Published online August 1, 2007
PEDIATRICS Vol. 120 No. 2 August 2007, pp. e291-e296 (doi:10.1542/peds.2006-3165)
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ARTICLE

Metabolic Correction Induced by Leptin Replacement Treatment in Young Children With Berardinelli-Seip Congenital Lipoatrophy

Jacques Beltrand, MDa, Marta Beregszaszi, MDa,b, Didier Chevenne, MDc, Guy Sebag, MD, PhDd, Marc De Kerdanet, MDe, Frédéric Huet, MDf, Michel Polak, MD, PhDg, Nadia Tubiana-Rufi, MDh, Didier Lacombe, MD, PhDi, Alex M. De Paoli, MDj and Claire Levy-Marchal, MDa

a Université Paris 7, INSERM U690, Paris, France
b Clinical Investigation Center
c Departments of Biochemistry
d Pediatric Radiology
h Pediatric Endocrinology and Diabetology, Robert Debré Hospital, Paris, France
e Pediatric Endocrinology and Diabetology Unit, South Hospital, Rennes, France
f Department of Pediatrics, Children Hospital, Dijon, France
g Department of Pediatric Endocrinology, Necker-Enfants Malades Hospital, INSERM EMI363, Paris, France
i Department of Medical Genetics, Pellegrin Children's Hospital, Bordeaux, France
j Amgen, Inc, Thousand Oaks, California


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 CONCLUSION
 REFERENCES
 
OBJECTIVE. Berardinelli-Seip syndrome is a rare congenital lipoatrophy with a severe prognosis and no efficient therapy. Children present with low leptin levels and severe metabolic complications (insulin resistance, elevated triglyceride levels, and hepatic steatosis). The objective of this study was to test safety and efficacy of recombinant-methionyl-human leptin replacement in children with Berardinelli-Seip syndrome before development of severe metabolic disease

METHODS. As part of an open trial, recombinant-methionyl-human leptin was given daily for 4 months to children who did not have diabetes and had Berardinelli-Seip congenital lipoatrophy and metabolic complications at a dosage that was meant to achieve physiologic levels. Six boys and 1 girl (age: 2.4–13.6 years), with a mean fasting insulin level of >15 mIU/L and hypertriglyceridemia, were included.

RESULTS. At the end of the recombinant-methionyl-human leptin treatment, a 63% reduction of fasting triglycerides level was achieved. A simultaneous 30% increase in insulin sensitivity was seen, and liver volume was reduced by 20.3%. More remarkable, values of insulin sensitivity and triglyceride level were in the reference range in 4 patients.

CONCLUSIONS. Leptin replacement is able to reverse metabolic complications in the majority of children with Berardinelli-Seip congenital lipoatrophy and with insulin resistance or dyslipidemia before the development of overt diabetes.

Key Words: leptin • Berardinelli-Seip syndrome • congenital lipoatrophy

Abbreviations: BSCL—Berardinelli-Seip congenital lipoatrophy • r-metHuLeptin—recombinant-methionyl-human leptin • FFA—free fatty acids • QUICKI—quantitative insulin sensitivity check index • ALAT—alanine transaminase • ASAT—aspartate transaminase

Berardinelli-Seip congenital lipoatrophy (BSCL; Berardinelli-Seip syndrome) is a rare but fatal congenital disease that is characterized by the lack of adipose tissue that leads to marked hypertriglyceridemia and abnormal storage of lipids.1,2 This ectopic fat storage induces triglyceride accumulation in the muscle, the liver, and the arterial wall, explaining most of the severe clinical complications: hepatic steatosis and cirrhosis, insulin resistance, diabetes, and arteriosclerosis. These complications appear very early in life and evolve to fatal events, generally in the third decade of life. There is no curative treatment but only symptomatic treatments of the complications, which are able only to slow the life-threatening progression of the disease.

Leptin is a protein that is secreted by the white adipose tissue and plays an important role in the balance of energetic metabolism, satiety, energy expenditure, and weight. Leptin acts through both central and peripheral mechanisms. As a direct consequence of the absence of subcutaneous adipocytes, circulating levels of leptin are nearly undetectable in children with BSCL, attesting to a profound defect in the secretion of this hormone.3

In adult patients who have congenital or acquired generalized lipodystrophy with overt diabetes and low leptin serum concentrations, leptin replacement treatment improved glycemic control to such an extent that antidiabetic treatment could be withdrawn.4,5 This beneficial effect was maintained for 1 year of treatment or more.6 Simultaneously, leptin treatment corrected hepatic steatosis and reversed insulin resistance in the same patients.7 Triglyceride levels decreased in these patients, and this effect also persisted over time. Altogether, these studies show that leptin treatment induces a marked and sustained improvement of the metabolic consequences of general lipodystrophy with overt diabetes.

The aims of this study were to test the metabolic effects of leptin replacement in children with BSCL before they develop overt and severe metabolic complications and to assess the effectiveness and tolerance of leptin replacement treatment in children.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 CONCLUSION
 REFERENCES
 
Patients and Study Design
Children with BSCL were recruited in several pediatric units all over France. Inclusion criteria were low leptin levels (<3 ng/mL), at least 1 abnormal feature (hyperinsulinemia >15 mIU/L and/or triglyceride level >2 mmol/L), but normal tolerance to glucose.

The study was a prospective, open-label, nonrandomized phase II trial. The treatment under evaluation was recombinant-methionyl-human leptin (r-metHuLeptin; Amgen, Thousand Oaks, CA) given at dosages that was meant to mimic physiologic levels. Children were kept under their recommended diet for the duration of the trial. The Paris/Saint-Louis Ethics committee approved the protocol.

Leptin Administration
R-metHuLeptin was administered once a day by subcutaneous injection at dosages that were predicted to achieve 50%, 100%, and 200% of normal leptin levels. The dosage of r-metHuLeptin needed to achieve a normal leptin concentration (100%) was calculated as 0.03 mg/kg lean body weight. Patients were started at 50% of this predicted dosage and maintained at this level for 1 month. The dosage was then increased to 100% of predicted dosage for another month and was further increased to 200% of the predicted dosage for the rest of the trial (2 months).

Methods of Investigation and Analytical Methods
Body composition was determined by dual-energy radiograph absorptiometry. Liver volume was evaluated using computed tomography scan under standardized procedure with measurements at the level of 0.1 cm. Insulin sensitivity was assessed as the peripheral glucose uptake at steady state of a 120-minute euglycemic-hyperinsulinemic clamp as originally described by De Fronzo et al.8 Insulin was infused throughout the test at 80 mIU/m2 per min. Peripheral glucose uptake was calculated as the average glucose infusion rate at steady state over 2 consecutive 20-minute periods. Glucose turnover rate was determined by the tracer dilution method. Hepatic glucose production was then calculated at baseline and at the steady state of the euglycemic clamp. Free fatty acids concentration (FFA) was measured in the plasma at baseline and at steady state under insulin infusion during the clamp. In 1 young patient (3 years of age), insulin sensitivity was assessed from parameters derived from oral glucose test tolerance. Quantitative insulin sensitivity check index (QUICKI) was calculated as follows: QUICKI = 1/[log (fasting insulin) + log (fasting glucose)].9

Plasma glucose concentrations during the clamp were measured by the glucose oxidase method on an on-site Beckman analyzer (Beckman Instruments, Fullerton, CA). Serum insulin concentrations were measured using an immunoradiometric assay (Bi-Insulin IRMA; Cis bio France, Gif sur Yvettes, France). Alanine transaminase (ALAT), aspartate transaminase (ASAT), {gamma}-glutamyl-transferase, and alkaline phosphatase were determined by enzymatic methods using an ADVIA analyzer (Bayer Diagnostics, Puteaux, France). Serum leptin concentrations were measured using a specific radioimmunoassay (Linco Research, St Charles, MO) as previously described by Maffei et al.10 Sensitivity of the assay is 0.4 ng/mL. Intra-assay and interassay coefficients of variation were 5.2% and 8.7%, respectively, at 2.3 ng/mL.

Statistical Considerations
This is an open, uncontrolled trial. Criteria for a positive response to treatment were defined as a reduction in liver volume of at least 30% and/or a 30% improvement in insulin sensitivity and/or a 40% reduction of fasting triglyceride level after 4 months of leptin replacement in comparison with baselines values. All data are given as means ± 1 SD. Comparisons between values at the end of the leptin treatment were performed using paired Wilcoxon's tests. P < .05 was considered as statistically significant.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 CONCLUSION
 REFERENCES
 
Patients Characteristics, Anthropometric Data, and Metabolic Status
Seven children were included and completed the trial. The characteristics of the patients at baseline are given in Table 1. There were 6 boys and 1 girl aged from 2.4 to 13.6 years. Tanner stages were between I and IV. Six patients had a mutation in the BSCL2 gene, and 1 (patient 7) had a mutation in the AGPAT2 gene.


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  		TABLE 1 Characteristics of the Patients Before Leptin Replacement and at the End of the Trial

 
As expected, fat mass was very low (2.04 ± 0.8 kg) in all patients. There were no significant changes in mean weight and fat mass after 4 months of leptin treatment (mean: 37.7 vs 37.4 and 2.04 vs 2.41; Table 2) . There were no changes in pubertal status either.


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  		TABLE 2 Metabolic Effects of Leptin Replacement Treatment

 
At baseline, all patients had normal fasting plasma glucose but high fasting plasma insulin (91.2–307.2 pmol/L; Table 1). Six patients showed a marked hypertriglyceridemia (z score: 6.11–13.0; Table 1) for age and gender. The mean fasting leptin level was 0.7 ± 0.4 ng/mL. At the second month of treatment (daily dosage: 0.03 mg/kg), mean serum leptin concentration was >2.5 ng/mL. At the end of the second treatment phase (daily dosage: 0.06 mg/kg), the mean leptin concentration was 41.8 ± 28.7 ng/mL, which is a supraphysiologic level.

There were no changes in fasting plasma glucose, but there was a mean 33% reduction in fasting insulin and a significant improvement in the fasting glucose to insulin ratio (mean: 135.2 vs 90.8 and 0.24 vs 1.04, respectively; Table 2). Six patients showed a marked reduction of triglyceride concentration with a mean 40% reduction of z score (from 6.84 to 2.49; Table 2, Fig 1). There were no changes in cholesterol or FFA concentrations; however, the values were within reference ranges at the beginning of the trial.


Figure 1
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  		FIGURE 1 Triglycerides before and at the end of the trial. M indicates peripheral glucose uptake. a Value is in the reference range for age. b Positive response to leptin.

 
Insulin Sensitivity
Insulin sensitivity was measured by the euglycemic-hyperinsulinemic clamp technique in 6 patients and with parameters derived when fasting in 1 patient (Fig 2). At baseline, 4 patients were clearly insulin resistant. Peripheral glucose uptake increased from 33% to 922% in 5 patients, whereas QUICKI increased by 46% in the younger boy (Table 1). In 1 boy (patient 6), insulin sensitivity did not improve; the increase in peripheral glucose uptake was modest (+11%) in another (patient 4). Under insulin infusion, hepatic glucose output was totally suppressed in all cases, and FFA concentration decreased by at least 50% in 3 of 7 patients.


Figure 2
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  		FIGURE 2 Insulin sensitivity before and at the end of the trial: peripheral glucose uptake during the euglycemic-hyperinsulinemic clamp in patients 2 through 7 and QUICKI in patient 1. The dotted line indicates reference value limits.16,17 M indicates peripheral glucose uptake. a Positive response to leptin.

 
Liver Volume and Function
Before therapy, 6 of 7 patients had an enlarged liver by clinical examination together with altered hepatic function. ALAT was increased in 6 patients (52–335 IU/L; reference range: 5–45 IU/L). ASAT was increased in 1 patient (140 UI/L; reference range: 5–45 UI/L). At the end of the trial, ASAT was normal in all patients and ALAT in 4 patients. ALAT remained elevated in 1 patient, although the value decreased by 79% (from 335 to 70 UI/L). Liver volume was measured at baseline and at 4 months in 4 patients and decreased by 17% to 24% in these patients. Simultaneously, mean waist circumference significantly reduced by 3.5 ± 1.75 cm (P = .02).

Efficacy Criteria and Adverse Effects
Metabolic effects of leptin replacement treatment are shown in Table 2. Therapy had a significant effect on triglycerides (P = .017), fasting glucose/insulin ratio (P = .04), and peripheral glucose uptake (P = .018). Liver volume, waist circumference, and hepatic enzymes were significantly reduced as well (P = .002, P = .02, P = .04, and P = .02, respectively). As indicated in Table 1, a positive response was achieved in 4 patients for insulin sensitivity and in 4 patients for triglycerides. An efficiency criteria was then present in 6 patients. We did not observe any adverse effects that were induced by the therapy, except for transitional and spontaneously resolved local reaction at the site of injection.


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
CONCLUSION
 REFERENCES
 
This is the first trial of replacement treatment by human recombinant leptin in young children with BSCL. This syndrome is a congenital disease whereby the total lack of adipose tissue induces hypertriglyceridemia and ectopic storage of lipids, promoting severe metabolic complications. Hypertriglyceridemia leads to cardiovascular diseases, steatosis leads to cirrhosis, and insulin resistance is the first step toward severe diabetes. The children included in the study were young but already showed marked hypertriglyceridemia, hepatic steatosis, and insulin resistance with normal tolerance to glucose. Leptin treatment induced an increase in insulin sensitivity in 5 patients and a decrease of triglyceride levels by >40% in 4 patients. A positive response to treatment was thus achieved in 5 of 7 patients according to the preestablished efficiency criteria. Moreover, this treatment was able to correct triglyceride concentration and insulin sensitivity in these patients.

We did not observe any adverse effects, particularly on pubertal development. There was no accelerated or induced puberty during the treatment phase. This aspect was of concern because leptin has been shown to be important in the development of puberty.11 The injections of r-metHuLeptin were well tolerated with no major local reaction or any other adverse effects. We used short needles (3 mm) adapted to the lack of subcutaneous tissue in these children.

The beneficial effect of leptin on insulin resistance has been demonstrated in rodents and humans.5–7,11 Muscle triglyceride content decreases in parallel to the increase of ß-oxidation via an adenosine monophosphate kinase–dependent pathway. In the liver, leptin decreases lipid synthesis and very low-density lipoprotein production and increases fatty acid oxidation by blocking stearoyl-coenzyme A desaturase-1.12

Replacement treatment with recombinant leptin has been available for some years. There were not any beneficial effects in obesity, where there is insulin resistance and leptin resistance with hyperleptinemia.13,14 In congenital leptin deficiency, characterized by severe obesity and insulin resistance, leptin replacement is efficient in reducing fat mass and the metabolic consequences.15 In adults with congenital lipodystrophy and overt diabetes, it induces the cessation of antidiabetic therapy and the reduction of hypertriglyceridemia and liver volume.

In this trial, leptin therapy not only induced but also reversed or normalized metabolic complications, because peripheral glucose uptake and triglyceridemia returned to normal values. This was followed by a drastic reduction of liver volume and by normalization of liver enzymes. Leptin injections might be the first therapy to slow the progression of the disease. This remarkable beneficial effect would indicate that leptin replacement treatment is more efficient when initiated at a younger age and more specific when severe complications have not yet developed. In support of this statement, the older boy (11 years; Tanner stage III) had the more incomplete response to the therapy despite his high leptin level at the end of the trial. He was insulin resistant, and his liver function was moderately altered at baseline. These criteria remained so at the end of the trial. By contrast, his younger brother, who had the same mutation and participated in the same trial, showed a positive response to leptin.

Four months may be a short period of time to obtain full reversal in all patients. Actually, indications for the need for a longer period of treatment have been documented for the improvement of hormonal metabolic and immune disorders in adults with lipodystrophy, where all beneficial effects are seen after 1 year but not 4 months of treatment. If the benefit of leptin replacement can be maintained on a long-term basis, then this hormonal treatment would be the first treatment that is able to reduce the progression of the severe metabolic complications by blocking ectopic fat storage in children with BSCL. If administered early in life and as a long-term and daily treatment, then leptin could be the first therapy in this disease and would be a new hope for patients.


   CONCLUSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 CONCLUSION
REFERENCES
 
We demonstrated that leptin treatment is efficient and well tolerated in young and very young children. It is able to reverse the metabolic disorders in young and very young children with BSCL, providing strong evidence that leptin deficiency is responsible for metabolic complications of this disease. It remains to be determined whether such a beneficial effect can be maintained on a long-term basis and whether it can prevent or at least delay the development of more severe complications, such as diabetes and liver cirrhosis, when instituted at a very young age.


   ACKNOWLEDGMENTS
 
This study was supported by a grant from INSERM (RBM 02–48) and from Amgen.

We thank the nursing staff and Christelle Daubrosse at the Clinical Investigation Center at the Robert Debré Hospital for careful supervision of the patients. We are grateful to Philippe Kiefer, MD, and Mireille Mur (Amgen, Neuilly sur Seine, France) for their continuous support and advice during this trial.


   FOOTNOTES
 
Accepted Jan 26, 2007.

Address correspondence to Jacques Beltrand, MD, INSERM U690, Hôpital Robert Debré, 48 Blvd Sérurier, 75019 Paris, France. E-mail: jacques.beltrand{at}rdebre.inserm.fr

Financial Disclosure: Dr De Paoli is an employee of Amgen.


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 CONCLUSION
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  7. Petersen KF, Oral EA, Dufour S, Befroy D, et al. Leptin reverses insulin resistance and hepatic steatosis in patients with severe lipodystrophy. J Clin Invest. 2002;109 :1345 –1350[CrossRef][ISI][Medline]
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  11. Oral EA, Ruiz E, Andewelt A, et al. Effect of leptin replacement on pituitary hormone regulation in patients with severe lipodystrophy. J Clin Endocrinol Metab. 2002;87 :3110 –3117[Abstract/Free Full Text]
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PEDIATRICS (ISSN 1098-4275). ©2007 by the American Academy of Pediatrics




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