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Original Studies |
Steno Diabetes Center and Hagedorn Research Institute (S.A.U., M.F., S.M.E., O.P.), Copenhagen; Copenhagen City Heart Study, National University Hospital (T.I.A.S., T.A., A.T-H.), Copenhagen; Danish Epidemiology Science Centre at the Institute of Preventive Medicine, Copenhagen University Hospital (T.I.A.S.), Copenhagen; Roskilde County Hospital (T.A.), Roskilde; Department of Clinical Biochemistry, Herlev University Hospital (A.T-H.), Copenhagen; and Center of Preventive Medicine, Glostrup University Hospital (J.O.C.), Copenhagen, Denmark
Address all correspondence and requests for reprints to: Søren A. Urhammer, Steno Diabetes Center, Niels Steensens Vej 2, DK-2820 Gentofte, Copenhagen, Denmark.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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g nucleotide polymorphism of the 5'-flanking
region of the UCP1 gene were examined for associations with accelerated
weight gain or reduced sensitivity to insulin in a cohort of 380 young
healthy Caucasians. The mutational analysis revealed five nucleotide substitutions that changed the sequence of UCP1, Arg/Trp40, Ala/Thr64, Val/Met137, Met/Leu229, and Lys/Asn257 and two nucleotide substitutions in the nontranslated region of exon 1. Among subjects with juvenile-onset obesity, the allelic frequencies of Ala/Thr64 and Met/Leu229 were both 8.2% (95% confidence interval: 5.1–11.3%) vs. 8.8% (6.0–11.6%) and 8.1% (5.3–10.9%), respectively, in the cohort of randomly selected control subjects. Among lean control subjects, the allelic frequencies of the polymorphisms were 8.2% (3.7–12.7%) and 5.6% (1.9–9.3%), respectively. In the cohort of young healthy subjects, measurements of obesity and insulin sensitivity did not differ between carriers of the Ala/Thr64 and Met/Leu229 variants and wild-type carriers. The Val/Met137 and Lys/Asn257 mutations were each found in one subject with juvenile-onset obesity, and the Arg/Trp40 mutation was found in two obese subjects and in one control subject. The allelic frequency of the nucleotide polymorphism of the 5'-flanking region of the UCP1 gene was 25.3% (22.2–28.4%) in the cohort of 380 young Danes. There were no differences in body mass index, fat mass, waist-to-hip ratio, or weight gain during childhood or adolescence between carriers and noncarriers of this nucleotide variant.
Although we cannot exclude an effect of the rare mutations in the UCP1 gene on susceptibility to juvenile-onset obesity, genetic variation of the coding region of the UCP1 gene is not a common factor contributing to obesity in Caucasian subjects of Danish ancestry.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Uncoupling protein 1 (UCP1), a 32-kilodalton protein located in
the inner mitochondrial membrane, is abundant in brown adipose tissue
(BAT). Through a regulation mainly by intracellular free fatty acids,
UCP1 dissipates the electrochemical gradient generated in the electron
transfer chain in the mitochondria and thereby uncouples the
respiration leading to heat production instead of ATP (6). Rodents with
genetic forms of obesity have decreased brown fat sympathetic activity
and decreased thermogenesis (7). Transgenic mice with decreased brown
fat mass are characterized by obesity, initially in the absence of
hyperphagia, but later in life they develop hyperphagia, indicating a
major role of brown fat on energy balance and food intake (8, 9).
Recently, it also has been demonstrated that these mice are glucose
intolerant and insulin resistant (10). Whereas human neonates possess
plenty of BAT, the amount in adult humans is low, and the functional
impact of BAT in adults is less clear. It has, however, been
hypothesized that BAT might be responsible for 1–2% of the energy
expenditure in humans, and that defects in BAT function of this
magnitude might lead to a weight gain of 1–2 kg/yr (11). Recently, a
ag polymorphism in the 5'-flanking region of the UCP1 gene has been
identified in Caucasian subjects (12). This UCP1 gene marker was
shown to be associated with an increased capacity for weight gain over
years (12).
The major objectives of the present study were 1) to examine for genetic variation in the coding region of the UCP1 gene; 2) to evaluate whether amino acid replacements were associated with juvenile-onset obesity; and 3) to examine whether the 5'-flanking polymorphism or genetic variants in the coding region of the gene were associated with present obesity, an accelerated weight gain during childhood and adolescence, or with impaired insulin sensitivity in a population-based sample of young healthy Danes.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The primary mutational analysis and subsequent association
studies of amino acid variants of UCP1 were performed in study groups
(Table 1
) selected from a population of
young Caucasian men of Danish ancestry, who at the age of 18–26 yr
were examined at the draft board, and who, in addition, had attended
school in the municipality of Copenhagen, where height and weight had
been measured as part of the school health examinations (13). The
population was further restricted to those who were examined at the
Copenhagen City Heart Study Program in 1981–1983 (14) and again in
1992–1994. The cohort of men with juvenile-onset obesity included 156
subjects who had a body mass index (BMI) 
31.0 kg/m2 at
the draft board examination. Their median BMI at the age of 7 yr was
18.1 kg/m2. From this obese cohort, 56 subjects were
randomly selected for the initial mutational screening. As a control
cohort, 205 draftees were selected at random as every hundredth from
the same population. Weight and height at different ages were recorded.
From this control cohort, 79 subjects who at the draft board
examination and at the examination in 1992–1994 had a BMI <25
kg/m2, were selected to comprise a lean control group. BMI
on average age 20 and 48 yr are listed in Table 1.
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Before participation, informed consent was obtained from all subjects. The study was approved by the Ethical Committee of Copenhagen and was in accordance with the principles of the Declaration of Helsinki II.
Biochemical studies
The concentration of fasting serum triglyceride, total cholesterol, and HDL cholesterol (Boehringer Mannheim Diagnostics, Mannheim, Germany) were analyzed using standardized methods.
Measurements of the insulin sensitivity index in a cohort of 380 young individuals
The insulin sensitivity index was estimated from an IV glucose tolerance test in combination with injection of IV tolbutamide as previously described (15).
Identification of intron sequences flanking exons
To amplify each exon, primers were designed in the flanking intron regions. Only a few bases of the introns flanking the 3'-end of exon 4, the 5'-end of exon 6, and both the 3'-end and the 5'-end of exon 5 were available from Genbank. Thus intron 4 and intron 5 were PCR amplified using 100 ng genomic DNA, 0.2 µM of each primer, 2 mM MgCl2 TaqPlus PCR kit (Stratagene, La Jolla, CA), and exon-derived primers; sense primer: 5'-gagctagtaacatatgatctaatgaaggag-3' and antisense primer: 5'-ctacatccaccggggaggacatagctgttg-3' and sense primer: 5'-gtgtgcccaactgtgcaatg-3' and antisense primer: 5'-gacgttccaggatccaagtcg-3', respectively. PCR conditions were denaturation at 94 C for 3 min followed by 40 cycles of denaturation for 30 s, annealing at 55 C for 30 s, and extension at 72 C for 30 s, with a final extension at 72 C for 9 min. Cycles were performed on a GeneAmp 9600 Thermocycler (Perkin-Elmer/Cetus, Norwalk, CT). Direct sequencing of approximately 100 base pair of the ends of these segments was performed using Thermo Sequenase Cycle Sequencing kit (Amersham Life Science, Cleveland, OH).
Identification of mutations in the UCP1 gene
Single-strand conformation polymorphism (SSCP) and heteroduplex
analysis was performed on the entire coding region, including
intron-exon boundaries of the UCP1 gene and on the noncoding region of
exon 1. PCR amplification of the 6 exons (7 segments) was carried out
in a volume of 25 µL, containing 100 ng genomic DNA, 0.2
µM of each primer, 0.313 u of Taq DNA
polymerase (Perkin-Elmer/Cetus), 1.5 mM MgCl2,
and 0.125 µL of a 37-MBq/mL solution
[
-32P]deoxycytidine triphosphate (Amersham,
Buckinghamshire, UK). Primers (Table 2)
were designed from the genomic UCP1 sequence (Genbank accession
numbers: X51952, X51953, X51954, and X51955) and from the partial
intron sequencing described above. PCR conditions were as described
above except for annealing at 56 C (exons 1, 3, and 5), 68 C (exon 2),
60 C (exon 4), and 58 C (exon 6).
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Screening for mutations and amino acid polymorphisms in the UCP1 gene
The DNA segment containing codon 40 was amplified as described
above, including primers used for amplification of the coding part of
exon 1 (Table 2
). Restriction fragment length polymorphisms (RFLPs)
were detected after digestion overnight with 2 U of HpaII.
Codon 64 and 137 were amplified as described, and RFLPs were detected
after digestion overnight with 1.5 U AciI and 2.5 U
NlaIII, respectively. The Met/Leu229 and Lys/Asn257 variants
were detected by RFLP-generating PCR using sense primers
5'-tatcgctggattttgcgcaacagcc-3' and 5'-aagtgtgcccaactgtgcaatgc-3',
respectively, and antisense primer (Table 2) and digestion with
NlaIII, as described. All fragments were resolved on a 3%
agarose gel and visualized by staining with ethidium bromide
Screening for ag nucleotide polymorphism of 5'-flanking
region
PCR amplification of the 5'-flanking region of the UCP1 gene was
carried out as described above (except for 2.5 mM
MgCl2) using sense primer: 5'-cttgggtagtgacaaagtat-3' and
antisense primer: 5'-ccaaagggtcagatttctac-3'. Conditions were as
described for the amplification of exons except for annealing at 55 C.
The ag polymorphism was detected after digestion with 2 U
BclI for 2 h. Fragments were resolved on a 3% agarose
gel.
Statistical analyses
-Square analysis and Fishers exact test when appropriate were
applied to test for significant differences in allele frequencies.
Differences in continuous variables between groups of subjects were
tested with Student’s t test when the distribution of the
variable or of the logarithmically transformed variable approached a
normal distribution, and the variances of the variables were equal in
the groups compared. Otherwise the Mann-Whitney rank sum test was used.
Data are medians (interquartile ranges). A P value <
0.05 (two-tailed) was considered significant. Statistical Package of
Social Science (SPSS) for Windows, version 7.0 was used for statistical
analysis.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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SSCP-heteroduplex scanning of the coding region including
exon-intron boundaries as well as the nontranslated region of exon 1 of
the UCP1 gene revealed seven nucleotide substitutions of which five
were located in the coding region and two in the nontranslated region
of exon 1 (Table 3).
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Two subjects with juvenile-onset obesity and one control subject had the Arg/Trp40 mutation on one allele. The Val/Met137 and Lys/Asn257 mutations were each found in one subject with juvenile-onset obesity (both were heterozygous carriers) and in none of the control subjects. With the exception of the Lys/Asn257 carrier, in which BMI increased from 19.4 kg/m2 to 50.8 kg/m2 from age 18 to 40 yr, whereas the median BMI of the obese wild-type carriers increased from 18.1 kg/m2 to 34.5 kg/m2 in the same follow-up period, the BMIs at age 7, 18, and 40 yr did not differ between homozygous or heterozygous carriers of a variant and wild- type carriers either within the obese cohort or within the cohort of control subjects (data not shown).
In the cohort of young healthy subjects, the allelic frequencies of the
Ala/Thr64 and Met/Leu229 variants were 8.7% (6.6–10.8%) and 10.4%
(8.2–12.6%), respectively. The genotypes observed in this cohort were
also in Hardy-Weinberg equilibrium. There was no association between
genotype and anthropometric or biochemical variables related to
obesity, weight gain during childhood or adolescence, or insulin
sensitivity for any of the polymorphisms examined (Table 4).
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We also genotyped 379 (of 380) young healthy subjects for
the previously reported ag nucleotide polymorphism in the 5'
flanking region of the UCP1 gene. The allelic frequency of this variant
was 25.3% (22.2–28.4%). The genotypes were in Hardy-Weinberg
equilibrium. There was no association between genotype of the
polymorphism and the variables listed, in particular no association
with BMI, fat mass, or weight gain during childhood and adolescence
(Table 5).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-helices of the protein (17) (Fig. 1). Furthermore, Thr (the mutated allele)
at codon 64 as well as Leu (the mutated allele) at codon 229 of the
UCP1 gene are conserved among rat, hamster, mouse, and rabbit (Fig. 1),
which suggests a potential for a functional importance of these
residues in rodents in which the UCP1 protein plays a critical role in
regulating body fat stores.
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). Furthermore,
both mutations are located within a mitochondrial carrier protein motif
of the UCP1 gene (Fig. 1) (18). Although the BMIs of the obese carriers
of the Arg/Trp40 and Val/Met137 mutations did not differ from the obese
wild-type carriers, the effect of these amino acid replacements is
unknown and needs to be examined directly. Also, segregation studies in
families may provide information on these variants. Similarly, any
functional impact of the Lys/Asn257 mutation, which was found in one
obese subject, needs to be examined directly. This mutation resides at
codon 257, which is not conserved in rodents or humans; on the other
hand, the mutation is located close to the putative nucleotide binding
site in the C-terminal region of UCP1 (Fig. 1). Furthermore, the obese
carrier of the Lys/Asn257 mutation showed a pronounced weight gain
between age 18 and 40 yr. In the present investigation we also demonstrated that the DNA polymorphism of the 5'-flanking region of the UCP1 gene is present in young healthy Danish subjects with an allelic frequency similar to that observed in unrelated Canadian subjects of French ancestry (12). The major finding of the Canadian study was a higher frequency of the rare allele of the nucleotide polymorphism among high gainers for percent body fat over a 12-yr period in adolescence compared with low gainers. In the present study, the polymorphism did not show any association with BMI, fat mass, waist-to-hip ratio, or weight gain during childhood or adolescence. The two studies are, however, not fully comparable, because the mean ages and the follow-up periods are different. Furthermore, measures of percent body fat in the present study were not available during the follow-up period. Nevertheless, our results suggest that this nucleotide polymorphism is not a marker of obesity in the Danish Caucasian population.
In conclusion, although we cannot exclude an effect of the rare mutations in the UCP1 gene on susceptibility to juvenile-onset obesity, the present study indicates that variation of the coding region of the UCP1 gene is not a common factor contributing to the development of obesity in Danish subjects. Recent studies have identified two new members of the UCP family, UCP2 and UCP3. Both of these proteins are supposed, as UCP1, to have mitochondrial uncoupling activity. Human UCP2 has a wide tissue distribution (18), whereas human UCP3 is preferable expressed in skeletal muscle (19, 20). Thus, mutational studies of the genes encoding these proteins are relevant and might provide clues as to the understanding of risk factors for the development of obesity in humans.
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Acknowledgments |
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Footnotes |
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Received June 13, 1997.
Revised August 11, 1997.
Accepted August 26, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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