Spontaneous single-gene loss-of-function mutations
The genetically obese ob/ob mouse is a classic case of a spontaneous single-gene loss-of-function mutation that generates massive obesity. Characterizing the genetic basis of this mutation revealed that the defect is a single base pair deletion, which results in a premature stop codon in a gene expressed almost exclusively in adipocytes. The gene product was called leptin (1). The gene is recessive, but mice that are heterozygotic (i.e. with one copy only, rather than two) for the defect produce reduced amounts of leptin and are moderately overweight. There are now at least 10 known single-gene loss-of-function defects that cause massive obesity and have been completely genetically characterized. In all the cases where such defects have been discovered, they have initially resulted from spontaneous mutational events in large breeding establishments.
Discovery of the defect in the first place has been down to sharp-eyed observers noticing an abnormal obese phenotype (a phenotype describes the observable physical and biochemical characteristics of an individual) and then selectively breeding to expose the responsible gene in homozygotes (which have two copies of the gene). In some cases, the gap between discovery of the original phenotype and its subsequent genetic and physiological characterization has been very long, for example, in the case of leptin, about 50 years. These delays were largely down to the fact that, prior to the 1980s, the tools were not available to clone genes and sequence them. The development of high-throughput sequencing capability, and the completion of the mouse and rat genomes in the early part of the new millennium, means that such delays between the discovery of a new mutant and its characterization are likely to get progressively shorter.
Nevertheless, there is an inherent randomness in the discovery of single-gene defects, which depends primarily on unusual animals being identified in routine colony checks. This means that genes with only minor effects on the heterozygote are unlikely to be discovered. This ability to only detect genetic defects that have major loss-of-function effects in heterozygotes means that, almost by definition, these defects will only affect a minor proportion of the population. Consequently, while the discovery of the ob/ob mouse and the leptin gene was a major leap forwards, genetic screens of the human population have revealed trivially small numbers of obese individuals that have loss-of-function mutations in this gene (2). The same is true for all the other genes that have been discovered as spontaneous single-gene defects and characterized genetically and functionally (3).
The real progress that study of these genes allows is to further our understanding of how the energy regulation system works. In fact, many of the genes that appear important in single-gene mutation events seem to be involved in a common pathway that includes leptin and insulin as signalling molecules (4). Our knowledge of this pathway has been crucially informed by characterization studies of spontaneous single-gene loss-of-function defects.
Artificially generated single-gene loss-of-function mutations
The arbitrary nature of relying on spontaneous mutational events resulting in major loss-of-function mutations of critical genes has led to attempts to accelerate the process by increasing the mutation rate artificially. This is performed by treating animals with mutagenic chemicals, or exposing them to radiation (5–7). Several countries have programmes of research that include such artificial mutagenesis studies (e.g. Gailus-Durner et al. (8)). The animals generated from these experiments may inform not only our understanding of energy regulation but many other aspects of animal function.
The major problem with this approach is the cost of phenotyping. As the mouse genome consists of around 30 000–40 000 genes and that a given mutation may only produce a loss-of-function effect in say 3–5% of cases, to discover the effect of loss of function in a given gene might require phenotyping over half a million animals. Just measuring body weight of animals on a single occasion in these sorts of numbers would be prohibitively expensive. Moreover, as with spontaneous genetic mutations, the effects still need to be present and of considerable effect in heterozygotes. Little progress has therefore been made from these studies in the context of understanding energy regulation.
An enormous number of transgenic models with obese or lean phenotypes have been created since the characterization of the first obesity genes (9,10). The 2005 update of the human obesity gene map cited 248 genes that, when mutated or expressed as transgenes in the mouse, result in phenotypes that affect body weight and adiposity (3). With traditional transgenic technologies, there was little control over where or how many copies of genes were introduced into the genome. However, current sophisticated gene-targeting strategies permit investigators to manipulate the genome in ways that essentially allow the introduction of virtually any desired change. Furthermore, advanced techniques allow alterations to the genome that act only at specific times, or that are expressed only in specific tissues or cell types (11).
Over-expression of target genes was the first widely used technique. The full-length coding sequence of the gene is cloned downstream of a promoter, which may provide global, or tissue-specific expression, resulting in transgenic offspring over-expressing the target gene. Although relatively straightforward and inexpensive, the level of gene and protein expression does not always generate a physiological effect. More predictable and reproducible than over-expression models are global-knockout models. Here, the phenotype is created through total ablation of the target gene in all tissues. These knockout models have often resulted in unpredicted effects of the target genes, which in some cases allow an unexpected insight into the action of the target. An example of an unexpected insight developed from a transgenic knockout is the axl mouse, originally developed to determine whether the tyrosine kinase receptor, axl, played a role in leukaemia, was found by to be associated with non-insulin-dependent diabetes mellitus.
In addition to global and tissue-specific knockouts, transgenic work has also involved ‘knock-in’ models, that is, replacing the endogenous gene with a mutated form. Knock-in mice have the ability to address more specific roles and can be used to determine the effects of subtle changes in protein structure or function. For example, the s/s mice created by Bates et al. were developed to investigate leptin signalling by introducing a knock-in mutation that disrupted the dominant intracellular signalling pathway through which leptin was believed to have its major action (the STAT3 pathway) (12).
An obvious problem with conventional transgenic knockouts is the potential that manipulating the gene may result in early embryonic death. A less obvious problem is that a genetic manipulation that acts over the whole life may be compensated for during the period of development. So, what is normally a key gene in the process of energy regulation may appear to have little importance when it is knocked out because its action has been taken over by compensatory mechanisms. A case in point is the neuropeptide Y (NPY) gene. When introduced directly into the brain, this neuropeptide is one of the most potent stimulators of feeding behaviour. When NPY was knocked out, however, the resultant mouse had no obvious abnormal phenotype (13–15).
The Cre/loxP system is a tool for tissue-specific and time-specific knockout of target genes, which permits investigation of such genes. This system involves two separate transgenic lines, one expressing Cre recombinase (Cre) and the other in which Cre recombinase recognition (loxP) sites are strategically positioned at either side of the target gene. When Cre is expressed in mice harbouring a loxP-containing target gene, the desired gene is excised. Depending on the tissue specificity and timing of recombinase expression, these modifications can be restricted to certain cell types or developmental stages (16). An example of the Cre/lox system is its use to create mice with disruptions of the insulin receptor in fat tissue (the FIRKO mouse) (17). The FIRKO mouse has a low-fat mass, loss of the normal relationship between plasma leptin and body weight, and appears protected against age-related and hypothalamic lesion-induced obesity.
It is clear that in most circumstances, obesity, and its related energetic precursors, are polygenic traits, resulting from the combined actions of many genes. In the light of this, several research programmes have turned to the study of the polygenic basis of obesity, and many of these have employed animal models. Although an animal’s phenotype is often easily measured, identifying the genes underlying this trait can be a laborious process. Compared with human studies, however, using animal models is often faster as enhanced techniques and some destructive methods (i.e. full body dissection) allow more accurate description of the phenotype. In addition, the power to detect quantitative trait loci (QTLs) using model animal species is improved because of the larger family size and formalized pedigree structure. Although several animal models have been used, such as rats (18), chickens (19) and pigs (20), the mouse is the most common genetic model species and recent advances in determining the mouse genetic map has driven their increased use. Currently, the molecular marker map for mouse consists of over 6500 PCR-based microsatellite markers (21). There are also a large number of different mouse populations, which offers a wide range of alternative genetic variances, and their short breeding cycle combined with a large litter size facilitates rapid proliferation of generations, which is necessary for inbreeding and selected line studies.
In the search to locate regions of the genome responsible for polygenic obese phenotypes, QTL mapping is a valuable tool that screens an organism’s genome for statistical associations between phenotypic and marker information. To date, over 200 obesity QTLs have been located in the mouse, although many of these are at the same chromosomal region and may therefore reflect the same gene(s) (22). A key advantage of this approach is that the technique requires no prior knowledge concerning the biological nature of the trait under examination (23). Obesity-related traits that have been divergently selected over many generations include body mass (24–26), body composition (27,28), food intake (27), heat loss (29,30) and spontaneous activity (31). A detailed list of those where QTL mapping has occurred is available in Brockmann and Bevova (32).
In addition to more traditional rodent models of obesity, there have been several studies that have used more exotic animal models. These models generally involve wild animals that undergo natural patterns of variation in their fat mass, for example, seals (33,34) and bats (35,36). In practice, the usefulness of such models is probably constrained in two ways. First, the tools to explore the genetic basis of such effects are a considerable way from being developed. Second, these animals pose significant challenges when it comes to establishing laboratory-based colonies that are amenable to investigation.
There are two areas, however, where such exotic models are probably of far more utility and will probably provide significant insights into aspects of the regulation of body fat storage. The first include studies of non-human primates. The second involves studies of ‘non-standard’ small rodents that undergo cycles of seasonally induced fat storage.
Separation of the primate and rodent lineages is a relatively ancient event (65–85 million years ago (37)). In contrast, the separation of the Hominoidea (humans and the other great apes) and the Cercopithecoidea (the Old World monkeys) occurred relatively recently (about 25 million years ago (38)). So, Old World monkeys (such as macaques, rhesus monkey and baboons) may provide a genetically more appropriate model for studying human obesity (39,40). It has been demonstrated that 10–15% of captive macaque and rhesus monkeys develop age-related obesity when maintained on a relatively low-fat (10% of energy) ad libitum diet (41). Interestingly, the reduced locomotor activity arising from caging appears not to be a key factor contributing to obesity in monkeys.
Seasonal models of obesity
Many small mammals exhibit annual cycles of reproduction that are accompanied by variations in body mass and adiposity (42–47). Many species (e.g. hamsters Phodopus sp. and Mesocricetus sp., voles Microtus sp. and lemmings Dicrostonyx sp.) rely on environmental cues such as increasing or decreasing day lengths to trigger these events (45,48,49). An advantage of this system is that the responses to photoperiod can be mimicked in the laboratory by acutely transferring animals between long-day (LD) and short-day (SD) photoperiods. This makes hamsters, voles and lemmings attractive study species for investigating the mechanisms underlying the regulation of body mass (45,50).
Although these robust rhythms of adiposity occur in a variety of mammalian species, they have been studied most extensively in the Siberian or Djungarian hamster (Phodopus sungorus), Syrian or golden hamster (Mesocricetus auratus) and the collared lemming (Dicrostonyx groenlandicus). However, seasonal changes in adiposity linked to photoperiod have also been demonstrated in several other species (51–55), which may also provide useful models. One species that has recently received considerable attention as a potential model of seasonal obesity is the field vole (Microtus agrestis), which is a non-hibernating arvicoline rodent. Cold-acclimated adult male voles transferred from SD (8-h light) to LD (16-h light) undergo a substantial increase in adiposity (56), which depends on photoperiod-triggered leptin resistance (57).