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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2004-1921
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The Journal of Clinical Endocrinology & Metabolism Vol. 90, No. 3 1618-1624
Copyright © 2005 by The Endocrine Society

Recombinant Methionyl Human Leptin Administration to Achieve High Physiologic or Pharmacologic Leptin Levels Does Not Alter Circulating Inflammatory Marker Levels in Humans with Leptin Sufficiency or Excess

Jean L. Chan, John Bullen, Violeta Stoyneva, Alex M. DePaoli, Carol Addy and Christos S. Mantzoros

Division of Endocrinology, Diabetes, and Metabolism (J.L.C., J.B., V.S., C.A., C.S.M.), Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215; and Amgen, Inc. (A.M.D.), Thousand Oaks, California 91320

Address all correspondence and requests for reprints to: Dr. Christos S. Mantzoros, Division of Endocrinology, Diabetes, and Metabolism, Beth Israel Deaconess Medical Center, Harvard Medical School, 330 Brookline Avenue, ST 816, Boston, Massachusetts 02215. E-mail: cmantzor{at}bidmc.harvard.edu.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
A role for high leptin levels in the proinflammatory state associated with obesity has been proposed on the basis of observational studies, but a recent interventional study employing administration of long-acting pegylated leptin resulting in very high pharmacologic levels in obese subjects did not support this idea. These interventional studies have not yet been independently confirmed, however, and varying levels and duration of hyperleptinemia as well as the presence of comorbidities such as diabetes have not yet been investigated as potential effect modifiers. We performed three interventional studies involving administration of recombinant methionyl human leptin (r-metHuLeptin) to lean, otherwise healthy obese, and obese diabetic subjects to investigate whether increasing circulating leptin levels over a wide spectrum of values (from low physiologic to high pharmacologic) would alter serum levels of inflammatory markers and other cytokines important in the T helper cell response. Increasing leptin levels from low physiologic to high physiologic in lean men and from higher physiologic to low pharmacologic in obese men over 3 d did not alter serum interferon-{gamma}, IL-10, TNF-{alpha}, monocyte chemoattractant protein-1, or soluble intercellular adhesion molecule-1. In obese subjects with type 2 diabetes mellitus, the administration of r-metHuLeptin for 4 or 16 wk, resulting in high pharmacologic leptin levels, did not activate the TNF-{alpha} system or increase cytokines or inflammatory markers, including IL-10, IL-6, C-reactive protein, monocyte chemoattractant protein-1, and soluble intercellular adhesion molecule-1. These findings do not support an etiopathogenic role for leptin in proinflammatory states associated with leptin excess such as obesity and have direct relevance for the potential future therapeutic use of r-metHuLeptin in humans.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
IT IS WELL recognized that human obesity is a proinflammatory state (1), and it has also been recently demonstrated that the accumulation of macrophages in adipose tissue results in up-regulation of inflammatory genes (2, 3). Up-regulation of these genes and increased levels of inflammatory markers contribute to the morbidity associated with obesity, including insulin resistance, diabetes mellitus, and cardiovascular disease (1, 4). The proinflammatory profile associated with obesity improves after weight loss, and this, in turn, contributes to the improvement in comorbidities (5, 6, 7).

Several hormones and other metabolically active molecules secreted by adipose tissue have been proposed as relevant to the pathogenesis of this inflammatory state (8). Leptin, an adipocyte-secreted hormone with structural similarity to cytokines, has emerged as a potential candidate for the link between obesity and the proinflammatory state. Studies in animal and human models of congenital complete leptin deficiency, which are characterized by morbid obesity and immune dysfunction, suggest a potentially important role for leptin in regulating immune function (9, 10, 11). Exogenously administered leptin reverses the immunosuppression associated with not only congenital, but also acquired, leptin deficiency induced by starvation in normal mice (9, 10). More specifically, leptin modulates T helper (Th) cells toward a Th cell type 1 (Th1) phenotype, with the secretion of proinflammatory cytokines such as interferon-{gamma} (IFN-{gamma}), and away from Th cell type 2 (Th2) regulatory cytokine production (10). Moreover, in two leptin-deficient children, recombinant methionyl human leptin (r-metHuLeptin) administration improved CD4+ Th cell counts, T cell proliferation, and cytokine release (11).

Data demonstrating that the complete absence of leptin is associated with clear deficits in immune function that are normalized by replacement dose r-metHuLeptin administration has formed the basis for the hypothesis that increased leptin levels at the other end of the energy homeostasis spectrum, i.e. human obesity, also play a pathogenic role in the development of the proinflammatory state. The evidence for a role for leptin in the latter has primarily been circumstantial, however. Circulating levels of leptin are high in human obesity (12) and correlate with increased levels of inflammatory markers in obese individuals (13), whereas weight loss results in a decrease in leptin levels and improvement in these markers (5, 6). These findings, derived from observational studies, are not free from potential bias due to uncontrolled confounding. Moreover, they are in contrast with the results of a recent placebo-controlled study on the effect of administration of long-acting pegylated recombinant leptin treatment (resulting in very high pharmacologic leptin levels) for 6 wk on inflammatory markers in 22 healthy overweight men (14). This study, which did not demonstrate any evidence for a pathogenic proinflammatory role for leptin (14), has not yet been confirmed. It could be argued, however, that the 500-fold higher leptin levels achieved with the pharmacologic doses of leptin employed in the prior study may not be of physiologic relevance for the obesity-associated proinflammatory state in which circulating leptin levels only rarely exceed 100 ng/ml. Also, the relatively small size of the prior study raises the possibility that a weak effect of leptin may have not achieved statistical significance and thus may have been missed due to inadequate power. Finally, whether there exists a threshold leptin level for changes in inflammatory markers to occur, and whether there is a differential response to high leptin levels based upon gender, body fat mass, or comorbidities, such as type 2 diabetes, remains unknown, but may have significant clinical relevance.

To address these questions, we performed three interventional studies in humans with administration of placebo or r-metHuLeptin to achieve high physiologic to pharmacologic levels. We first evaluated whether raising circulating leptin levels in lean subjects from their normal levels to higher physiologic levels, such as those seen in obese subjects, over the course of a few days can change cytokines important in the Th cell response as well as inflammatory and adhesion molecules such as monocyte chemoattractant protein-1 (MCP-1) and soluble intercellular adhesion molecule-1 (sICAM-1). Similarly, we evaluated the effect of raising serum leptin levels in obese individuals from higher physiologic to supraphysiologic or low pharmacologic ranges on serum levels of the same cytokines and inflammatory molecules. Finally, in the context of a randomized, placebo-controlled study of 117 obese patients with type 2 diabetes mellitus, we studied whether increasing leptin levels for a longer period of time, i.e. 4 and 16 wk, with pharmacologic dose r-metHuLeptin administration would alter cytokines (e.g. IL-10) or several of the inflammatory markers most important in insulin resistance (including the TNF-{alpha} system) and/or cardiovascular disease [including IL-6, C-reactive protein (CRP), MCP-1, and sICAM-1].


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Short-term studies: effect of short-term r-metHuLeptin on serum cytokine levels and inflammatory markers in healthy men fasted for 3 d

Five lean and five obese, otherwise healthy subjects gave written informed consent to participate in a study protocol approved by the institutional review board of the Beth Israel Deaconess Medical Center (BIDMC). Clinical quality r-metHuLeptin was supplied by Amgen, Inc. (Thousand Oaks, CA), and administered under an investigational new drug application approved by the Food and Drug Administration (to C.S.M.). All subjects were healthy, had no evidence of immunologic or endocrine disease based on physical examination and routine blood tests, and had no history of recent infection.

r-metHuLeptin administration to increase leptin levels from low physiologic to high physiologic levels. Five lean men [age, 22.2 ± 2.0 yr; body mass index (BMI), 22.0 ± 1.0 kg/m2 (mean ± ] were studied at the BIDMC General Clinical Research Center (GCRC) during a 3-d fasting protocol. Subjects were admitted the night before the first study day and received only caffeine- and calorie-free liquids, NaCl (500 mg), KCl (40 meq), and a standard multivitamin with minerals daily for 3 d. Subjects received a single dose of r-metHuLeptin (0.3 mg/kg) by sc injection at 0800 h once daily for 3 consecutive days. Blood samples for measurement of leptin, TNF-{alpha}, IFN-{gamma}, IL-10, MCP-1, and sICAM-1 levels were obtained at baseline (0800 h, just before the dose of r-metHuLeptin) and 4 and 8 h after the 0800 h dose on the third day.

R-metHuLeptin administration to increase leptin levels from higher physiologic to pharmacologic levels. Five obese men (age, 23.4 ± 3.4 yr; BMI, 32.0 ± 2.3 kg/m2) were studied at the BIDMC GCRC during a 3-d fasting protocol similar to that described above. Subjects received a single dose of r-metHuLeptin (0.3 mg/kg) by sc injection at 0800 h once daily for 3 consecutive days, with measurement of leptin, cytokine, and inflammatory marker levels as described above at 0800 h (baseline) and 4 and 8 h after the 0800 h dose on the third day.

Long-term study: effect of pharmacologic-dose r-metHuLeptin for 16 wk on inflammatory markers in hyperleptinemic obese subjects with diabetes mellitus

One hundred and seventeen obese subjects (62 males and 55 females; age, 53.3 ± 11.4; BMI, 33.2 ± 3.8 kg/m2) with diet-controlled type 2 diabetes mellitus gave written informed consent to participate in a study protocol approved by the University of California-Los Angeles Medical Center human subjects protection committee. Subjects were randomized in a 2:1 ratio to receive either r-metHuLeptin at a dose of 10 mg or matching placebo, respectively, twice daily (morning and evening) by sc injection for 16 wk (resulting in a total daily dose of 20 mg r-metHuLeptin, i.e. ~0.2 mg/kg). Criteria for entry into the study included hemoglobin A1c between 7–11%, BMI between 27–40 kg/m2, and adherence to a stable weight-maintaining diet for at least 4 wk before the screening evaluation. Subjects who were pharmacologically treated for diabetes could not have taken oral hypoglycemic agents in the 12 wk before screening. Blood samples for measurement of leptin, soluble TNF receptor type I (sTNF-RI, 55 kDa), sTNF-RII (75 kDa), MCP-1, sICAM-1, CRP, IL-6, and IL-10 were obtained at baseline (before treatment with r-metHuLeptin or placebo) and after 4 and 16 wk of treatment (except in nine subjects who received r-metHuLeptin and six subjects who received placebo due to insufficient serum).

Measurements. Leptin was measured using RIA for study 1 [Linco Research, Inc., St. Charles, MO; sensitivity, 0.5 ng/ml; coefficient of variation (CV), 6–7%] and by immunoradiometric assay for study 2 [Diagnostics Systems Laboratory (DSL), Webster, TX; sensitivity, 0.1 ng/ml; CV, 3.7–6.6%]. The Linco and DSL assays for leptin demonstrated a high correlation with each other (for leptin level <100 ng/ml by DSL: r = 0.91, n = 262, and P < 0.001; for leptin level up to 800 ng/ml by DSL: r = 0.85, n = 517, and P < 0.001) (Mantzoros, C. S., unpublished observations). Cytokines and inflammatory markers were measured in serum or plasma using commercially available ELISAs: TNF-{alpha} (Quantikine HS, R&D Systems, Minneapolis, MN; sensitivity, 0.06 pg/ml; CV, 5.3–8.8%), sTNF-RI (Quantikine, R&D Systems; sensitivity, 3 pg/ml; CV, 2.7–6.9%), sTNF-RII (Quantikine, R&D Systems; sensitivity, <1 pg/ml; CV, 1.6–2.5%), IFN-{gamma} (Amersham Biosciences, Piscataway, NJ; high sensitivity, 0.1 pg/ml; CV, <10%), IL-10 (Amersham Biosciences; high sensitivity, 0.1 pg/ml; CV, <10%), MCP-1 (Quantikine, R&D Systems; sensitivity, <5.0 pg/ml; CV, 7.8–8.7%), sICAM-1 (Quantikine, R&D Systems; sensitivity, 0.35 ng/ml; CV, 4.8–10.1%), CRP (DSL; sensitivity, 1.6 ng/ml; CV, 1.7–3.9%), and IL-6 (Quantikine, R&D Systems; sensitivity, 0.04 pg/ml; CV, 6.9–7.8%).

Statistical analysis

Data are presented as the mean ± SD. Cytokine levels and inflammatory markers were compared between time points (0, 4, and 8 h or 0, 4, or 16 wk) using repeated measures ANOVA with post hoc tests by least significant difference as primary analysis as well as one-way ANOVA, with similar results obtained except where noted. ANOVA was performed with and without multivariate adjustment for treatment group, BMI, glycemic control, and covariates that differed between active treatment and placebo groups or with stratification for gender for the long-term study. {chi}2 tests were used to compare actual with expected rates of gender, aspirin, and statin medication use between active treatment and placebo groups. Analyses were carried out using StatView 5 (SAS Institute, Inc., Cary, NC) and/or SPSS 8.0 (SPSS, Inc., Chicago, IL). The long-term study had sufficient power (80% at the conventional {alpha} = 0.05 level) to detect a 3-fold smaller difference in mean values compared with the previous interventional leptin study (14). Subgroup analysis (after stratification for gender) resulted in equal or higher power compared with that of the only prior interventional study (14).


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Effect of short-term r-metHuLeptin on serum cytokine levels and inflammatory markers

We first evaluated whether raising circulating levels of leptin from normal to high physiologic levels (similar to levels in obese subjects) in lean men could affect cytokines important in the Th cell response, including IFN-{gamma}, a proinflammatory Th1 cytokine involved in Th1 differentiation of CD4+ naive T cells; IL-10, a regulatory cytokine important in Th1 and Th2 responses; and TNF-{alpha}, a proinflammatory cytokine previously associated with obesity and insulin resistance that represents an important link between specific immune and systemic inflammatory responses and has a known interaction with leptin in animal models and humans (15, 16, 17). We also evaluated inflammatory and adhesion molecules previously linked to obesity and insulin resistance, such as MCP-1 and sICAM-1, which have been shown to change over a similar time frame in response to administration of insulin (18). After a 0.3 mg/kg dose of r-metHuLeptin was administered to lean men, serum leptin levels increased 50-fold at 4 h to levels similar to those in obese subjects (from 1.6 ± 0.3 to 84.7 ± 13.8 ng/ml) and then decreased to 53.0 ± 11.0 ng/ml at 8 h, although still remaining at high physiologic levels. Over this time course of high physiologic leptin levels, serum levels of IL-10, TNF-{alpha}, MCP-1, and sICAM-1 did not change (Table 1Go). Serum IFN-{gamma} showed a slight decrease at 4 h, followed by a slight increase at 8 h (P = 0.003, by repeated measures ANOVA; P = 0.73, by one-way ANOVA), although the absolute magnitude of change was quite small. Fasting over this time frame resulted in a small, but significant, decrease in body weight (66.2 ± 5.8 to 63.5 ± 5.9 kg; P = 0.04).


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  		TABLE 1. Serum leptin and cytokine levels (mean ± SD) at baseline and 4 and 8 h after administration of a pharmacologic dose of r-metHuLeptin (0.3 mg/kg) for five lean men and five obese men

 
We then evaluated whether raising serum leptin levels from higher physiologic levels (as usually seen in obesity) to supraphysiologic or low pharmacologic (as typically only seen in extreme obesity) could alter the same cytokines and inflammatory markers. In obese men who received a 0.3 mg/kg dose of r-metHuLeptin, serum leptin levels increased 20-fold to low pharmacologic levels (from 13.7 ± 1.3 ng/ml at baseline to 274.3 ± 37.4 ng/ml at 4 h) and then decreased to 206.4 ± 25.1 ng/ml at 8 h, although still remaining at supraphysiologic levels. Over this time course of higher physiologic and pharmacologic serum leptin levels, there were no significant changes in IFN-{gamma}, IL-10, TNF-{alpha}, MCP-1, or sICAM-1 levels (Table 1Go). In obese men, body weight decreased similarly by a small, but significant, amount in response to fasting (104.9 ± 11.7 to 101.8 ± 11.5 kg; P = 0.04).

Effect of more long-term, pharmacologic dose r-metHuLeptin on serum inflammatory markers in hyperleptinemic obese subjects with diabetes mellitus

We then evaluated whether more long-term increases in leptin levels at pharmacologic ranges in obese diabetic subjects would increase the activity of the TNF-{alpha} system or affect levels of inflammatory markers important in cardiovascular disease, including IL-6, CRP, MCP-1, and sICAM-1. At baseline, the characteristics of subjects receiving r-metHuLeptin (n = 79) were similar to those of subjects receiving placebo (n = 37) with respect to BMI (r-metHuLeptin, 33.3 ± 3.9 kg/m2; placebo, 33.2 ± 3.6 kg/m2; P = 0.92), glycemic control (hemoglobin A1c: r-metHuLeptin, 8.1 ± 0.9%; placebo, 8.1 ± 0.8%; P = 1.0), and gender (male/female ratio, 39:40 for r-metHuLeptin; 18:19 for placebo; P = 0.94). Use of a statin medication for lipid lowering was similar in the two groups [n = 15 (19.5%) for r-metHuLeptin; n = 4 (11.1%) for placebo; P = 0.27], but the placebo group had a higher rate of aspirin use [n = 15 (19.5%) for r-metHuLeptin; n = 14 (38.9%) for placebo; P = 0.03]. Baseline leptin levels and inflammatory markers were similar between the r-metHuLeptin and placebo groups, except for MCP-1, which was higher at baseline in the placebo group (P = 0.04).

As expected, serum leptin levels remained unchanged in the 37 obese diabetic subjects who received placebo during the study (39.1 ± 23.9 ng/ml at baseline, 60.7 ± 122.8 ng/ml at 4 wk, and 60.9 ± 114.9 ng/ml at 16 wk; P = 0.63), and there were no changes in sTNF-{alpha} receptors, IL-10, IL-6, CRP, MCP-1, or sICAM-1 in these subjects (Table 2Go). In contrast, serum leptin levels increased from 43.9 ± 37.0 ng/ml at baseline to 405.9 ± 334.0 ng/ml at 4 wk and 920.0 ± 379.9 ng/ml at 16 wk (P < 0.001) in the 79 obese diabetic subjects who received pharmacologic doses of r-metHuLeptin (10 mg twice daily). Over a 4- or 16-wk time course of high pharmacologic leptin levels, cytokines and inflammatory markers did not change (Table 2Go). Similar results were obtained after stratification for gender and in univariate analysis of all subjects with adjustment for treatment group, and there was no effect of body weight, glycemic control, or aspirin use in multivariate analysis (data not shown).


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  		TABLE 2. Serum leptin levels and inflammatory markers (mean ± SD) at baseline and after 4 and 16 wk of placebo treatment (n = 37) or r-metHuLeptin (10 mg twice daily) treatment (n = 79) in obese subjects with type 2 diabetes mellitus

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
A role for leptin in modulating immune function has been established in models of leptin deficiency in animals and humans. Specifically, leptin reverses the immunosuppression associated with low leptin states. Because human obesity is generally a high leptin state and is associated with a proinflammatory tendency, prior observations in leptin-deficient states have been extrapolated to suggest that this proinflammatory state is due to high leptin levels. Although observational studies in humans have implicated leptin as possibly playing a role in the pathogenesis of the obesity-related pro-inflammatory state, a recent 6-wk interventional study employing very high pharmacologic doses of exogenous leptin did not support this idea. This concept has critical importance to the extensive literature identifying inflammation as an integral component of obesity-related states such as diabetes and cardiovascular disease, which contributes to morbidity and mortality (1, 4, 19). In these short- and long-term interventional r-metHuLeptin administration studies in lean and otherwise healthy obese subjects as well as in 117 obese diabetic subjects, respectively, we found that increasing serum leptin levels to high physiologic and pharmacologic ranges over 4 and 16 wk did not alter several inflammatory markers known to be important in insulin resistance and cardiovascular disease. Thus, our data do not support a pathogenic role for leptin in the proinflammatory state associated with obesity.

The realization that adipose tissue is a highly active endocrine organ that produces a vast array of hormones, cytokines, and other molecules relevant to metabolic function and inflammatory processes (8, 20) has fostered a proliferation of studies attempting to elucidate how inflammation develops in the context of excess adipose stores. Research has focused on molecules that are increased with obesity and/or are of potential pathophysiologic importance, such as TNF-{alpha}, IL-6, CRP, MCP-1, sICAM-1, and leptin among others, because they are associated with diabetes and cardiovascular disease. Activation of the TNF-{alpha} system is an important component of the systemic inflammatory response and correlates with important cardiovascular risk factors, such as insulin resistance, dyslipidemia, and prothrombotic tendencies (8, 21, 22). Soluble TNF-{alpha} receptors act as a buffer to prolong the biologic effects of TNF-{alpha} and thus serve as an accurate marker of activation of the TNF-{alpha} system in clinical research studies (16, 23). In addition, TNF-{alpha} increases the production of IL-6, (21), a systemic, proinflammatory cytokine that acts as a powerful inducer of the acute phase response and largely regulates the hepatic synthesis and secretion of CRP (24, 25), a well established and important indicator of cardiovascular risk (26). MCP-1, a chemokine that recruits monocytes to sites of inflammation and decreases insulin-stimulated glucose uptake, is increased in obesity and may contribute to insulin resistance (20, 27). Increased levels of inflammatory mediators induce systemic endothelial dysfunction, as reflected in elevated levels of adhesion markers such as sICAM-1, E-selectin, and vascular cell adhesion molecule-1 (28, 29). All of these inflammatory markers are associated with morbidity and mortality from diabetes and/or cardiovascular disease, (1, 17, 19, 21, 27, 30, 31), and this proinflammatory state is now considered part of the constellation of metabolic risk factors termed the metabolic syndrome (32), which has an alarmingly high prevalence of nearly 25% in the United States (33).

In observational human studies, leptin has a significant relationship with the TNF-{alpha} system. Leptin is independently associated with sTNF-{alpha} receptors after adjustment for BMI, gender, and/or insulin levels in healthy and diabetic subjects, (13, 16) and was a significant predictor of sTNF-{alpha} receptors in normal weight and obese women (17). In animals and humans, administration of TNF-{alpha} increases leptin levels (15, 34). Thus, we evaluated whether administration of r-metHuLeptin would similarly affect the TNF-{alpha} system, but did not find evidence that high pharmacologic leptin levels increase sTNF-{alpha} receptor levels. These findings are consistent with prior interventional studies of the effect of extremely high pharmacologic leptin levels on soluble TNF-{alpha} receptors (14, 35). Taken together, this suggests a threshold effect of leptin similar to that observed in neuroendocrine function (36) to normalize the TNF-{alpha} system when leptin levels are replaced in low leptin states, whereas increasing leptin levels to high physiologic or pharmacologic levels does not activate the TNF-{alpha} system further.

Leptin has also been shown to correlate with several other inflammatory markers (e.g. CRP, lipopolysaccharide-binding protein, serum amyloid A, {alpha}-acid glycoprotein, and plasminogen activator inhibitor-1) in healthy subjects with a wide range of BMIs; however, these correlations become nonsignificant after adjustment for BMI and gender (13). Although an independent association between leptin and CRP has been reported, the data are conflicting regarding whether this association is independent of adiposity (13, 37, 38). Leptin was not associated with MCP-1 in a large population of women with varying degrees of insulin sensitivity, and although insulin had an independent association with 7-yr cardiovascular disease mortality, leptin actually had a protective effect (31). The data from these observational human studies suggest that except for TNF-{alpha}, the association between leptin and inflammatory markers may be due to the correlation between leptin and fat mass or another, as yet unidentified, obesity-associated factor rather than an independent effect of leptin per se. However, the possibility remains that true associations between leptin and inflammatory markers may have been missed because of uncontrolled confounding or improperly performed multivariate analyses, possibly due to adjustment for fat mass per se (because leptin is produced by adipose tissue and correlates closely to fat mass) (12). In this respect, interventional studies are necessary to prove or disprove a potential causal effect of leptin.

Only one other group has previously used an interventional design to investigate whether high leptin levels (achieved through administration of long-acting pegylated leptin for weight loss) can alter inflammatory markers (14, 35). In that study, 28 subjects received either leptin or placebo for 8 wk, with no effect of leptin on sTNF-{alpha} receptors or CRP (35), and in a more recent study, 22 subjects received leptin or placebo concurrently with a very low calorie diet (14). In the latter, exogenous leptin administration had no major effect on several inflammatory markers, including IL-1, IL-6, TNF-{alpha}, sTNF-{alpha} receptors, CRP, fibrinogen, plasminogen activator inhibitor-1, tissue plasminogen activator, von Willebrand factor, and ICAM-1, except for a minor increase in CRP (14). In both previous studies, exogenous leptin administration resulted in extremely high serum leptin levels, ranging from approximately 2500–4000 ng/ml, i.e. more than 500 times normal levels.

We evaluated the effect of increasing leptin levels from 1) low physiologic (~2 ng/ml) to high physiologic (up to ~85 ng/ml) in lean men over the short term, 2) intermediate physiologic (~14 ng/ml) to low pharmacologic (up to ~275 ng/ml) in obese men over the short term, and 3) higher physiologic (~40 ng/ml) to intermediate pharmacologic (~400 ng/ml) and high pharmacologic (~920 ng/ml) in obese men over a longer time frame to determine whether there were any threshold levels with respect to duration or dose of leptin on inflammatory markers. In contrast to another study that found an effect of insulin administration to decrease MCP-1 and sICAM-1 levels after only several hours (18), high physiologic and pharmacologic leptin levels over a period of days to weeks did not have a major effect on cytokines important in the Th lymphocyte response or other inflammatory cytokines after a few days, with the exception of a slight, but statistically significant, decrease in IFN-{gamma} (by repeated measures ANOVA only). However, the small magnitude of the change, the decrease, rather than increase as might be expected based on prior in vivo studies in leptin-deficient humans, (11), and the lack of robustness due to lack of significance using different statistical tests (in contrast to the concordance between statistical analyses for all other markers) make this finding probably a statistical artifact. Importantly, more chronic administration of r-metHuLeptin at pharmacologic levels, i.e. to a maximum of approximately 20 times normal, for 16 wk had no effect on these inflammatory markers or on other molecules known to be important in the development of insulin resistance, diabetes, and/or cardiovascular disease. By performing these more detailed dose-response studies, we determined whether leptin has an effect on increasing inflammatory markers.

In the short-term study, we studied cytokines important in the T cell response, given previous findings of a positive effect of leptin replacement on T cell function in low leptin states (10, 11), as well as markers important to the inflammatory response and endothelial function that are known to change over a comparable time frame (18). For the long-term study in obese diabetic subjects, we focused mainly on markers known to be important in insulin resistance and cardiovascular disease, which are more relevant to evaluate over this time frame, but we also measured IL-10 as a marker of T cell function. These data have implications for the safety of exogenously administered r-metHuLeptin in the context of clinical trials and/or clinical use. These findings could also be of physiologic and pathophysiologic importance in terms of whether the presence of obesity resulting in higher secretion and baseline leptin levels can increase cytokines and inflammatory markers through altered secretion of leptin.

Limitations of the study include the lack of a fasting placebo control in the short-term study, but the measurement of cytokines just before r-metHuLeptin administration provides baseline levels in the fasting state, and the close mean and SD of the cytokine measurements performed over an 8-h time frame when leptin levels reached pharmacologic ranges provide reassurance that an effect of fasting (e.g. to decrease cytokine levels) was not missed. In addition, despite the inclusion of a placebo control group for the long-term study, direct comparison with a hormone (e.g. insulin) that might be expected to have effects on inflammatory markers (i.e. a positive control) was not made, but available data from several previous studies involving insulin administration provide sufficient support with which to make reliable comparisons. The strengths of this study include 1) the randomized, interventional design of these studies that eliminates bias from uncontrolled confounding; 2) statistical adjustment and/or stratification for potential effect modifiers, such as gender and adiposity; 3) the increased power provided by the number of subjects studied in the randomized, placebo-controlled, long-term study (and its subgroups) compared with the previous interventional studies (14, 35); 4) the wide spectrum of leptin levels achieved and evaluated through graded increases in leptin doses, ranging from low physiologic to high pharmacologic (i.e. the entire range of physiologically and therapeutically important leptin levels), which maximizes the possibility of detecting a dose-response and/or a threshold effect(s) of leptin on inflammatory markers; 5) the novel evaluation of high leptin levels on inflammatory markers in subjects with diabetes mellitus, an important obesity-related comorbidity; and 6) the evaluation of an array of markers important in Th cell responses, insulin resistance, and/or cardiovascular disease to more fully elucidate the role of leptin in immune function and systemic inflammatory responses.

In summary, our data do not support a role for leptin in regulating immune function in leptin-sufficient states or an etiologic role for high leptin levels in the pathogenesis of the inflammatory state associated with obesity. These findings stand in distinct contrast to the beneficial effect of physiologic dose leptin replacement to improve the immunosuppression associated with congenital leptin deficiency in humans (11) and suggest a threshold effect of leptin on immune function, i.e. although leptin deficiency may cause immunologic defects, high levels of leptin do not cause the converse. By excluding leptin as a candidate for the potential link between obesity and the associated proinflammatory state, this finding has considerable clinical relevance for the mechanism by which the proinflammatory state develops in obesity and contributes to major associated comorbidities, such as insulin resistance, diabetes, and atherosclerotic disease. These findings may also have significant relevance for the potential future therapeutic use of r-metHuLeptin, i.e. when r-metHuLeptin gains a place in the therapeutic armamentarium for the severe insulin resistance and metabolic abnormalities associated with congenital lipoatrophy (39) and/or the reproductive and other neuroendocrine dysfunctions present in disease states associated with hypothalamic amenorrhea (40).


   Acknowledgments
 
We thank the General Clinical Research Center (GCRC) nurses at Beth Israel Deaconess Medical Center for collecting the samples for this research and the GCRC nutritionists for their help with the subjects’ diets.


   Footnotes
 
This work was supported by National Institutes of Health (NIH) Grants MO1-RR-01032 and R01-58785 a grant from Amgen, Inc. (to C.S.M.), and NIH Grant K23-RR-018860 (to J.L.C.).

First Published Online December 21, 2004

Abbreviations: BMI, Body mass index; CRP, C-reactive protein; CV, coefficient of variation; IFN-{gamma}, interferon-{gamma}; MCP-1, monocyte chemoattractant protein-1; r-metHuLeptin, recombinant methionyl human leptin; sICAM-1, soluble intercellular adhesion molecule-1; sTNF-R, soluble TNF receptor type I; Th, T helper; Th1, Th cell type 1; Th2, Th cell type 2.

Received September 29, 2004.

Accepted December 8, 2004.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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