Leptin, Ghrelin, and Adiponectin in the Metabolic Adjustment to Burn Injury in Children

Objective. Leptin, adiponectin, and ghrelin have diverse roles in the control of inflammation and metabolism in a normal state as well as in a chronic disease state. The aim of this study was to evaluate their role in the extreme metabolic and proinflammatory state after burn injury and during the initial weeks of recovery. Methods. A prospective descriptive study in a tertiary care center was undertaken. Patients were comprised of 5 children aged 20-108 months with severe burn injury; burn size ranged from 15%-36% of total body surface area. Early enteral feeding, according to estimated energy expenditure, was initiated as 150% of the recommended dietary allowance and in accordance with the patients’ nitrogen balance. Seven blood samples were collected sequentially, approximately 5 days apart, during the first 65 days after the burn injury. Samples were tested for leptin, ghrelin, and adiponectin. Results. Leptin, ghrelin, and adiponectin had a similar trajectory of concentration over time: low levels at the beginning, increasing until 2-3 weeks post-burn, where they reached a plateau at 5 weeks post-injury. The typical inverse correlations of ghrelin and adiponectin with leptin were absent. Interleukin-6 was negatively associated with ghrelin and adiponectin and was not associated with leptin. Insulin-like growth factor-1 (IGF-1) had a positive association with the 3 hormones; however, their profiles differ in their relationship to the expected concentration based on a literature review. Ghrelin and adiponectin were higher, leptin and IGF-1 were lower than expected. Conclusions. In the early weeks after burn injury, the hypermetabolic state and inflammation have a major effect on leptin, ghrelin, and adiponectin. The concurrent and similar change of the 3 hormones serves the parallel anabolic and catabolic processes during the recovery from burn injury.

Introduction

Leptin, adiponectin, and ghrelin have diverse roles in the control of inflammation and metabolism. Most of the data regarding the interrelationship between these hormones and both processes were collected in chronic pathological states.^1-5 The current study investigated the changes in the profile of leptin, adiponectin, and ghrelin after burn injury, which is an acute inflammatory and hypermetabolic state.

Leptin is an adipocytokine produced mainly by adipose tissue and involved in the regulation of appetite and energy expenditure.¹ Its deficiency results in morbid obesity in mice and humans.⁶ Leptin is effective both as a long-acting signal stimulated by an increase of body mass index (BMI) or white adipose tissue (WAT), and a short-acting signal triggered by food intake. It activates the Proopiomelanocortin (POMC) and cocaine- and amphetamine-regulated transcript (CART) neurons, and inhibits the neuropeptide Y (NPY)/Agouti-related peptide (AgRP) neurons in the arcuate nucleus, resulting in satiety, weight loss, and increased energy expenditure.^1,2 Leptin is also a mediator of the inflammatory response, modulating the activity of both the innate and the adaptive immune systems. Mice lacking the leptin gene (ob/ob) or its receptor (db/db), were found to be immunodeficient^3,7; leptin-deficient humans have profound abnormalities of T cell number and function⁸; and leptin increases the production of proinflammatory cytokines such as interleukin-1 and interleukin-6 (IL-6).

Adiponectin is an adipocytokine with structural homology to collagen, complement factors, and tumor necrosis factor a (TNFa). Unlike leptin, adiponectin knockout mice have normal body weight, but exhibit various manifestations of the metabolic syndrome.⁹ Adiponectin is inversely correlated with BMI and WAT. Its metabolic effects are mainly enhancing insulin sensitivity and, to a lesser extent, inducing proliferation and differentiation of adipocytes. Adiponectin also has an anti-inflammatory role consisting of downregulation of B and T lymphocytes and the production of stimulating interleukins, such as IL-6, TNFa, and endothelial adhesion molecules.^3,7 Recent studies found that stimulation of the adiponectin receptor AdipoR1 in the arcuate nucleus enhanced food intake and decreased energy expenditure.¹⁰

Ghrelin is secreted mainly from the stomach, but also from the intestine, placenta, and other organs. Its concentrations are inversely correlated with BMI and WAT, and it has both long- and short-acting effects opposing the effect of leptin. The long-term effects are weight gain, adipogenesis, and reduction of energy expenditure, while its main short-term effect is orexogenic, as a result of activation of NPY/AgRP neurons in the arcuate nucleus. Other areas that regulate hunger and express the ghrelin receptor are components of the dorsal vagal complex and the mesolimbic dopaminergic system.^11,12 Ghrelin also induces secretion of growth hormone and, to a lesser extent, adrenocorticosteroid releasing hormone (ACTH) and prolactin,⁴ and may inhibit thyroid stimulating hormone (TSH) secretion from the pituitary gland.13 Ghrelin has an anti-inflammatory effect by inhibiting the secretion of IL-6 and TNFa from monocytes and T cells.^5,14 Recently, ghrelin was shown to ameliorate the hemodynamic and metabolic disturbances and to attenuate cancer, cardiac, and burn-induced cachexia, and to protect against burn-induced multi-organ injury.¹⁵

Previous studies demonstrated that leptin is negatively correlated with adiponectin and ghrelin. However, most of them were conducted under normal conditions or chronic pathological metabolic and inflammatory states such as obesity, diabetes type II, anorexia nervosa, inflammatory bowel disease, or ankylosing spondylitis.^16-19 In the current study, the effect of burn injury on the 3 hormones was investigated. Burn injury induces acute and severe inflammation and a hypermetabolic state which are strongly correlated to the size of the burn.²⁰ The inflammatory process reaches a peak during the first week post-burn and persists to a lesser extent throughout convalescence.²¹ The hypermetabolic state begins 5 days post-burn, and may last up to 1 year after the injury, with energy requirements reaching 150%-200% of basal metabolic rate.²²

This study investigated the effect of the extreme metabolic and inflammatory conditions after burn injury on leptin, adiponectin, and ghrelin. Their secretion was followed for 5 weeks post-burn to evaluate the immediate effect as well as subsequent changes during the healing period. A second question addressed in the current study is in which of the processes, metabolic or inflammatory, the 3 hormones are predominantly involved. This was done by comparing the profiles of their concentrations over time to those of IL-6 as a marker of inflammation and IGF-1, as the metabolic mediator in burn injury.^21,22

Methods

Patients and Protocol. Patients were recruited from the pediatric intensive care unit (PICU) in The Edmond and Lily Safra Children’s Hospital, Tel Hashomer, Israel. The study protocol was approved by the institution’s ethical committee and informed consent was obtained from all parents of patients.

Demographic data including age, sex, percent of total body surface area (TBSA) burned, nutritional treatment, and pertinent laboratory results was obtained from the patients’ charts. Weight was recorded within the first 6 hours after admission and weekly thereafter. Height was measured during the first week in the PICU.

All patients were first treated with the standard resuscitation protocol of the PICU and afterwards with early enteral feeding according to their estimated energy expenditure, determined as 150% of their recommended dietary allowance (RDA) and in accordance with their nitrogen balance.

Blood samples for leptin, ghrelin, adiponectin, IL-6, cortisol, and IGF-1 were drawn at least twice during the first 3 days and thereafter every week for at least 5 weeks following admission. The sampling time was between 6 am and 12 pm, while the patient was fasting. The samples were grouped by days in the following way: days 1-3, 4-7, 8-14, 15-21, 22-28, 29-35, and 36 until the end of the hospitalization.

Biochemical Analysis. Leptin was measured by a human leptin radioimmunoassay (RIA) kit (Linco Research Incorporation, St. Louis, MO). The sensitivity of the assay is 0.5 µg/l. The within- and between-assay variation was 4.98% and 4.5%, respectively.

Total ghrelin was determined using RIA kits. The sensitivity of the total ghrelin assay was 30 pmol/l and the inter-assay coefficient of variation (CV) ranged from 15% to 20%.

Adiponectin was determined using RIA kits. The sensitivity of the adiponectin assay was 1 ng/ml and the inter-assay CV ranged from 6.9% to 9.3%.

Interleukin-6 was measured by enzyme immunochemiluminescent assay (Immulite, Diagnostic Products Corp, Los Angeles, CA). The sensitivity of the assay is 5 pg/mL and the mean inter-assay CV was 8%. According to the manufacturer’s profile, the antibody is highly specific for IL-6.

Insulin-like growth factor-1 was measured by a chemiluminescent immunometric method (Immulite 2000, Diagnostic Products Corp, Los Angeles, CA). The sensitivity of the assay was 2.26 nmol/l and the inter-assay CV ranged from 3% to 6 %.

A review of the literature helped determine the normal range of the hormones and cytokines: adiponectin, 8-11 µg/ml23-25; leptin, 4-9 µg/l26, 27; ghrelin, 45-105 pmol/l28-31; and IGF-1 10-20 nmol/l32; IL-6 2-4 pg /ml.^21,25

The results are presented as mean ± SEM (standard error of the mean). Adjusted correlation between the hormones was tested using mixed effect linear regression. In each comparison, 1 hormone (dependent variable) was correlated with another hormone (independent variable). Sampling time and patients were random effect parameters (STATA, Statacorp, College station, TX).

Results

Patients’ characteristics. Five patients (3 males, 2 females) were recruited during an 18-month period. They were all younger than 10 years of age (Table 1 and continued). All of the burn injuries were third degree and the range of burn size was 15%-36% of TBSA. Patients 2-5 had major burn injuries and patient 1 had a moderate burn injury, according to the severity scale of the American Burn Association.³³ Their Z-scores of height, weight, weight-for-stature, and BMI were all within 2 standard deviations of the mean for their age (Table 2). There was no clear trend of change in patients’ weight during the hospitalization (Table 2).

There was considerable clinical variability between the patients. Patients 3 and 4 had 20% TBSA burns; however they also endured inhalation injury and experienced complications while in the hospital. Patients 1, 2, and 5 had a milder recovery period, spending most of the time in a regular pediatric ward. Enteral nutrition was started in all patients within the first 48 hours. There was a large variability in the time each patient achieved complete enteral and complete oral feeding.

Correlations between hormones concentrations and patients’ characteristics. Correlations of the concentrations of leptin, adiponectin, and ghrelin during the first 3 days and over the whole study period with patients’ characteristics were examined. Contrary to previous studies,16-18,26,28,29,31 none of the 3 hormones had a significant correlation with patients’ age, weight, or weight Z-score at admission or at discharge. No correlations were found between the size of the burns and the concentration of the 3 hormones in these periods. This study did not look at the correlation of the 3 hormones with BMI, because BMI is used as a measure of adiposity only in children > 2 years of age,³⁴ and 2 of the patients were younger.

The profiles of the concentrations of leptin, adiponectin, and ghrelin. Figure 1 depicts the time course of the average concentrations of the peptides. The study compared the averaged profile of the 3 hormones to their normal range as reported previously. The concentration of ghrelin and adiponectin were in the higher normal range in the first sample and above the normal range in subsequent samples. All of leptin average concentrations were within the normal range, changing from the lower to the higher range as time progressed.

All 3 hormones had a similar profile. The concentrations immediately after the burn were the lowest with a rise of ghrelin and adiponectin levels during the following 2 weeks, and a rise of leptin up to 3 weeks post-burn. Subsequently, levels of all 3 hormones reached a plateau at the end of the week 5 after admission.

Analysis of the correlation between the hormones, using mixed effect linear regression, revealed a nonsignificant correlation between leptin and ghrelin (coefficient = 7.60, P = 0.38), a significant positive correlation between leptin and adiponectin (coefficient = 0.30, P = 0.02), and a positive trend for correlation between ghrelin and adiponectin (coefficient = 0.001, P = 0.15).

The profiles of the concentrations of IL-6 and IGF-1. IL-6 had a peak in week 1 post-burn that was considerably higher than the expected normal level and subsequently declined to a lower mean level that was still higher than the normal range (Figure 1). The profile of IGF-1 levels was lower than the expected normal level throughout its course, with the lowest concentration in the first days after the burn (Figure 1). There was a negative correlation of IL-6 with adiponectin (coefficient = -0.02, P = 0.02) and ghrelin (coefficient = -0.5, P = 0.01), but not with IGF-1 (coefficient = -0.01 P = 0.25) and leptin (coefficient = 0.01, P = 0.20). Insulin-like growth factor-1 had a positive correlation with ghrelin (coefficient = 30.1, P < 0.01) and leptin (coefficient = 0.75, P < 0.01), and a trend toward positive correlation with adiponectin (coefficient = 0.58, P = 0.1).

Discussion

Burn injuries impose a profound change in metabolism as well as severe inflammation.^20,22,35,36 In this study, the authors investigated their effect on the secretion of leptin, ghrelin, and adiponectin, 3 hormones involved in the regulation of metabolism and inflammation after burn injury.

The burn injury in this cohort in comparison to previous studies. This cohort is small and burn size was less than 40% TBSA in all patients. Jeschke et al²⁰ have shown that patients with this burn size are in a hypermetabolic and proinflammatory state but to a lesser extent than children with larger burn injuries. The pattern of the profiles of IGF-1 and IL-6 in this study is similar to previous studies. Interleukin-6 is increased in the first week after burn as part of the pro-inflamatory state.^20,21 During the subacute period, IL-6 levels are lower but still significantly higher than normal concentrations. No association was found between the catabolic process of hypermetabolism and inflammation for milder burn injuries in Jeschke et al.²⁰ However, even in their study, children with milder burn injury had IGF-1 levels lower than its expected range. Lower-than-normal IGF-1 levels were found in the current study as well as in a study in adults³⁶ comparing burn with and without inhalation injury. As suggested, inflammation after mild burn injury tends to suppress the anabolic metabolism mediated by IGF-1.²⁰

The profile of concentrations of leptin, ghrelin, and adiponectin. The 3 hormones had the same pattern of changes of concentrations over time. Though levels of the 3 hormones in the first days fell within the normal concentration range, the concentrations of the 3 hormones after 2-3 weeks reached a peak and remained at that level until week 5. The peak level and the plateau of adiponectin and ghrelin were higher than the normal range, and similar in range to the adiponectin and ghrelin levels of other pathological conditions in children and adults such as obesity,²⁵ Prader-Willi syndrome,³¹ anorexia nervosa,¹⁷ and inflammatory bowel disease.¹⁸ The corresponding profile of leptin was within normal range concentrations. However, it is higher than expected; in most pathological conditions, leptin concentration was lower than the normal range.^18,37

One of the interesting findings in this study is the similarity of the profiles of the 3 hormones. It stands in contrast with previous studies that demonstrated inverse correlation between leptin and adiponectin or ghrelin under many pathological conditions in children and adults such as obesity,²⁵ anorexia nervosa,¹⁷ ankylosing spondylitis,¹⁶ and inflammatory bowel disease.³⁸ One explanation could be that all measurements in this study were done during the acute and subacute phase after burn and the inverse association between the hormones would appear once a convalescent equilibrium is achieved as in the chronic diseases. In addition, the results of the studies of chronic diseases were based on 1 or 2 samples of the hormones (ie, before and after a change in weight). As such, these studies may have missed the dynamics of the hormones during the stabilization in the new energy equilibrium. These results are in accordance with previous studies that investigated the changes of leptin and ghrelin after physiologic stress such as sepsis, endotoxemia, or surgery.^37,39-41 All of these studies demonstrated a rise in leptin concentration after 24-120 hours, and the one that measured ghrelin reported nonsignificant correlation between the hormones. Another study followed leptin and adiponectin during refeeding of adolescents with anorexia nervosa, showing a simultaneous increase in the levels of both hormones in the first month of refeeding, similar to the results of the current study. The inverse correlation between adiponectin and leptin was restored after the first month.⁴² These studies, as well as the absence of association of leptin, adiponectin, and ghrelin with age and weight, suggest that the classical response of these hormones is altered when the physiological equilibrium is markedly disrupted.

The profiles of leptin, ghrelin, and adiponectin reflect the metabolic process rather than the inflammation. Leptin, adiponectin, and ghrelin have been reported to have both metabolic and inflammatory effects.^1-5 In order to establish if leptin, ghrelin, and adiponectin better reflect metabolic or inflammatory processes, the authors compared them to IL-6, which promotes inflammation, and IGF-1 which is an anabolic mediator. The profiles of the average concentrations of the 3 hormones were positively associated with IGF-1 and negatively associated, or not associated, with IL-6. The association of ghrelin, adiponectin, and IGF-1 was expected as it has been described before; they were shown to have a lower concentrations during acute pathological conditions as sepsis.^25,43-45 Ghrelin and adiponectin were also shown to be negatively correlated with IL-6 while simultaneously positively correlated with IGF-1.⁴⁶ In the current study, although ghrelin, adiponectin, and IGF-1 had similar profiles, they differed in their relationship to the expected hormone level. Insulin-like growth factor-1 concentrations were lower; while ghrelin and adiponectin were higher than the expected level. This finding may suggest that ghrelin and adiponectin are more effected by the anabolic requirements and are not suppressed by the inflammation after burn injury.

The authors thought a surprising finding was the positive association of leptin with IGF-1 and lack of association with IL-6. Leptin is considered a proinflammatory mediator,^3,7,47 in concert with other adipocytokines such as TNF-a and IL-6.^48,49 A direct increase of leptin in response to TNF-a has been shown in animal models.⁵⁰ However, there is controversy between different studies regarding the association of leptin and IL-6.^51-53 In the current study there was no correlation between leptin and IL-6 levels. Moreover, from a metabolic perspective the authors expected leptin levels to be high during the initial catabolic state and to decrease with time as enteral feeding is initiated and the anabolic state ensues,^1,2 however, the opposite was found. A similar association of leptin and IGF-1 was found by other investigators during fasting conditions, but the mechanism of this phenomenon is not clear.^54,55 Adding these findings to the results of the current study, the authors conclude that leptin level and dynamics did not reflect inflammation but rather the metabolic state, similar to ghrelin and adiponectin. As the 3 hormones have different and partially contradicting effects on metabolism, a possible explanation for the similar change of the 3 hormones is the concurrent anabolic and catabolic process. This concurrent and similar change over time may serve to aid in the breakdown of endogenous substrate from one tissue for build up of the damaged tissue, or as futile energy used for raising core temperature in the period after burn injury.

Limitations

The study comprised a small number of patients. Nevertheless, since the patterns of changes in hormones were highly consistent between patients, and the results were statistically significant, the authors believe the results are valid. The fact that the size of the burns was below 40% TBSA could limit the generalization of the findings to more severe burn injury. However, the authors think that the findings are a result of extreme disruption of the physiological metabolic and inflammatory equilibrium post burn, as reflected by cortisol and IL-6 levels.

Conclusion

This is the first study to follow adiponectin, leptin, and ghrelin concurrently and consecutively after burn injury in children. Based on the findings of this study, the authors suggest there are 2 modes of action of these hormones: one mode under normal physiological conditions or chronic disease states, and a second mode after acute severe physiological stress such as burn injury.^16-19 The concurrent change of the hormone concentrations can be explained by an override of the stress response post-burn that exceeds the normal association between the 3 hormones to better serve the inflammatory state and metabolic needs of the patient after burn injury.

Acknowledgments

Omer Bar-Yosef, MD, PhD; Josef Haik, MD, MPH; Ran Levy, MD; Ori Efrati, MD; Yoram Bujanover, MD; Brigitte Kochavi, RD, BS; Clara Pariente, MSc; Hannah Kanety, PhD; Oren Weissman, MD; and Dalit Modan-Moses, MD are from the Chaim Sheba Medical Center, Tel Hashomer, Israel. Ohad Hilly, MD is from the Chaim Sheba Medical Center, Tel Hashomer, Israel; and Rabin Medical Center, Petah Tiqwa, Israel.

Address correspondence to:
Oren Weissman, MD
Department of Plastic & Reconstructive Surgery
Sheba Medical Center
Tel Hashomer, Israel
Orenweissman@gmail.com

Disclosure: The authors disclose no financial or other conflicts of interest.

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