Beneficial effects of leptin on obesity, T cell hyporesponsiveness, and neuroendocrine/metabolic dysfunction of human congenital leptin deficiency

Abstract

The wide range of phenotypic abnormalities seen in the leptin-deficient ob/ob mouse and their reversibility by leptin administration provide compelling evidence for the existence of multiple physiological functions of this hormone in rodents. In contrast, information regarding the roles of this hormone in humans is limited. Three morbidly obese children, who were congenitally deficient in leptin, were treated with daily subcutaneous injections of recombinant human leptin for up to 4 years with sustained, beneficial effects on appetite, fat mass, hyperinsulinemia, and hyperlipidemia. Leptin therapy resulted in a rapid and sustained increase in plasma thyroid hormone levels and, through its age-dependent effects on gonadotropin secretion, facilitated appropriately timed pubertal development. Leptin deficiency was associated with reduced numbers of circulating CD4(+) T cells and impaired T cell proliferation and cytokine release, all of which were reversed by recombinant human leptin administration. The subcutaneous administration of recombinant human leptin has major and sustained beneficial effects on the multiple phenotypic abnormalities associated with congenital human leptin deficiency.

Full text

Introduction
Lacking the adipocyte hormone leptin, ob/ob mice devel-
op severe obesity as a result of a combination of
increased food intake and diminished energy expendi-
ture (1). In addition to obesity, the congenital deficiency
of leptin in mice results in a wide range of other pheno-
typic abnormalities. The ob/ob mice are infertile as a
result of hypogonadotropic hypogonadism (2). They
have high circulating glucocorticoid levels that may con-
tribute, by suppressive effects on growth hormone secre-
tion, to their diminished linear growth (3). Leptin-defi-
cient mice adapt poorly to cold exposure (4) and have
impaired regulation of the hypothalamo-pituitary-thy-
roid axis (5). They develop hyperinsulinemia and often
progress to diabetes (6). Additionally, they show marked
abnormalities in cellular immune function with changes
in the number and function of circulating T cells (7). All
of these abnormalities are reversible by the subcutaneous
administration of leptin (8–12).
In 1997 we reported two children (child A and child
B), first cousins of Pakistani origin, who were homozy-
gous for a frameshift mutation in the ob gene
that resulted in undetectable circulating leptin and
a syndrome of hyperphagia and severe obesity (13).
We subsequently reported marked improvements in
hyperphagia and body fat mass in child A after
1 year of subcutaneous recombinant human leptin
(r-metHuLeptin) therapy (14). We recently identified
Beneficial effects of leptin on obesity, T cell
hyporesponsiveness, and neuroendocrine/metabolic
dysfunction of human congenital leptin deficiency
I. Sadaf Farooqi,1Giuseppe Matarese,2Graham M. Lord,3Julia M. Keogh,1
Elizabeth Lawrence,4Chizo Agwu,5Veronica Sanna,2Susan A. Jebb,6Francesco Perna,7
Silvia Fontana,2Robert I. Lechler,3Alex M. DePaoli,4and Stephen O’Rahilly1
1University Department of Medicine and Department of Clinical Biochemistry, Addenbrooke’s Hospital, Cambridge,
United Kingdom
2Centro di Endocrinologia ed Oncologia Sperimentale, Consiglio Nazionale delle Ricerche (CNR-CEOS), Naples, Italy
3Department of Immunology, Imperial College School of Medicine, Hammersmith Hospital, London, United Kingdom
4AMGEN Inc., Thousand Oaks, California, USA
5Department of Paediatrics, Sandwell Hospital, West Midlands, United Kingdom
6Medical Research Council, Human Nutrition Research, Cambridge, United Kingdom
7Cattedra di Malattie dell’Apparato Respiratorio, Dipartimento di Medicina Clinica e Sperimantale, Universita di Napoli
Frederico II, Naples, Italy
The wide range of phenotypic abnormalities seen in the leptin-deficient ob/ob mouse and their
reversibility by leptin administration provide compelling evidence for the existence of multiple phys-
iological functions of this hormone in rodents. In contrast, information regarding the roles of this
hormone in humans is limited. Three morbidly obese children, who were congenitally deficient in
leptin, were treated with daily subcutaneous injections of recombinant human leptin for up to 4 years
with sustained, beneficial effects on appetite, fat mass, hyperinsulinemia, and hyperlipidemia. Lep-
tin therapy resulted in a rapid and sustained increase in plasma thyroid hormone levels and, through
its age-dependent effects on gonadotropin secretion, facilitated appropriately timed pubertal devel-
opment. Leptin deficiency was associated with reduced numbers of circulating CD4+T cells and
impaired T cell proliferation and cytokine release, all of which were reversed by recombinant human
leptin administration. The subcutaneous administration of recombinant human leptin has major
and sustained beneficial effects on the multiple phenotypic abnormalities associated with congeni-
tal human leptin deficiency.
This article was published online in advance of the print edition. The date of publication is available
from the JCI website, http://www.jci.org. J. Clin. Invest. 110:1093–1103 (2002). doi:10.1172/JCI200215693.
Received for publication April 15, 2002, and accepted in revised form
August 13, 2002.
Address correspondence to: I. Sadaf Farooqi, University
Department of Medicine and Department of Clinical
Biochemistry, Addenbrooke’s Hospital, Cambridge, United
Kingdom. Phone: 44 1223 762634; Fax: 44 1223 762657;
E-mail: ifarooqi@hgmp.mrc.ac.uk.
Giuseppe Matarese and Graham Lord contributed equally
to this work.
Conflict of interest declared: Elizabeth Lawrence and
Alex M. DePaoli are employees of AMGEN Inc.
Nonstandard abbreviations used: recombinant human leptin
(r-metHuLeptin); lean body weight (LBW); dual-energy x-ray
absorptiometry (DXA); basal metabolic rate (BMR); follicle-
stimulating hormone (FSH); luteinizing hormone (LH);
murine IL-3 (mIL-3); phytohemagglutinin (PHA); total energy
expenditure (TEE); SD score (SDS); bone mineral content (BMC);
bone mineral density (BMD); free tri-iodothyronine (fT3);
free thyroxine (fT4); thyrotropin (TSH); tuberculin purified
protein derivitive (PPD).
The Journal of Clinical Investigation | October 2002 | Volume 110 | Number 8 1093

another unrelated leptin-deficient child (child C), also
from a consanguineous family of Pakistani origin living
in the United Kingdom. This child was homozygous for
the same frameshift mutation (∆G133) that was report-
ed previously in child A and B. The two families identi-
fied by us as carrying this mutation were not known to
be related over at least five generations. We now report
the results of leptin replacement therapy in these three
children who were treated for periods ranging from 10
to 50 months. We have undertaken assessments of a
range of physiological, endocrine, metabolic, and
immune parameters, which allow cross-species com-
parison of the pleiotropic effects of leptin replacement.
Methods
Leptin administration and dosage. All studies were
approved by the local ethical committee and were con-
ducted in accordance with Declaration of Helsinki
principles. All studies had full informed consent from
the parents and children involved. R-metHuLeptin was
administered as a once-daily subcutaneous injection.
The initial dose was calculated to achieve 10% predict-
ed serum leptin concentration based on age, gender,
and percentage of body fat (calculations based on in-
house pharmacodynamic and pharmacokinetic data
from AMGEN Inc., Thousand Oaks, California, USA).
The dose administered (milligram per kilogram of lean
body weight [LBW]) remained the same if weight sta-
bilized or was reduced. If weight increased over two suc-
cessive 2-month periods, the dose was increased to
achieve 20% and subsequently to 50, 100, and 150%
predicted serum leptin concentration.
Body composition. Body composition was measured in all
three subjects using whole-body dual-energy x-ray
absorptiometry (DXA) (QDR 1000W; Hologic Inc.,
Waltham, Massachusetts, USA) to determine bone min-
eral content, lean mass, and fat mass every 2 months (15).
Energy intake and expenditure. To assess the impact of
leptin on ingestive behavior, child A, B, and C were
given an ad libitum test meal after an overnight fast.
Although we have reported previously an effect of lep-
tin on ad libitum energy intake in child A, this was
almost certainly an underestimate because before treat-
ment the child consumed the entire 7-MJ (1,670 kcal)
meal (14). For this reason all further ad libitum intake
studies were undertaken with an 18-MJ (4,400 kcal)
meal presented after an overnight fast. The contents of
the ad libitum test meal were covertly weighed before
and after consumption and total energy intake and
macronutrient composition calculated using standard
tables (16). Energy intake was expressed per kilogram
LBW as a means of comparing intake between subjects
of different age and body size.
Basal metabolic rate (BMR) was measured by indirect
calorimetry (Europa Gas Exchange Monitor; Nutren
Technology Ltd., Manchester, United Kingdom) after
an overnight fast in a thermoneutral environment.
Total energy expenditure was measured using doubly
labeled (2H18O) water (17).
Hormonal and metabolite assays. Blood samples were
obtained after an overnight fast and analyzed for
cholesterol, triglycerides, HDL cholesterol, LDL cho-
lesterol, glucose, and insulin (Dade Behring Inc.,
Newark, Delaware, USA). Thyroid-stimulating hor-
mone, free thyroxine, free tri-iodothyronine, adreno-
corticotrophic hormone, cortisol measured at 0900,
follicle-stimulating hormone (FSH), luteinizing hor-
mone (LH), estradiol, and testosterone were measured
using standard assays (Wallac Oy, Turku, Finland).
R-metHuLeptin pharmacokinetics. Serum leptin was
measured using a solid-phase sandwich ELISA
(AMGEN Inc.). Samples were taken before injection
and at 4 hours after injection every 2 months.
Anti–r-metHuLeptin-neutralizing Ab bioassay. Samples
identified as reactive in a screening immunoassay
were tested for the presence of neutralizing Ab’s to
r-metHuleptin. The bioassay used the murine cell line
32D clone 4 that naturally expresses the murine IL-3
receptor and was engineered to express a chimeric
receptor consisting of the extracellular leptin-binding
domain and the transmembrane- and intracellular-
signaling domains of the EPO receptor (32Dcl4
OBECA). Both leptin and murine IL-3 (mIL-3) inde-
pendently induced proliferation of the OBECA cells.
Samples were partially purified by using a 50-K
molecular-weight cut-off Microcon centrifugal filter
device (Millipore Corp., Bedford, Massachusetts,
USA) and were diluted 1:20 with assay media (RPMI-
1640 containing 2% FBS and 1×Pen-Strep/gluta-
mine). The samples were centrifuged at 12,900 gfor
15 minutes at 25°C. The retentate was returned to the
starting volume by addition of assay media. This cen-
trifugation step was repeated. Fifty microliters of the
final retentate were added to OBECA cells in the pres-
ence of 0.5 ng/ml leptin in the wells of a 96-well
microplate. The final concentration of human serum
in the well was 1.25%. Samples and controls were
allowed to incubate with the OBECA cells for 48
hours at 37°C at 5% CO2before addition of tritiated
thymidine. The plates were incubated for an addi-
tional 4 hours before being harvested onto filter mats
for counting on a Packard Matrix beta counter.
Samples that demonstrated more than 50% inhibi-
tion of the response of the cells to 0.5 ng/ml leptin were
considered reactive and tested for their ability to inhib-
it the response of the cells to 160 pg/ml recombinant
mIL-3. A patient was considered positive for the devel-
opment of neutralizing Ab’s if (a) the post-dose sample
inhibited the leptin-induced proliferation by equal to
or greater than 50%; (b) the ratio of pre-dose
counts/post-dose counts was equal to or greater than
2.0; and (c) the post-dose sample did not inhibit the
response of the cells to mIL-3.
Lymphocyte function. Flow cytometry was performed as
described previously using a FACS scan flow cytometer
and appropriate directly conjugated mAb’s (Becton
Dickinson-PharMingen, San Diego, California, USA).
A panel of 15 normal 2- to 4-year-old children was used
1094 The Journal of Clinical Investigation | October 2002 | Volume 110 | Number 8

as the control population, and our data were found to
be consistent with observations published previously.
Analyses were performed on fresh lymphocytes at the
time of sampling. T cell cultures were performed in
triplicate in microtiter 96-well round-bottomed plates
(BD Biosciences, San Jose, California, USA). After pass-
ing blood through a Ficoll gradient (Pharmacia Biotech
AB, Uppsala, Sweden) peripheral blood leukocytes
(2 ×105/well) were stimulated in parallel with the fol-
lowing stimuli: OKT3 0.1 µg/ml (Ortho Diagnostic
Systems Inc., Raritan, New Jersey, USA); 2 µg/ml phy-
tohemagglutinin (PHA; Sigma Chemical, Milan, Italy);
0.01 µM PMA and 0.5 µM Ionomycin (Iono; Sigma
Chemical); 10 µg/ml tuberculin purified protein deriv-
itive (PPD) (Northern Serumin Institut, Copenhagen,
Denmark). T cell viability was assessed before each pro-
liferative assay by trypan blue staining and was between
85 and 95%. Lymphocytes were incubated for 72 hours
at 37°C in RPMI-1640 medium (Gibco-BRL; Life Tech-
nologies Inc., Gaithersburg, Maryland, USA), supple-
mented with 2% (vol/vol) of patient’s autologous
serum or autologous serum for controls, 2 mM L-glut-
amine (Life Technologies Inc.), 0.1 mM nonessential
amino acids (Life Technologies Inc.), 1 mM sodium
pyruvate (Life Technologies Inc.), 100 U/ml penicillin,
100 µg/ml streptomycin (Life Technologies Inc.). Forty-
eight to sixty hours after initiation cell culture, super-
natants (100 µl) were removed and frozen at –80°C for
cytokine assay. IFN-γ, IL-4, IL-10, and TGF- βwere
measured by ELISA (PharMingen, San Diego, Califor-
nia, USA). The lower limits of detection for each assay
were less than 2 pg/ml for IFN-γ, less than 0.6 pg/ml for
IL-4, and less than 1 pg/ml for IL-10 and TGF-β. The
remaining cells were incubated for an additional 16
hours, pulsed with 0.5 µCi/well of [3H]thymidine
(Amersham-Pharmacia Biotech, Buckinghamshire,
United Kingdom), harvested on glass-fiber filters using
a Tomtec Inc. (Orange, Connecticut, USA) 96-well cell
harvester, and counted in a 1205 Betaplate liquid scin-
tillation counter (Wallac Inc., Gaithersburg, Maryland,
USA). Results are expressed as mean counts per minute
plus or minus SD from triplicate cultures.
Results
Leptin induces sustained weight loss due to loss of fat mass. All
three subjects lost weight within 2 weeks of initiation
of r-metHuLeptin therapy (Figure 1a). Weight loss con-
tinued in all subjects throughout the trial, albeit with
some refractory periods, which were overcome by
increases in r-metHuLeptin dose (Figure 1b). At all time
points more than 98% of the weight lost was represent-
ed by fat mass (Table 1). Lean mass increased in all chil-
dren over the study period, in keeping with their
increase in linear growth (18) (Table 1). This was most
marked in the two youngest children, in whom the
amount of fat-free mass that accrued was in keeping
with that seen in normal children of this age (18, 19).
Leptin effects on energy intake and expenditure. In all three
children there was a marked reduction in energy intake
(range 45–84%) at the test meal after 2 months of
r-metHuLeptin therapy (Figure 2a). These findings are
supported by parental reports of a marked ameliora-
tion in their hyperphagia.
With the continuation of r-metHuLeptin therapy, in
all three children there were episodes during which fat
mass was regained. In all instances this was preceded by
Figure 1
Effects of r-metHuLeptin on
weight in three children with con-
genital leptin deficiency. (a)
Weights of child A compared
with normal centiles for girls and
of child B and child C compared
with normal centiles for boys.
Arrows indicate the start of
r-metHuLeptin therapy. (b) Clin-
ical photographs of child B
before (height = 107 cm) and 24
months after r-metHuLeptin ther-
apy (height = 124 cm) (repro-
duced with the permission of the
child’s parents).
The Journal of Clinical Investigation | October 2002 | Volume 110 | Number 8 1095

Table 1
Effects of r-metHuLeptin therapy on body composition
Months of 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48
treatment
Weight A 94.4 89.2 87.2 83.6 81.2 79.7 78.0 78.8 78.8 77.4 77.6 77.0 76.6 75.2 75.2 75.4 76.6 76.2 77.2 76.4 78.6 78.2 – 77.4 –
(kg) B 41.0 39.0 38.0 36.8 36.8 38.4 40.8 39.6 37.4 35.0 33.2 31.6 32.7 39.5 45.3 47.6 44.5 40.7 36.2
C 38.8 37.3 35.6 37.5
Height A 140 141 141 142 142 143 143 144 145 145 147 149 149 150 150 150 152 152 153 153 154 154 154 155 155
(cm) B 107 107 111 112 114 116 116 117 119 120 121 123 124 125 126 128 128 129 130
C 100 102 103 105
Fat mass A 55.9 53.4 49.9 47.0 42.5 42.0 40.3 40.7 38.3 37.8 38.8 38.1 38.2 35.3 36.2 36.8 37.1 36.8 37.0 36.2 37.5 37.0 37.3 37.9 37.0
(kg) B 23.8 20.3 21.0 17.3 16.4 19.3 21.9 19.9 18.2 15.6 13.6 12.2 11.7 16.9 22.2 24.6 21.7 18.4 13.1
C 21.9 20.6 18.9 19.7
Fat-free A 38.5 36.0 37.3 36.8 38.7 37.5 37.7 37.9 39.2 39.5 38.1 38.6 37.0 39.7 38.1 38.3 38.7 38.3 39.2 40.1 40.4 40.7 38.4 40.3 39.2
mass (kg) B 17.9 19.5 16.8 19.7 21.3 19.3 19.4 20.1 19.4 19.4 19.8 19.7 21.2 22.7 22.9 22.7 23.0 22.1 22.9
C 17.6 17.2 18.1 1 7.7
Body fat A 59.2 59.3 57.2 56.1 52.4 52.9 51.7 51.8 49.4 48.9 50.5 49.6 50.8 47.1 48.7 49.0 48.9 49.0 48.6 47.5 48.2 47.6 49.3 47.9 48.6
(%) B 57.1 51.0 55.6 46.7 43.5 50.0 53.0 49.7 48.4 44.6 40.7 38.2 35.5 42.7 49.2 52.0 48.5 45.4 36.3
C 55.4 53.2 51.7 51.3
Table 2
Pharmacokinetics and Ab formation
Months of treatment 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42
Child A
Body fat (%) 59.2 59.3 57.2 56.1 52.4 52.9 51.7 51.8 49.4 48.9 50.5 49.6 50.8 47.1 48.7 49.0 48.9 49.0 48.6 47.5 48.2 47.6
Dose (mg/kg LBW) 0.027 0.027 0.027 0.027 0.027 0.027 0.027 0.027 0.027 0.027 0.027 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07
Basal leptin (ng/ml) – 3.1 7.6 51.9 19.8 10.4 7.7 8.3 13.7 30.4 18.8 15.8 27.9 85.2 49.0 31.2 34.2 25.2 NA NA NA 17.1
Peak leptin (ng/ml) – 20.1 23.0 80.6 38.0 75.2 23.4 NA NA 101 87.5 38.0 77.8 98.1 146 92.7 NA 98.3 NA NA NA NA
Ab’s – + + + + + + + + + + + + + + + + + NA NA NA +
Neutralizing function – – – – – – – – – – – – – – – – – – NA NA NA –
Child B
Body fat (%) 57.1 51.0 55.6 46.7 43.5 50.0 53.0 49.7 48.4 44.6 40.7 38.2 35.5 42.7 49.2 52.0 48.5 45.4 36.3
Dose (mg/kg LBW) 0.017 0.017 0.017 0.017 0.017 0.021 0.060 0.060 0.060 0.060 0.060 0.060 0.060 0.059 0.151 0.151 0.151 0.151 0.151
Basal leptin (ng/ml) – 0.2 4.1 10.5 10.7 4.4 LD 15.2 NA 21.3 NA 18.1 NA LD LD 10.7 85.5 NA 131
Peak leptin (ng/ml) – NA NA NA NA 23.8 5.0 NA NA NA NA NA NA LD NA 193 282 NA NA
Ab’s – + + + + + + + NA + NA + NA + + + + NA +
Neutralizing function ––––––––NA–NA–NA++– –NA–
Child C
Body fat (%) 55.4 53.2 51.7 51.3
Dose (mg/kg LBW) 0.014 0.014 0.014 0.014
Basal leptin (ng/ml) – LD 6.5 LD
Peak leptin (ng/ml) – 5.8 NA 6.4
Ab’s – + + +
Daily dose expressed in milligram per kilogram LBW. Serum leptin concentration measured before injection (basal) and 4 hours after injection (peak). Ab’s (+/–) indicate presence/absence. Neutralizing function denoted as
+/– for present/absent. LD, limit of detection; NA, not available.
1096 The Journal of Clinical Investigation | October 2002 | Volume 110 | Number 8

parental reports of the return of hyperphagia 2 to 4
weeks before the documented weight gain. In each
instance the reported hyperphagia was supported by the
results at the ad libitum test meal (Figure 2b; data for
child B). In at least one instance an episode of refrac-
toriness to therapy coincided with the transient appear-
ance of neutralizing Ab’s (see below). In each case an
increase in dose of leptin (Table 2) led to a decrease in
hyperphagia, reduced food intake at the test meal, and
loss of fat mass after a period of approximately 4 weeks.
Measurements of energy expenditure were performed
on the two children (child A and child B) who were able
to tolerate the methods used. There was no significant
change in BMR adjusted for lean mass between baseline
and 1 or 2 months of r-metHuLeptin therapy in child A
or child B (Table 3). Adjusted BMR remained unchanged
over the subsequent 42 months in child A and 28
months in child B. To assess whether leptin might have
acute effects on metabolic rate, BMR was measured daily
for the first 3 days after the start of r-metHuLeptin ther-
apy in child B with no differences from baseline values
being observed (data not shown). Free-living or total
energy expenditure (TEE) was measured using doubly
labeled water in these two children. In child A, there was
no change in TEE adjusted per kilogram of lean mass
before and after r-metHuLeptin treatment; similar obser-
vations were made in child B after adjusting for changes
in body composition (Table 3).
Metabolic and endocrine effects of r-metHuLeptin therapy.
All three children had normal fasting plasma glucose
values before treatment but all had hyperinsulinemia,
which was consistent with their age and degree of obe-
sity (ranges for severely obese children established in
more than 400 subjects: 29–183 pmol/l for 3 year olds
Figure 2
Effects of r-metHuLeptin therapy on energy intake. (a) Energy intake at an ad libitum test meal before (black bars) and 2 months after (white
bars) r-metHuLeptin therapy in child A, B, and C. Energy intake (KJ) expressed per kilogram lean body mass to compare intake of subjects
of different age and body size. (b) Changes in body mass index SDS (BMI SDS) (filled symbols) and energy intake at an 18-MJ ad libitum
test meal (gray bars) during 36 months of treatment in child B. Panels indicate duration of r-metHuLeptin dose expressed as a percentage
of predicted serum leptin concentration based on age, gender, and body composition.
Table 3
Effects of r-metHuLeptin therapy on energy expenditure
Months of 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42
treatment
Child A
BMR 7.7 7.1 7.4 6.8 6.9 6.8 6.3 6.4 – – – 6.0 6.5 6.4 7.0 – – – 6.8 6.9 6.8 6.9
(MJ/day)
BMR/kg LBW 0.21 0.21 0.21 0.19 0.19 0.19 0.17 0.18 – – – 0.16 0.19 0.17 0.19 – – – 0.18 0.18 0.18 0.18
(MJ/kg/day)
TEE (MJ/day) 12.4 – – 11.1 – – 12.3 – – 12.2 – – – 11.5 – 13.5 – – 10.1 – – –
TEE/kg LBW 0.33 – – 0.32 – – 0.34 – – 0.33 – – – 0.30 – 0.37 – – 0.26 – – –
(MJ/kg/day)
Child B
BMR 4.5 4.7 3.9 5.0 5.0 5.2 5.4 5.1 4.1 3.7 5.2 3.8 4.5 4.9 5.0
(MJ/day)
BMR/kg LBW 0.26 0.25 0.24 0.26 0.26 0.28 0.29 0.27 0.22 0.20 0.27 0.20 0.22 0.23 0.23
(MJ/kg/day)
TEE (MJ/day) 7.4 – – 6.4 – – 7.4 – – – 6.4
TEE/kg LBW 0.42 – – 0.33 – – 0.40 – – – 0.34
(MJ/kg/day)
The Journal of Clinical Investigation | October 2002 | Volume 110 | Number 8 1097

with a BMI SD score [SDS] = 5–7; 46–244 pmol/l for 9
year olds with BMI SDS = 5) (personal observations).
Plasma insulin concentrations did not fall acutely after
initiation of r-metHuLeptin treatment, but rather a
steady and consistent reduction in fasting plasma
insulin was observed in keeping with the gradual
loss of fat mass (Table 4). Similarly, in response to
r-metHuLeptin therapy, serum cholesterol, triglycerides,
and LDL cholesterol levels gradually reduced and serum
HDL cholesterol increased in all three subjects.
In all three children growth was not stunted in the
untreated state and continued along predicted centiles
throughout the course of treatment (Table 1 and data
not shown). Plasma IGF-1 levels were within the
normal age-related reference range before treatment
and increased appropriately with age (Table 5). Whole-
body bone mineral content (BMC) and bone mineral
density (BMD) were appropriate for age and gender in
the leptin-deficient state (data not shown), although
skeletal maturation (assessed using radiographs of the
left hand and wrist) was increased by a mean of 2.1
years. After treatment, BMC, BMD, and skeletal matu-
ration increased in line with normal age-related devel-
opment in all three children (data not shown).
Urinary free cortisol levels were normal and not sig-
nificantly changed by leptin administration in child A.
The two younger children were unable to undertake
24-hour urine collections, but 0900-hour cortisol
Table 4
Effects of r-metHuLeptin therapy on metabolic parameters
Months of treatment 0 6 12 18 24 30 36 42 48
Insulin (0–60 pmol/l) A 291 381 86 83 66 – – 33 –
B 162 90 – 86 46 – 60
C 201 147
Glucose (3.5–5.5 mmol/l) A 4.3 5.0 4.7 4.6 4.5 4.6 4.5 5.0 4.5
B 4.0 4.6 5.2 4.8 4.3 – 4.3
C 4.0 4.4
Total cholesterol (mmol/l) A 5.0 4.3 4.7 4.7 4.3 4.3 4.2 – –
B 4.4 4.2 3.8 3.4 3.9 – 2.9
C 5.2 4.4
LDL cholesterol (mmol/l) A 3.4 3.1 2.9 3.0 2.8 2.8 2.3 – –
B 3.1 2.4 2.2 2.2 2.1 – 1.8
C 3.4 2.5
Triglycerides (mmol/l) A 1.3 1.0 1.5 0.9 0.8 0.7 1.2 – –
B 1.3 2.1 1.6 0.7 0.7 – 0.6
C 2.0 2.1
HDL cholesterol (mmol/l) A 1.1 0.8 1.2 1.3 1.2 1.2 1.4 – –
B 0.8 0.9 0.9 0.9 1.5 – 0.9
C1.01.0
Figure 3
Leptin therapy results in pubertal development at an appropriate developmental age. (a) Pulsatile secretion of LH and FSH in child A after
12 and 24 months of r-metHuLeptin therapy. (b) No pulsatile secretion after 12 months of treatment in child B.
1098 The Journal of Clinical Investigation | October 2002 | Volume 110 | Number 8

measurements were in the normal range in child A, B,
and C before and after r-metHuLeptin administration
(Table 5). Although thyroid function tests were within
the normal range before the start of r-metHuLeptin
therapy, free thyroxine levels were significantly
increased compared with baseline in all three children
at their first post-treatment measurement (2 months)
and subsequently remained constant (Table 5). The
mean (± SD) concentrations of free thyroxine pre- and
post–r-metHuLeptin therapy were 11.2 (± 1.1) and 14.3
(± 1.1) pmol/l, respectively (P< 0.05, paired Student t
test). Plasma tri-iodothyronine (fT3) concentrations
rose after leptin administration in child B and C but
not in child A. There was no significant change in plas-
ma concentrations of thyrotropin (TSH) (Table 5).
In the two prepubertal children, basal FSH and LH
concentrations and sex steroid concentrations
remained in the prepubertal range after a maximum
of 36 months of r-metHuLeptin therapy (Table 5). In
contrast, there was a gradual increase in gon-
adotropins and estradiol in child A, and after 24
months of r-metHuLeptin therapy, child A (age 11
Table 5
Effects of r-metHuLeptin therapy on endocrine physiology
Months of 0 2 6 12 18 24 30 36 42 48
treatment
TSH A 2.8 3.2 4.5 2.3 3.2 2.7 1.3 3.1 2.2 1.3
(0.4–4.0 mU/l) B 2.7 4.0 3.9 3.9 – 3.9 – 3.3
C 3.3 5.2 3.8
fT4 A 11.8 13.8 14.1 14.0 15.2 14.1 13.8 14.3 14.4 12.1
(9–20 pmol/l) B 12.0 15.4 15.5 14.7 – 16.4 – 14.2
C 9.9 12.0 12.3
fT3 A 6.5 7.2 5.5 5.7 7.4 7.6 7.7 7.4 – 5.5
(3.0–7.5 pmol/l) B 5.5 9.4 8.5 9.2 8.5 6.6
C 6.6 10.0 7.8
0900 cortisol A 420 388 484 290 337 295 178 211 – 175
(280–650 nmol/l) B 156 328 218 462 192 171
C 195 142 158
IGF-1 (IU/l) A 19.1 – 24.0 28.0 – 44.0 – 75.0 – –
B 8.2 – – 13.9 – 15.9 – 21.0
C 5.0 – –
FSH (mU/l) A 0.2 0.7 2.8 2.9 4.3 5.3 4.9 4.9 – –
B 0.2 – 0.2 0.2 – 0.2 – 0.2 – –
C 0.2 – 0.2
LH (mU/l) A 0.2 0.2 0.3 0.3 1.7 2.4 4.5 6.4 – –
B 0.2 0.2 0.2 0.2 – 0.2 – 0.2
C 0.2 – 0.2
Estradiol A 67 34 68 51 60 57 108 126 105 –
(pmol/l)
Testosterone B 0.2 – 0.2 0.2 – 0.2 – 0.2
(nmol/l) C 0.2 – 0.2
Age-specific ranges for IGF-1: 2.5–20 IU/l (0–7 years), 4.5–37.5 IU/l (7–10 years), 7–50 IU/l (10–11 years), 8.5–60 IU/l (11–12 years), 10–75 IU/l (12–13 years).
Prepubertal ranges of gonadotropins and sex steroids: FSH (<0.2 mU/l), LH (<0.2 mU/l), estradiol (<80 pmol/l), and testosterone (<0.5 nmol/l). Females in
the follicular phase of the menstrual cycle: FSH (2.9–8.4 mU/l), LH (1.3–8.4 mU/l), estradiol (100–750 pmol/l).
Table 6
Effects of r-metHuLeptin replacement therapy on T cell subpopulations
Cell type –10 –6 0 +2 +6 +10 Normal range
(cell/mm3)(cell/mm3)
(%) (%)
CD3+2,328 2,686 2,416 1,893 3,262 3,087 1,200–2,500
(56) (49) (58) (51) (72) (66) (65–82)
CD4+815 750 866 927 1,468 1,358 1,000–2,000
(19) (18) (21) (24) (31) (29) (35–50)
CD8+1,410 1,836 1,450 850 1,468 1,543 240–1,000
(25) (26) (25) (22) (31) (33) (25–35)
CD19+1,560 2,189 1,525 1,468 1,247 1,216 200–400
(35) (31) (37) (38) (26) (26) (8–15)
CD3–/CD16+/CD56+282 636 289 464 240 374 212–318
(5) (9) (7) (12) (5) (8) (7–23)
CD4+/CD8+ratio 0.57 0.40 0.59 1.09 1.0 0.88 1.0–2.6
Data represent the absolute number (relative percentage) of T cell subpopulations before (–) and after (+) starting leptin treatment in child C.
The Journal of Clinical Investigation | October 2002 | Volume 110 | Number 8 1099

years) had multiple synchronous nocturnal pulses of
LH and FSH (Figure 3a). She subsequently pro-
gressed through the clinical stages of pubertal devel-
opment, which was associated with a growth spurt,
behavioral changes associated with pubertal devel-
opment, enlargement of the ovaries on ultrasound
with observation of follicles, and an increase in uter-
ine size. She had her first menstrual period at 12.1
years and now has regular menstrual cycles; at age
13.6 years she has reached Tanner stage III. In con-
trast, after 12 months of r-metHuLeptin therapy,
there was no evidence for pulsatile secretion of
gonadotropins in child B at age 4.9 years (Figure 3b).
The effect of leptin on T cell subset number and function.
In all three children there was a clinical history before
treatment of frequent childhood infections (predom-
inantly of the upper respiratory tract) when compared
with their wild-type siblings. There were no gross
abnormalities of thermoregulation reported, with the
children being able to mount a pyrexial response to
infection. Of note, prior to treatment all children were
reported to show the normal reduction in appetite
when suffering from an acute infective illness. We
analyzed the immunophenotype and T cell re-
sponses in freshly isolated cells before and after
r-metHuLeptin therapy in child C and undertook
similar studies in frozen lymphocytes from child B.
Child C had a normal total lymphocyte number and
a normal number of CD3+T cells, but there was a
reduction in CD4+T cell number and an increase in
CD8+and B cells, causing a marked reduction in the
CD4+/CD8+ratio (Table 6). The absolute number of
naive (CD4+CD45RA+) T cells was reduced
(193.6 ± 16.3, 17.0% ± 3.0%, vs. 1,789.0 ± 219.0,
80.4% ± 8.7%, in controls)and consistently lower than
that of memory (CD4+CD45RO+) T cells (676.6 ± 31.3,
82.1% ± 8.1%, vs.366.6 ± 219.4, 17.8% ± 9.0%, in con-
trols), as was the naive/memory T cell ratio (0.28 vs.
4.88 in controls). R-metHuLeptin therapy normalized
the immunophenotype in child C. Thus, CD4+T cell
number was increased to a normal level as was the
CD4+/CD8+T cell ratio, while the number of CD8+and
CD19+B cells was reduced (Table 6). During the period
of r-metHuLeptin therapy, the number of naive
CD4+CD45RA+T cells and the naive/memory T cell
ratio were increased (410.6 ±78.4 and 0.48, respective-
ly). Finally, the proportion of NK cells, as defined by
CD3–/CD16+/CD56+expression, was normal and
maintained constant before and after leptin treatment
(Table 6), as was the expression of the γδ+T cells (range
3–5%, data not shown).
Prior to r-metHuLeptin therapy, lymphocytes from
both patients showed reduced proliferative responses
and lower production of cytokines to a variety of poly-
clonal stimuli such as OKT3, PHA, PMA/Iono, and the
recall antigen PPD (Figure 4a for child C; child B data
not shown). The T cell hyporesponsiveness persisted
even when further stimuli were added (IL-2, anti-CD28
mAb, allogeneic stimulator cells). Immunoglobulin lev-
els were within the normal age-related range before
treatment, with slightly increased IgM (data not
shown), which is in agreement with data from ob/ob
mice (20). Similar observations were made for child B
(data not shown).
In the leptin-deficient state, secretion of the proin-
flammatory cytokine IFN-γwas completely sup-
pressed. The absence of this Th1 cytokine was accom-
panied by impaired but detectable secretion of the
Th2/regulatory cytokines IL-4, IL-10, and elevation of
TGF-β. Intracellular cytofluorimetric (FACS) analysis
of T cells after PMA/Iono stimulation confirmed IL-4
and IL-10 secretion before treatment and undetectable
staining for IFN-γ(not shown). Chronic leptin replace-
ment increased the proliferative responses and
cytokine production of the patient’s lymphocytes in all
assays, in some even to a level comparable with lym-
phocytes from age-matched controls (Figure 4b). The
most significant and best-maintained increases after
treatment were observed in the production of IFN-γ,
which was restored to a level similar to that of control
cells (Figure 4c). Significant increases in the levels of
IL-4 and IL-10 were also observed after 2 months of
Figure 4
Effect of r-metHuLeptin on T cell proliferation and cytokine produc-
tion in child C. (a) Proliferative responses of peripheral lymphocytes to
T cell–specific stimuli at three different time points before (–) and after
(+) recombinant leptin treatment. All data are from triplicate cultures
and expressed as mean ± SD. Proliferative responses from normal age-
matched controls were measured in parallel experiments (mean shown
as single point ± SD). (b) Cytokine profiles in child C at three different
time points before (–) and after (+) recombinant leptin treatment.
Cytokine measurements from normal age-matched controls were
measured in parallel experiments (mean shown as single point ± SEM).
1100 The Journal of Clinical Investigation | October 2002 | Volume 110 | Number 8

r-metHuLeptin therapy, although the levels were not
always maintained (Figure 4c). TGF-βsecretion was
completely suppressed to normal levels following the
commencement of r-metHuLeptin therapy. Similar
observations were made for child B (data not shown).
Discussion
These studies demonstrate that the subcutaneous
administration of r-metHuLeptin is an effective, long-
term therapy for children with congenital leptin defi-
ciency. Leptin treatment in ob/ob mice results in
dramatic and selective loss of fat mass due to a combi-
nation of decreased food intake and increased energy
expenditure (8–10). In humans, a similarly selective
loss of fat mass is seen, with beneficial clinical and
social outcomes for the treated children. The major
impact of leptin on human energy balance was medi-
ated by its suppressive effects on food intake, with a
marked reduction in caloric consumption during a
test meal. These effects were seen at a dose equivalent
to 10% predicted serum leptin concentration (0.01
mg/kg LBW) in contrast to the supraphysiological
doses (0.1–0.3 mg/kg body weight) required to induce
weight loss in leptin-sufficient obesity (21). The ob/ob
mice are more sensitive to the anorectic effects of lep-
tin compared with diet-induced obese mice, an effect
that may be explained by increased hypothalamic
expression of the signaling form of the leptin receptor
(22). The effects of leptin were highly selective for fat
mass, and the treated children grew normally and
showed normal increases in lean mass during the
course of the treatment. Leptin therapy produced a
marked decrease in the amount of food ingested at an
ad libitum test meal in all children, and this was asso-
ciated with parental reports of a near normalization of
eating behavior in the domestic setting. Fluctuations
in body fat mass during therapy correlated strongly
with alterations in food intake.
In contrast to the dramatic effects of leptin on
human energy intake, we were unable to demonstrate
a major effect of leptin on basal metabolic rate or
free-living energy expenditure. Because weight loss by
other means is reported to be associated with a fall in
BMR (23, 24), the failure of decline in energy expen-
diture in these subjects may, in itself, be significant.
Activity-related energy expenditure, the energy
expended during physical activity and skeletal mus-
cle efficiency, which are altered in normal obese sub-
jects after weight loss (25), were not measured. We
cannot exclude effects on these parameters and on
diet-induced thermogenesis, which may be signifi-
cant at the levels of energy intake seen in untreated
leptin deficiency. While the absence of measurable
direct effects on energy expenditure may reflect an
innate difference between humans and rodents, it is
noteworthy that even in rodents the effects of leptin
deficiency on energy expenditure and thermogenesis
are highly dependent on the thermal conditions
under which these measurements are taken (4). For
ethical reasons we were unable to explore the effects
of cold challenge in human leptin deficiency, either
in the treated or untreated state.
The administration of r-metHuLeptin resulted in a
steady and marked fall in plasma insulin concentrations
and a reduction in serum cholesterol and triglycerides
in all three children, whereas HDL-cholesterol concen-
trations increased. Although, in rodents, leptin admin-
istration has been demonstrated to have acute benefi-
cial effects on insulin sensitivity (26), the fall in plasma
insulin in these children was delayed and appeared to
parallel the reduction in fat mass. The improvement
in serum lipids was similarly gradual. While these
observations do not exclude an acute effect of leptin per
se on insulin sensitivity and lipid metabolism, the effect
of loss of fat mass appears to dominate in humans.
We have commented previously on the marked differ-
ence between mice and humans in the effects of leptin
deficiency on both the hypothalamic-pituitary-adrenal
axis and on linear growth (14). The extended data set
represented by the studies reported herein confirms the
lack of any major impact of leptin deficiency on these
endocrine functions in humans, in contrast with mice.
Our findings do, however, support the notion that the
hypothalamic-pituitary thyroidal axis can be influenced
by leptin in a relatively acute manner. Thus, free thy-
roxine levels, although being in the normal range before
treatment, consistently showed an increase at the earli-
est post-treatment time point and subsequently stabi-
lized at that new state. In contrast, weight loss by other
means is well recognized to be associated with a fall in
concentrations of thyroid hormones (27). These find-
ings are consistent with evidence from animal models
that leptin profoundly influences TRH release from the
hypothalamus (28, 29) and with recent data that leptin
administration can blunt the fall in free thyroxine con-
centrations induced by weight loss in previously weight-
stable subjects (30). Leptin may also regulate TSH pul-
satility as has been suggested by a previous study of an
adult leptin-deficient subject (31). The rapid increase in
fT4 was accompanied by a similar increase in fT3 in the
two youngest, but not the oldest child. Currently, we
cannot explain this divergent response, but it may con-
ceivably relate to age-dependent effects of leptin on thy-
roid hormone deiodination (32).
The presence of adequate circulating levels of leptin
are clearly essential for the activation of the hypothal-
amic-pituitary-gonadal axis at puberty because adults
with congenital leptin deficiency (33, 34) and leptin
receptor mutations (35) fail to undergo pubertal devel-
opment. Our findings in child A, who, prior to treat-
ment, was prepubertal despite a bone age of 12.5 years,
demonstrates conclusively that leptin replacement can
facilitate the entire repertoire of pubertal development.
Some studies have suggested that an increment of plas-
ma leptin may be involved in the initiation of puberty
(36). There was, therefore, at least a theoretical concern
that leptin replacement in young leptin-deficient chil-
dren might initiate precocious puberty. There was no
The Journal of Clinical Investigation | October 2002 | Volume 110 | Number 8 1101

clinical or biochemical evidence for this in the younger
children in this study. In particular, the detailed stud-
ies of LH and FSH pulsatility undertaken in child B
after 12 months of leptin therapy demonstrated the
maintenance of an entirely prepubertal pattern of
gonadotropin secretion. Our findings in humans are
therefore consistent with the notion that leptin acts as
a permissive factor, or so-called “metabolic gate” (37),
for the development of puberty, permitting its progress
only when other leptin-independent signals affecting
the timing of the onset of puberty are present.
Prior to treatment, the two patients tested (B and C)
showed selective CD4+T cell lymphopenia and severely
impaired lymphocyte function, comparable with that
observed in anergic-hyporesponsive T cells. This stage of
hyporesponsivness could, at least in part, be explained
by the moderate to severe CD4+T cell lymphopenia and
the secretion of Th2/regulatory cytokines such as IL-4,
IL-10, and TGF-β, observed before treatment in the
absence of any consistent IFN-γproduction. After
recombinant leptin replacement, both the immunophe-
notype and the T cell responsiveness were significantly
improved and in some experiments reached the levels
observed in normal age-matched controls. The reduced
responsiveness to T cell receptor–specific stimuli OKT3
and PPD observed after 6 months of r-metHuLeptin
treatment in patient C could be related to the produc-
tion of anti–r-metHuLeptin neutralizing Ab’s. These
results suggest that leptin is a key molecule in both CD4+
T cell development and function in humans, as testified
by the increased proportion of CD4+naive T cells and
the restored IFN-γsecretion, respectively.
Ab’s to r-metHuLeptin developed in all children after
approximately 6 weeks and markedly altered the phar-
macokinetics of the injected peptide, thus hampering
interpretation of serum leptin levels. Because full-length
leptin represents a novel antigen to these children, the
development of an Ab response was not unexpected.
The fluctuating nature of the anti–r-metHuLeptin Ab’s
may reflect the complicating factor that leptin defi-
ciency is itself an immunodeficient state and that leptin
administration leads to a switch from the secretion of
predominantly Th2 to Th1 cytokines, which may direct-
ly influence Ab production. Ab’s that were capable of
neutralizing leptin activity in a bioassay appeared tran-
siently in child B. The appearance of this activity coin-
cided with a relapse of hyperphagia and weight gain in
child B between 24 and 28 months, and its disappear-
ance coincided with the return of a clinical response.
Similarly neutralizing Ab’s were detected in child C after
6 months of r-metHuLeptin therapy and were associat-
ed with hyperphagia and weight gain. Whether or not
these Ab’s will eventually lead to a complete blockade of
therapeutic efficacy is currently uncertain.
A major question concerning leptin relates to whether
it is principally involved in the “switch” from the fed to
the starved state (38), with maximal sensitivities to
changes in its concentration occurring at very low plas-
ma levels, or whether plasma leptin concentrations
varying across the range of concentrations seen in nor-
mal humans actually continue to exert a dose-related
effect in the control of fat mass. While our study should
theoretically have been able to provide unprecedented
information regarding the relationship between plasma
leptin concentration and its biological effects, the rapid
development of circulating Ab’s to leptin in the children
made meaningful interpretation of serum leptin con-
centrations difficult.
In a recent study of leptin-sufficient subjects, admin-
istration of twice-daily recombinant human leptin pre-
vented the fall in energy expenditure and thyroid hor-
mones seen after 10% weight loss (30). This study
provides strong support for the notion that the meta-
bolic and neuroendocrine changes associated with
weight loss in normal humans are the result of a fall in
serum leptin concentrations. The ability of leptin
replacement (to physiological levels) to prevent these
adaptive changes and thereby prevent rebound weight
gain, are consistent with our observations in congeni-
tal leptin deficiency. The weight-reduced state may
thus be considered a state of relative leptin deficiency.
The therapeutic response to r-metHuLeptin admin-
istration in these three children confirms the impor-
tance of leptin in the regulation of human body weight,
fat mass, and appetite. The sustained beneficial effects
seen in these subjects (continued for over 4 years in one
subject) have rarely been reported with other pharma-
cotherapies for obesity.
Leptin administration permits the full progression of
appropriately timed puberty, but does not appear to
cause precocious activation of the pubertal process in
younger children. Leptin therapy results in gradual but
sustained improvements in hyperinsulinemia and lipid
profile. Of particular note is the relatively acute effects
on circulating thyroid hormone concentrations, pro-
viding compelling evidence in humans that leptin is a
regulator of the hypothalamic-pituitary-thyroidal axis.
Finally, we have described, we believe for the first time,
the immunodeficient state associated with human lep-
tin deficiency and its reversal by leptin therapy. It is
plausible that leptin’s effects on human neuroen-
docrine function and T cell–mediated immunity may
extend beyond the paradigm of congenital leptin defi-
ciency and that leptin therapy may be more generally
applicable in other disorders characterized by
hypoleptinemia. This has been demonstrated recently
by the sustained metabolic improvements seen with
leptin therapy in human lipodystrophic disorders (39).
Given the immunoregulatory properties of leptin on
CD4+T cells, increasing clinical applications of this
hormone can be hypothesized. Indeed, in immunode-
ficiencies associated with anorexia nervosa and HIV-1
infection, leptin levels are dramatically reduced (40), as
are CD4+T cell number and function. Leptin adminis-
tration could conceivably be a useful therapeutic tool
to help the immunoreconstitution process in such
patients, considering its effects on the thymic output
of T cells and cell-mediated immune response (40–42).
1102 The Journal of Clinical Investigation | October 2002 | Volume 110 | Number 8

Acknowledgments
We thank C. Cheetham and A. Earley for help with clin-
ical studies, S. Jones for body composition analysis, and
A. Wright and A. Coward for doubly labeled water
analysis. This work is supported by grants from The
Wellcome Trust (to I.S. Farooqi), Medical Research
Council UK (to S. O’Rahilly), and a grant from
AMGEN Inc. for clinical studies.
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The Journal of Clinical Investigation | October 2002 | Volume 110 | Number 8 1103