C, Paramore D, Brothers J, Landt M. 1996 Plasma leptin and insulin relationships in obese and nonobese humans. Diabetes. 45:695– 698. 4. Zhang Y, Proenca R, Maffei M, Barone M, Leopold L, Friedman JM. 1994 Positional cloning of the mouse obese gene and its human homologue. Nature. 372:427– 432. 5. Considine RV, Considine EL, Williams CJ, et al. 1995 Evidence against either a premature stop codon or the absence of obese gene mRNA in human obesity. J Clin Invest. 95:2986 –2988. 6. Masuzaki H, Ogawa Y, Isse N, et al. 1995 Human obese gene expression. Adipocyte-specific expression and regional differences in the adipose tissue. Diabetes. 44:855– 858. 7. Pelleymounter MA, Cullen MJ, Baker MB, et al. 1995 Effects of obese gene product on body weight regulation in ob/ob mice. Science. 269:540 –543.
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