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1 Department of Pediatrics, In an effort to understand the genetics of
human obesity, we have studied the physiology and molecular genetics of
rodent models with monogenetic forms of obesity including the leptin gene-defective
(Lepob/Lepob)
and leptin receptor gene-defective
(Leprdb/Leprdb)
mouse. In the experiments reported here, we investigated the effects of
heterozygosity at
Lepob and
Leprdb on body
composition and circulating leptin concentration in +/+, Leprdb/+, and
Lepob/+ adult
mice to identify possible gene dosage effects of these mutations that
might elucidate their physiology. Adult mice heterozygous for the
Lepob or
Leprdb allele
had equivalent fat mass and percentage body fat, which was increased
27-47% and 23-35%, respectively, relative to +/+ littermates. Plasma leptin concentrations adjusted for fat mass were
6.5 ng/ml in the
Lepob /+, 9.6 ng/ml in the +/+, and 11.5 ng/ml in the
Leprdb/+ mice.
Sex had no effect on plasma leptin after controlling for fat mass.
These data, and data from a small number of mice heterozygous at both
Lepob and
Leprdb (compound
heterozygotes), suggest that leptin protein produced per mass of body
fat is reduced in
Lepob/+ mice and
that body fat is increased in
Lepob/+ mice
until plasma leptin concentrations reach that of a normal +/+ mouse.
The elevated plasma leptin concentration in the
Leprdb/+ mice
suggests that LEPR may mediate autocrine suppression of Lep expression. These results raise
the possibility that human mutations that have even subtle effects on
the leptin/leptin receptor system in either the homozygous or
heterozygous state may have significant effects on adiposity.
leptin receptor; fat mass; obesity; diabetes
OBESITY IS A MAJOR HEALTH problem in the western world
due to its contributions to cardiovascular disease,
non-insulin-dependent diabetes mellitus, and cancer (16). The high
concordance rates for obesity in identical twins (44, 45), familial
clustering of increased body fat (23, 29), and single gene syndromes associated with obesity including Prader-Willi (37), Bardet-Biedel (3,
5), Alstrom (1), and Cohen syndromes (15) suggest a genetic component
to obesity in humans.
Molecular cloning of the genes defective in the
Lepob/Lepob
and
Leprdb/Leprdb
mice, leptin (Lep) (52) and leptin
receptor (Lepr) (8, 9, 30, 47),
respectively, has identified a novel hormone-hormone receptor system by
which quantities of stored somatic triglycerides can be signaled to the
hypothalamus and thereby influence energy intake, expenditure, calorie
partitioning, and various endocrine functions (31). Recent
identification of human subjects with a loss-of-function mutation in
Lep (34) and
Lepr (K. Clement, C. Vaissel, N. Lahlou, S. Cabrol, V. Pelloux, D. Cassutol, M. Gourmelen, C. Dina, J. Chambaz, J.-M. Lacorte, A. Basdevant, P. Bougneres, Y. Lebouc, P. Froguel, and B. Guy-Grandl, unpublished observations) suggests that
leptin participates in the regulation of body fat in humans.
Statistical linkage of the Lep genomic region with extreme obesity was independently observed in two Caucasian
populations (13, 38), and polymorphisms within
Lepr have been associated with
increased adiposity in multiple populations (48; P. Behn, W. K. Chung,
C. Iannotti, K. Clement, M. Province, C. Welling, J. McGill, S. Dagogo-Jack, P. Froguel, R. L. Leibel, and M. Permutt; Y. Chagnon, W. K. Chung, L. Perusse, M. Chagnon, S. Roy, R. L. Leibel, and C. Bouchard; and M. Karvonen, U. Pesonen, S. Leal, R. Sipilainen, A. Rissanen, H. Naukkarinen, W. K. Chung, R. L. Leibel, M. Laakso, M. Uusitupa, and M. Koulu, unpublished observations).
Given the high prevalence of obesity in western societies and the
relative rarity (despite extensive searches) of subjects homozygous for
loss-of-function mutations in genes associated with monogenetic rodent
obesity (6, 11, 19, 32), it is unlikely that a significant proportion
of the variation in adiposity in humans will be explained by
homozygosity for gross alterations in
LEP or
LEPR. However, more subtle and common
allelic variants, or heterozygosity for loss-of-function alleles at
these and other genes, may act in concert to determine genetic
predisposition to increased adiposity within a permissive environment.
Subtle heterozygous phenotypes, such as increased length of survival during a fast (17) in
Lepob/+ and
Leprdb/+ mice
and increased adiposity in 7-day-old
Leprfa/+ pups
(49), have been described.
We produced +/+,
Lepob/+,
Leprdb/+, and
Lepob/+
Leprdb/+ mice to
determine whether differences in circulating leptin and body
composition could be detected in adult mice that were heterozygous for
Lepob
and/or
Leprdb. Such
differences might provide insights into the regulation of leptin
metabolism and identify a mechanism whereby heterozygosity for sequence
variants of this ligand/receptor system might contribute to human
obesity.
Animals. Eighteen pairs of C57BL/6J
Leprdb/+ or
C57BL/6J Lepob/+
males and C57BL/6J +/+ females (Jackson Laboratories, Bar Harbor, ME)
were bred. Preliminary data on compound heterozygotes
(Leprdb /+,
Lepob/+) were
also generated by breeding
Leprdb/+
C57BL/6J males and
Lepob/+ C57BL/6J
females. The offspring were weaned at 21 days of age and separated into
same-sex cages. All animals were kept in a pathogen-free environment at
25°C. Autoclaved water and irradiated chow (Pico Lab mouse breed
chow 5058, 9% fat; Purina Mills, St. Louis, MO) were provided ad
libitum. The mice were on a 12:12-h light-dark cycle. After weaning,
weekly body weight and nasoanal length were determined. Mice were
killed by carbon dioxide asphyxiation between the ages of 39 and 120 days after a 2-h fast. At death, sex, body weight, and nasoanal length
were determined. Body mass index (BMI) was calculated as body weight
divided by nasoanal length squared. Approximately 1.0 ml of blood was
removed by cardiac puncture and mixed with 50 µl of 82 µM EDTA
anticoagulant. The spleen was removed and frozen at Genotyping. Genomic DNA was prepared
from the frozen spleens by phenol-chloroform extraction (2). Genotypes
at Lepob and
Leprdb were
determined as previously described (10).
Leptin quantitation. Fasting plasma
leptin concentrations were determined with a solid-phase sandwich
enzyme immunoassay using an affinity-purified polyvalent antibody
immobilized in microtiter wells as previously described (40).
Body composition analysis. Carcasses
were autoclaved at 125°C in 50-60 ml distilled water for 30 min, cooled, and homogenized in a Polytron for 7-10 min.
Twenty-five-milliliter aliquots were stored at Statistical analysis. The progeny were
grouped by genotype at Lep
and/or Lepr, and these
genotypic groups were compared [by analysis of covariance
(ANCOVA)] for the dependent variables: body fat mass, percent
body fat, and plasma leptin concentration. For the fat mass and percent
body fat ANCOVAs, age and sex were treated as covariates. For the
plasma leptin ANCOVAs, the covariates were fat mass or age and sex. The
ANCOVA requirement for parallelism of regression was found not to be
violated at the P < 0.05 level for
any of these analyses. Scheffé's test for unequal
n values was employed in making all
post hoc pairwise comparisons between the adjusted means of the
respective genotypic groups. All statistical analyses were conducted
using Statistica/W software (Statsoft, Tulsa, OK).
One hundred thirty-five progeny were generated (mean age = 67.5 days ± 22.3 days SD): 64 +/+, 51 Leprdb/+, 14 Lepob/+, and 6 Leprdb/+
Lepob/+. Sexual
dimorphism was evident, with females within each genotypic class having
slightly lower BMI (Table 1). There was no
statistically significant difference in BMI between genotypic classes
of the same sex. Females in both the +/+ and
Leprdb/+
genotypic classes had higher percentage body fat than the corresponding males. There was no sex effect on circulating leptin concentration adjusted for fat mass (P = 0.28).
ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References
METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References
80°C for
DNA extraction. Gastrointestinal contents were removed from each mouse
to reduce error in body composition determination. Plasma was decanted
and stored at 80°C. Carcasses were stored at
80°C.
10°C before
chemical analysis. Total body water was determined by drying duplicate
1-g samples of homogenate overnight at 90°C to stable weight. Total
carcass lipid was determined in triplicate by chloroform:methanol
extraction of homogenate samples (22). Nitrogen was determined by an
adaptation of the Kjeldahl method (20, 36) and was protein calculated,
assuming a nitrogen-to-protein ratio of 0.16.
RESULTS
Top
Abstract
Introduction
Methods
Results
Discussion
References
Table 1.
Phenotypic data by sex and genotypic class
Fat mass, adjusted for age and sex, was 47.3% higher in the Leprdb/+ animals than +/+ (P = 0.0000001) and 26.7% higher in the Lepob/+ than +/+ animals (P = 0.03), with genotype accounting for a significant difference in fat mass among the three genotypes (P = 0.0000001) (Table 2). Adjusted fat mass was not statistically different between Leprdb/+ and Lepob/+ animals.
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Percentage body fat adjusted for age and sex was 35.2% higher in Leprdb/+ animals than +/+ (P = 0.0000001) and 23.5% higher in the Lepob/+ than +/+ animals (P = 0.007), with genotype accounting for a significant difference in percent body fat among the three genotypes (P = 0.0000001) (Table 2). Percentage body fat was not statistically different between Leprdb/+ and Lepob/+ animals.
Mean plasma leptin concentration was twice as high in the Leprdb/+ animals of each sex relative to the +/+ class (12.4 vs. 6.6 ng/ml for the males; 11.8 vs. 6.2 ng/ml for the females), whereas mean plasma leptin concentrations in the +/+ and Lepob/+ mice were comparable (6.6 vs. 8.8 ng/ml for males; 6.3 vs. 5.2 ng/ml for females) (Table 1). When plasma leptin concentrations were adjusted for fat mass, leptin concentration was 19.8% higher in the Leprdb/+ animals relative to the +/+ (P = 0.03), 32.8% lower in the Lepob/+ animals relative to +/+ (P = 0.03), and 43.9% lower in the Lepob/+ relative to the Leprdb/+ (P = 0.0001) (Table 2 and Fig. 1). Genotype accounted for a significant difference in plasma leptin concentrations adjusted for fat mass (P = 0.000003).
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DISCUSSION |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Adult mice heterozygous for the Lepob or Leprdb allele had equivalent fat mass and percentage body fat. These phenotypes were increased 27-47% and 23-35%, respectively, relative to +/+ littermates. Several other phenotypes have been associated with heterozygosity at Lepob or Leprdb when genotype assignment was determined by test crosses. These phenotypes include increased epididymal fat cell size in Lepob/+ mice (26), prolonged survival of a fast in 7- to 9-mo-old Lepob/+ and Leprdb/+ mice (17), more effective conversion of acetone to lactate in Lepob/+ and Leprdb/+ mice (18), slower glucose oxidation in fat pads of Lepob/+ mice relative to +/+ (50), 40% less cholesterol in the brains of Lepob/+ relative to +/+ mice (41), hyperinsulinemia in Lepob/+ mice relative to +/+ controls, and increased (44%) glucose and increased (88%) insulin during glucose tolerance tests in Lepob/+ mice relative to +/+ controls (21). The existence of a phenotype attributable to heterozygosity for these mutations has potentially important implications regarding the genetics of human obesity. Although there have been reports of human subjects with profound, early onset obesity caused by mutations in Lep (34), Lepr (K. Clement, C. Vaissel, N. Lahlou, S. Cabrol, V. Pelloux, D. Cassutol, M. Gourmelen, C. Dina, J. Chambaz, J.-M. Lacorte, A. Basdevant, P. Bougneres, Y. Lebouc, P. Froguel, and B. Guy-Grandl, unpublished observations), and proconvertase 1 (PC-1), an enzyme active in the same proneuropeptide processing pathway as carboxypeptidase E that is mutated in the Cpefat/Cpefat mouse (25), the number of subjects with either homozygous or compound heterozygous loss-of-function mutations in any of these genes has to date been limited to five individuals (three families) despite intensive searches (11, 19, 32, 48; K. Clement, C. Vaissel, N. Lahlou, S. Cabrol, V. Pelloux, D. Cassutol, M. Gourmelen, C. Dina, J. Chambaz, J.-M. Lacorte, A. Basdevant, P. Bougneres, Y. Lebouc, P. Froguel, and B. Guy-Grandl, unpublished observations). However, heterozygosity for these and yet-to-be-described mutations relating to obesity will, of course, be much more common.
Preliminary data on six individual mice heterozygous for both Lepob and Leprdb demonstrate further increases in sex- and age-adjusted fat mass (3.84 g Lepob/+, Leprdb/+; 3.58 g Leprdb /+; 3.08 g Lepob/+) and age- and sex-adjusted percentage body fat (16.7% Lepob/+, Leprdb/+; 15.5% Leprdb/+; 14.2% Lepob/+) over either single heterozygote. The increased adiposity associated with single allele loss of two obesity-related genes also has implications for the genetics of human obesity. Human obesity is a complex trait that in most instances is probably related to the interaction of allelic variation in several obesity-related genes and the environment. Because obesity is a convergent phenotype "resolving" multiple effects on energy intake and expenditure, there is likely to be genetic heterogeneity for obesity susceptibility (39). The responsible genes will likely vary not only among populations but among individuals within a population. Heterozygosity at several loci for alleles with relatively subtle effects on gene expression and function is likely to be a common mechanism for increased adiposity.
Plasma leptin concentration adjusted for fat mass is lowest in the Lepob/+, intermediate in +/+, and highest in Leprdb/+ mice. Preliminary data indicate that mice doubly heterozygous for Lepob and Leprdb have leptin concentrations adjusted for fat mass that are indistinguishable from those of Lepob/+ mice (6.2 vs. 6.5 ng/ml, respectively). These data suggest several things about the regulation of circulating leptin concentration. The decreased plasma leptin concentrations (adjusted for fat mass) in the Lepob/+ mice relative to the +/+ suggest that induction of the single normal Lep allele in Lepob/+ mice is not sufficient to compensate completely for the defective allele, which produces none of the full-length hormone. In contrast, Leprdb/+ mice have higher plasma leptin concentrations (adjusted for fat mass) than +/+ mice, suggesting that they have a defect in feedback regulation of leptin production. Such dysregulation could be due to attenuation of autocrine effects of leptin that are conveyed by the leptin receptor, differences in fat cell size, number or anatomic distribution, or effects mediated by an unidentified feedback mechanism acting through, e.g., the hypothalamus. Similar data demonstrating equivalently elevated circulating leptin concentrations in suckling Leprfa/+ (51) and Leprfak/+ rats (M. Boschmann, unpublished data) suggest that the "long" form of the receptor that includes the complete intracellular signaling domain, the only splice form defective in the Leprdb /+ mouse, is the splice form responsible for dysregulated leptin production. The apparent equivalence of fat mass-adjusted leptin concentrations in the Lepob/+ and Lepob/+, Leprdb/+ (doubly heterozygous) mice suggests that leptin is being maximally produced by the single normal Lep allele in the Lepob/+ mouse because leptin concentrations are not increased in the Lepob/+, Leprdb/+ mice despite the tendency to increase leptin production/release demonstrated by the Leprdb/+ mice. The increase in circulating leptin concentrations in the Leprdb/+ mice is unlikely to be attributable to decreased clearance; otherwise Lepob/+, Leprdb/+ mice would have circulating leptin concentrations higher than Lepob/+ mice. Lep expression data from Zucker rat pups with a defect in Lepr demonstrate increased Lep mRNA in adipose tissue of Lepfa/+ relative to +/+ pups (51) and support the hypothesis that dysregulation of leptin metabolism in rodents with heterozygous defects in Lepr is the result of increased leptin production.
Animals with only a single normal leptin allele (Lepob/+) have sex- and age-adjusted plasma leptin concentrations comparable to +/+ animals. However, these "normal" plasma leptin concentrations in the Lepob/+ mice occur in the context of a 26.7% increase in fat mass. These data support the hypothesis that a minimal threshold concentration of leptin in plasma is necessary to sustain eumetabolism: i.e., normal thermoregulation, energy expenditure, and fertility (39, 43). The Lepob/+ animals are not capable of producing as much leptin per gram of fat as the wild-type animals and therefore, in a teleological sense, increase their total body fat stores to achieve a "normal" plasma leptin concentration. Thus a single defective Lep allele is associated in this mouse model with what might be regarded operationally as an increased "set point" of somatic fat mass. Similarly, animals with a single defective Lepr allele display increased adiposity despite increased fat-adjusted leptin concentrations, suggesting that Leprdb/+ mice also increase total body fat stores to achieve a "normal" intensity of postreceptor leptin signaling in relevant target organs (e.g., hypothalamus) (8, 30). The leptin signal is apparently attenuated in the Leprdb/+ animals as a result of a reduced number of molecules of the intact long receptor isoform (30).
These data, obtained in single and compound heterozygotes for mutations in Lep and Lepr, indicate that these mutations are not fully recessive with regard to either somatic fat mass or the production of leptin by adipose tissue. The former has implications for the complex genetics of human obesity, the latter for the molecular physiological mechanisms underlying the regulation of the "adipostat" (24).
Finally, in none of the genotype groups in the mice was there a significant effect of sex on plasma leptin concentration adjusted for fat mass. This finding is in contrast to the two- to threefold higher plasma leptin per unit fat mass in adult female humans compared with adult males (40). Menopausal status in human females has some influence on leptin per unit fat mass, but postmenopausal women still have higher circulating leptin concentration per unit fat mass than men (40). In humans, there are fat depot-specific differences in leptin expression, with subcutaneous being higher than intra-abdominal fat (35). Exogenous gonadal steroids influence circulating leptin concentrations in rats and humans. Estrogen administration increases leptin production in rats and humans (42). Leptin concentrations decline in human males as puberty (and testosterone production) progresses (12, 33). The lack of sex-related differences in leptin and fat mass in adult mice suggests that the higher concentrations in human females may be due primarily to their relatively greater subcutaneous adipose mass (28, 46) rather than to direct effects of gonadal steroids on leptin expression in adipose tissue.
Perspectives
On the basis of the linkage studies and mutation analyses of candidate genes (e.g., the
3-adrenergic
receptor, leptin, the leptin receptor) performed to date, it appears
unlikely that a significant fraction of human obesity will be
explicable by major functional disruption of any single gene. More
likely, the conflation of subtle variations in coding and promoter
sequences of several genes will be the usual mechanism. The present
study shows that, in this context, classical notions of genetic
recessivity may be contravened. That is, that recessivity is not
absolute with regard to some phenotypes. Mice heterozygous for a
"recessive" mutation in either
Lep or
Lepr mutations are more obese than the homozygous wild type. Thus, in searching for the genetic bases of
complex phenotypes such as obesity, we should not exclude heterozygotes for sequence variations in recessive candidate genes from
consideration.
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ACKNOWLEDGEMENTS |
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The authors gratefully acknowledge the animal care provided by the staff at the Laboratory Animal Research Center at The Rockefeller University, technical assistance from Lena Basilo, and the assistance with manuscript preparation provided by Mary Prudden.
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FOOTNOTES |
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This work was supported in part by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-52431, P30-DK-26687, T32-DK-07559, and DK-49853 to C. N. Boozer.
Address for reprint requests: R. L. Leibel, 1230 York Ave., Box 181, New York, NY 10021.
Received 7 November 1997; accepted in final form 23 December 1997.
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References
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