Simopoulos AP, Childs B (eds): Genetic Variation and Nutrition. World Rev Nutr Diet. Basel, Karger, 1990, νο1 63, pp 90-101

Genetic Variation and Nutrition in Obesity: Approaches to the Molecular Genetics of Obesity1 Rudolph L. Leibel, Nathan Bahary, Jeffrey Μ. Friedman The Rockefeller University, New York, N.Y., USA

Definition of Obesity

1 This work was supported by NIH Grants DK30583 and DK 41096.

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Obesity is the most prevalent nutritional disturbance in modern western societies. Depending upon the diagnostic criteria employed, it has been estimated that as many as 20% of children and 30% of adults in the United States are obese [1]. In functional terms, obesity is a maladaptive increase in the size of the adipose organ relative to lean body mass. Optimal adipose tissue content is dependent upon a variety of factors which include age, health status, genotype and environment. There are clearly physiologic and environmental states — for example, early pregnancy and incipient famine — when it is advantageous for an individual to have extra calories stored as fat. In other circumstances, such as late gestation or in an environment of readily available food, this extra fat may actually constitute a health hazard. Thus, the definition of obesity is context dependent. A corollary of this argument is that this phenotype will be very sensitive to environmental and other nongenetic factors. As will be emphasized below, this very sensitivity of body composition phenotype to environment has confounded efforts to quantify the heritability of adiposity. For this reason the genetic study of body composition in mice has numerous experimental advantages. Body composition is determined by the balance between energy input and energy output. When input and output are of equal magnitude, body composition (degree of adiposity) remains constant. An imbalance be-

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tween energy intake and expenditure results in a gain or loss of stored energy (mainly as fat) until a new equilibrium position is achieved. The components of energy input (food intake) and energy output (resting metabolic rate, physical activity, thermic effect of feeding) are clear [2]. The cellular events which regulate these two aspects of energy homeostasis are poorly understood, and a major problem in the study of these systems is that their controls are interlocked is such a way as to make the experimental disarticulation of these processes extremely difficult. For example, a decrease in food intake to the point of weight loss is accompanied by a decline in resting metabolic rate whose magnitude exceeds that explicable by the loss of body mass [3].

There has been an ongoing debate concerning the respective relative contributions of genotype and environment to obesity in humans. Clear examples exist in mice and rats of obesity transmitted by single autosomal recessive genes (e.g. db, ob, fa), single dominant alleles (AY), and by `polygenes' (e.g. IZO) [4]. In no instance is the specific biochemical `lesion' underlying any of these genetic obesities known. The situation in man is a great deal more complex, with obesity reflecting the complex interaction of genotype with shifting environmental forces. Studies of twin pairs as well as adopted children have been performed in an effort to quantify the relative contributions of genotype and environment to body composition [5]. Comparisons of body composition between mono- and dizygous twins suggest that as much as 80% of the variance in skinfold thickness or weight-for-height may be attributable to genotype [6, 7]. The calculation of heritability (Η2) in such studies assumes comparable similarity of environment between mono- and dizygous twin pairs. Such an assumption is not necessarily valid [8]. In adoption studies, the body compositions of adopted children are compared to those of their biologic and adoptive parents. Such studies have found significant correlations of adiposity between female adoptees and their biologic but not adoptive mothers [9]. Other investigators, however, have failed to find differential correlations of skinfold thickness between adoptees and their biologic and adoptive parents [l0]. In the aggregate, such studies suggest that there is a substantial genetic influence on body composition and fat distribution [11] in both children

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Genetics of Human Obesity

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and adults. Efforts to identify the defects responsible for obesity in humans have been impeded by experimental difficulties in precisely quantifying food intake and energy expenditure, as well as the apparent polygenic control of body composition in man. These considerations have stimulated interest in the simpler models of obesity occurring in certain inbred animal strains, particularly mice.

The mouse has a number of distinct advantages over other mammals as a model for studying human diseases: (1) a fast breeding rate (1 litter every 6-8 weeks); (2) large average litter size (5-7 offspring), and (3) availability of highly inbred strains, ensuring genetic homogeneity within strains. Many mouse mutant models of human disease are available, spanning the range of physiological abnormalities from obesity and diabetes to neurological and immunological disturbances. Cloning genes whose phenotype is known, but whose protein product is unidentified (so-called `reverse genetics'), is simpler in mice than in humans because the mutant allele can be `fixed' on a homogeneous genetic background and large genetic crosses can be generated to permit fine mapping of the gene relative to other genetic markers such as restriction fragment length polymorphisms (RFLPs; see section `Use of RFLPs'). Additionally, mice with defects in specific genes can be created by homologous recombination techniques which direct DNA insertions to specific regions of the genome of mouse embryo-derived stem cells. These stem cells (ES) are then injected into mouse blastocysts to generate germ-line chimeras [ 12]. Thus, mouse models of human diseases for which a portion of the relevant human genomic sequence is available may be created and studied. To date, more than 1,000 loci (mainly RFLPs) have been positioned on the mouse genome [13] (compared to nearly 4,000 in man) [14]. During the past 5 years the direct mapping of gene sequences by RFLP analysis has greatly increased the diversity of markers on the mouse genetic map. Because RFLPs are both genetic and physical markers, the availability of a dense genetic map of RFLPs greatly accelerates efforts to clone mutant genes by providing information regarding the precise position of the mutant gene on the chromosome. Among the mutant loci which have been mapped in mice are several forms of genetically transmitted obesity and diabetes. Autosomal domi-

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Mouse Models of Obesity

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nant variants in mouse include the yellow obese mouse (Ay, A°y, AiY) (chromosome 2), and the adipose (Ad) (chromosome 7) mouse. Autosomal recessive mutants include fat (fa), tubby (tu, chromosome 7), obese (ob, chromosome 6) and diabetes (db, chromosome 4) and its related alleles. Polygenic inheritance is responsible for the obesity noted in certain strains of pigs (Large White and Berkshire), rat (Osborne-Mendel) and mouse (ΝΖΟ, KK, and `Wellesley'). The coexistence of hyperphagia, enhanced metabolic efficiency, and hyperinsulinemic diabetes mellitus in homozygous ob and db mice makes these animals particularly interesting models relative to human obesity.

The ob mutation in mouse was originally described in 1950 by Snell and co-workers [15] as a recessive mutation that arose spontaneously on a noninbred mouse and which was subsequently transferred to the inbred mouse strain C57BL/6J. Animals homozygous for the mutation are easily distinguishable as obese by several weeks of age, and demonstrate glucose intolerance and diabetes, whose severity depends upon the background strain upon which the mutant allele is engrafted [ 16]. The ob gene was localized to proximal chromosome 6 using classical genetic linkage studies. Since then, numerous studies [ 17] have characterized many of the biochemical, physiologic and behavioral aspects of the ob/ob animal, but the primary genetic defect in ob/ob mice remains unidentified. Diabetes (db) is an autosomal recessive mutation located on chromosome 4 which arose spontaneously in the C57BL/KsJ (BL/Ks) strain [ 18]. At least five separate spontaneous mutations allelic to db — db2J, dbad, db3", db4J, and dbpasreur, all with identical phenotypes, have been described [ 16]. Homozygous db/db mice exhibit a diabetes-obesity syndrome, which like ob, varies in severity depending upon the background strain onto which the mutant allele is bred. The db/db phenotype, including susceptibility to diabetes, is similar to the phenotype of ob/ob mice on every inbred strain of mouse that has been tested [ 19]. The earliest phenotypic evidence of homozygosity for both ob and db is enhanced fat deposition by 2 weeks of age, despite a normal energy intake. Hyperphagia and elevated plasma [glucose], [insulin] and triglycerides can be seen at 4 weeks of age accompanying an almost 3-fold increase in the content of carcass fat. Ob/ob and db/db animals not only consume

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The ob and db Mutations

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more calories than their lean littermates, but pair-fed obese animals deposit a higher fraction of ingested calories as fat than do their lean littermates [18]. Homozygous animals also demonstrate an impairment in heat-generating capacity, which causes them to become hypothermic in a cold environment [20]. Other metabolic disturbances noted in db/db and ob/ob mice include infertility, hyperinsulinemia, hyperglycemia, and a diminished metabolic rate. Many of the relevant endocrine and autonomic axes are coordinately controlled in the hypothalamus, leading some investigators to suggest that the primary defect in these mutants is located in the hypothalamus [ 17]. Neel [21 ] suggested that the harsh environment in which our forbears evolved may have conferred a survival advantage on individuals of high metabolic efficiency (`thrifty genotype'). The positive selection pressure on the relevant genes, and their relative overrepresentation in modern populations, may lead to a predisposition to obesity and diabetes in environmental circumstances characterized by ready access to calorically dense foods. Coleman has reported that ob/+ and db/+ animals survive a fast 2-3 days longer than their +/+ littermates [22], and that this advantage may be due to enhanced rates of conversion of ketones (acetone) to gluconeogenic precursors (lactate) [23].

As noted, the genetic `background' (strain) onto which these mutant alleles have been bred has a potent effect on the phenotype of these mutations. Homozygosity for the ob or db genes on the C57ΒL/Κs background results in hyperphagia, polydipsia, polyuria, hyperinsulinemia, sustained hyperglucagonemia, severe hyperglycemia, and peripheral insulin resistance. The concentration of plasma insulin is elevated by a factor of approximately 10 by 2-3 months of age. Necrosis of islet beta-cells, insulopenia, and hyperglycemia occur by 5-8 months of age, resulting in the death of the animal [18]. On the C57ΒL/6J background, both mutations result in massive obesity, but the severity of the accompanying diabetes mellitus is greatly diminished compared to the mutant phenotype on the C57ΒL/Κs background. Homozygous C57ΒL/6J db/db animals demonstrate hyperglycemia with compensating beta-cell hypertrophy and only a mildly reduced life span [ 19]. Because inbred strains differ in their susceptibility to the diabetes-obesity syndrome produced by homozygosity for

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Effects of Background Genome

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the ob and db alleles, examination of the distribution of metabolic phenotypes of offspring from inter-strain crosses permits estimates of the number of background genes influencing the mutant phenotype. Used in conjunction with chromosome-specific RFLP's between the strains employed, such a strategy may permit the assignment to specific chromosomes of the phenotype-influencing genes. Since the db/db and ob/ob phenotypes are indistinguishable in mice of identical genetic background, it is possible that the two genes may code for components/regulators of a single biochemical or regulatory pathway. Parabiosis experiments in which animal pairs exchange blood through a capillary anastomosis (- 3% of cardiac output), suggest that ob animals are missing a circulating satiety factor that suppresses appetite, and that db animals are missing the receptor for that factor [ 18].

The obese phenotype of the ob/ob and db/db animal itself exerts powerful and experimentally confounding effects on those metabolic and neural pathways which are regulators of energy homeostasis. As noted, these considerations have — to date — made it impossible to identify the primary biologic mechanisms responsible for obesity. In order to circumvent these problems, we are attempting to isolate the ob and db genes, utilizing the technique of `reverse genetics'. An important aspect of this approach is that it requires no knowledge of, or assumptions concerning, the biological nature of the defect being pursued. The technique has been used successfully to clone the genes for Duchenne's muscular dystrophy [24], chronic granulomatous disease [25], and `testis determining factor' [26], and has provided the foundation for physical maps (and ultimate cloning) of the regions surrounding the loci for cystic fibrosis [27]. Huntington's chorea [28], and Wilms' tumor/aniridia [29]. Before the advent of modern molecular biological techniques, the ability to map mammalian genes was limited to those genes which encoded a recognizable physical or biochemical phenotype which could be used to discriminate one individual's genotype from another's. By analyzing suitable genetic crosses, cosegregating phenotypes were assigned to a `linkage group'. By creating an overlapping set of such linkage groups, maps were created which, with occasional exceptions, contained few physical landmarks. Such landmarks were obtained primarily by: (1) analysis of those

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Strategy for Molecular Cloning of Mouse Mutations

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phenotypes associated with a chromosomal deletion and (2) hamster/human hybrids which contained a limited amount of stably propagated human DNA encoding an expressed protein which could be distinguished from its hamster analogue. Using these techniques, occasional genes (and their linkage group) could be localized to a specific chromosome. These procedures, however, did not allow the positioning of many `markers' on the genome, since most genes do not have an associated phenotype that can be tracked. Deisseroth et al. [30] first used a DNA marker to assign a gene to a specific mammalian chromosome. A cDNA sequence for the alpha-globin gene was localized to human chromosome 16 by hybridization to a panel of somatic cell hybrids containing known human chromosomes. The technique of somatic cell hybridization is cumbersome and does not position probes with sufficient physical accuracy to generate detailed genetic maps of a mammalian genome.

In 1980, Botstein et al. [31 ] proposed a method whereby RFLPs could be used as genetic markers. Restriction enzymes are sequence-specific endonucleases which `recognize' specific nucleotide sequences in DNA and incise (`restrict') the DNA within that sequence or at a point nearby. These enzymes `recognize' DNA sequences ranging in length from 4 to 8 nucleotides, and thus will cut, theoretically, at intervals of 44 (256) to 48 (65,536) base pairs. The actual frequency of restriction (which may be as slow as 1/106 base pairs) is dependent on regional variations in the abundance of G-C nucleotide pairs, and their methylation pattern. Only a small portion of genomic DNA actually codes for protein. In the relatively vast amount of noncoding (intronic) DNA, sequence differences amongst individuals occur as often as every 50-100 base pairs [31]. This high degree of variability in sequence generates interindividual differences in the frequency and location of restriction sites. Such sequence `polymorphisms' (RFLPs) can be detected by Southern blotting of enzymecleaved, size-fractionated genomic DNA using labelled cloned DNA as probe. Historically, such probes have usually been nucleotide sequences coding for all or part of a known gene. However, any unique sequence for which a polymorphism is demonstrable can be used. In fact, some of the most valuable sequences in this context are so-called `variable number

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Use of RFLPs

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tandem repeats' (VNTR's). The size variability of these loci results from their constitution as sets of tandem repeats of short oligonucleotide sequences (11-60 bp). The resulting size variability in DNA restriction fragments produces a highly heterozygous polymorphism which can be used in linkage analysis [32]. The elegance of the RFLP-linkage technique resides in the fact that potentially many thousands such RFLP's with high heterozygosity exist in man, and can be exploited using otherwise classical genetic techniques. The availability of genetic linkage maps consisting of DNA polymorphisms (typically RFLPs) greatly increases the analytic power of molecular genetics in higher organisms. Since hundreds of genetic markers can be scored in a single genetic cross it is easy to undertake high resolution genetic mapping of single gene traits, and it is feasible to carry out genetic dissection of polygenic traits into discrete Mendelian factors. For example, Paterson et al. [33], in an interspecific backcross of tomato, were able to map 6 quantitative trait loci (QTLs) controlling fruit weight, 4 QTLs for the concentration of soluble solids, and 5 QTLs for fruit pH, to a resolution of 20-30 cM (1 cM = 1 % recombination). Polygenic obesity in mice and humans could be similarly dissected when high resolution linkage maps of the mouse and human genomes are available. In addition, since the genetic marker loci are defined by cloned DNA fragments, they can be used as starting points for long range physical mapping, and for `chromosome walks' to clone the genes contributing to the obese phenotype [34, 35].

In the mouse, mapping has been carried out using inbred strains of the common house mouse, Mus musculus domesticus. Inbred mice are created by sequential brother-sister matings of wild mice which removes significant genetic heterogeneity between individual animals after a sufficient number of generations (usually 20 or more). Heterogeneity between inbred strains depends upon the timing of their evolutionary divergence as wild strains. The genetic or molecular mapping of mouse chromosomes has been greatly facilitated by the use of interspecific M. m. domesticus/Mus spretus crosses. The greater evolutionary distance between these two strains (— 1 million years) as compared to pairs of M. m. domesticus inbred strains, greatly increases the likelihood of heterozygosity (and hence RFLP poly-

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Intra- and Interspecific Crosses

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morphism) at any genetic locus [36]. The progeny of such an interspecific cross can therefore be more easily typed for the inheritance of many RFLPs to generate detailed genetic maps. This approach has already been used to generate fairly detailed genetic maps of mouse chromosomes 1, 12 and X. We have used an interspecific M. spretus cross to generate moderately detailed genetic maps of regions of mouse chromosomes 4 and 6, where db and ob, respectively, have been previously mapped. In order to understand the genetic basis of obesity at the molecular level, we are attempting the molecular cloning of two mouse mutations, ob and db, using the techniques of reverse genetics. We have generated 12 animals from intraspecific backcrosses segregating the ob and db mutations and have mapped a large number of probes relative to these mutations [37]. From the published maps of syntenic linkage groups between mouse and man, and the flanking markers described for ob and db, it appears that a human homologue to ob would be located on human 7q, near the cystic fibrosis locus, and that a human homologue to db would be found on either human 1p or 9p [37].

The mouse is also an excellent model for the examination of polygenic disturbances — such as obesity — in man. Just as described earlier for the tomato, the availability of a 5-10 cM map of the mouse genome will permit the delineation of the number and approximate location of alleles influencing continuous quantitative traits such as body composition. The high degree of linkage and synteny homology between mouse and human genomes [38] suggests that phenotype-linked sequences obtained from mouse will be `mappable' relative to the analogous phenotype in human pedigrees. Thus, genes (or linkage relationships to RFLP's) identified in controlled mouse crosses may provide insights into the genetic interactions which dictate human phenotypes, and can provide invaluable reagents for the isolation of relevant human genomic sequences. The mouse is thus uniquely suited as a model system for studying obesity, and for cloning and characterizing those genes which contribute to the obese phenotype. The similarity between the obesity syndromes produced as a result of mutations in the mouse and obesity in humans suggests that the normal human homologues of these mutant alleles may be playing an important role in the physiology of body composition in man. This

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Polygenic Models of Obesity

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possibility can be tested by scoring the inheritance of RFLPs from human chromosomes 7q (which is syntenic to ob) and human 1p and 9p (which appear syntenic to db) on human pedigrees segregating an obese phenotype. The cloning of the family of mutant mouse genes which cause obesity syndromes in mice, and studies of inter-strain background modifiers in the metabolic parameters altered by the obese phenotype in the inbred mouse, will undoubtedly provide insights into the metabolic control of body composition in man.

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Rudolph L. Leibel, MD, Rockefeller University, New York, NY 10021 (USA)

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Genetic variation and nutrition in obesity: approaches to the molecular genetics of obesity.

Simopoulos AP, Childs B (eds): Genetic Variation and Nutrition. World Rev Nutr Diet. Basel, Karger, 1990, νο1 63, pp 90-101 Genetic Variation and Nut...
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