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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online November 1, 2002 as doi:10.1096/fj.02-0111fje. |
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Institute of Cell, Animal and Population Biology, University of Edinburgh, UK;
* Fachhochschule Lippe, University of Applied Sciences, Lemgo, Germany;
Biotechnical Faculty, Zootechnical Department, University of Ljubljana, Slovenia;
Aberdeen Centre for Energy Regulation and Obesity (ACERO), Department of Zoology, University of Aberdeen, UK;
ACERO, Division of Appetite and Energy Balance, Rowett Research Institute, Bucksburn, Aberdeen, UK; and
|| Animal Science Complex, University of Georgia, Athens, Georgia, USA
2Correspondence: Institute of Cell, Animal and Population Biology, University of Edinburgh, West Mains Road, Edinburgh, EH9 3JT, UK. E-mail: Lutz.Bunger{at}ed.ac.uk
SPECIFIC AIM
Long-term divergent selection in mice on fatness for > 60 generations produced a fat (F) and a lean (L) line. These lines are a unique resource for dissecting the genetic basis underlying line divergence, elucidating genes causing the variation in fatness in unselected mouse populations and via sequence homology in other species, including humans. The objective of this experiment was to elucidate the importance of the leptin regulatory feedback loop in the genetic changes produced by this selection. The recessive mutations Lepob and Leprdb, causing leptin production and leptin receptor deficiency, respectively, were introgressed separately by repeated backcrossing into the F and L lines. Mating of heterozygous mice was set up to generate families segregating for all three genotypes. Thus, we had available F-line mice, designated Fob/ob, Fob/+, Fdb/db, Fdb/+, that were homozygous or heterozygous for the respective mutation, and wild-type F+/+ mice. The same groups were available for the L line. To investigate the effects of both deficiencies on the line differences, we assessed body fatness, circulating leptin, food intake, and body temperature and calculated energy budgets in these segregating litters.
PRINCIPAL FINDINGS
1. Polygenic fat and lean mouse lines developed by divergent long-term selection are useful models for study of the polygenic basis of obesity
Before this study, we developed polygenic fat and lean lines of mice by > 60 generations of divergent long-term selection on fatness at 70 and 98 days of age. Two highly divergent lines were generated: the fat line (F) and the lean line (L), with 
22% and 4% total body fat in males, respectively. The heritability for fatness was estimated at 0.5, which is near the average of values from selection experiments for body composition in rodents, confirming that body fatness has a strong genetic basis. Since in human populations the genetic basis of obesity is predominantly polygenic, our mouse lines present a potential good model for uncovering a molecular genetic basis for obesity in humans.
2. The line divergence in amount of fat produced by long-term selection is independent of leptin production and receptor deficiencies, indicating that regulatory mechanisms largely independent of the leptin system are responsible for the genetic changes caused by selection
The female wild-type animals from F and L lines differed in body fatness at 106 days by on average 3.9 g (6.3 g fat for F+/+ and 2.4 g for L+/+) and the males by 7.2 g (8.5 g for F+/+ and 1.3 g for L+/+). In leptin receptor-deficient mice (Fdb/db and Ldb/db), the body fat content of both lines increased significantly (P>0.05), but the difference between the lines was preserved. In females, the fat amount was 5.4 g higher in Fdb/db than Ldb/db; in males, amount of fat was 4.5 g higher in Fdb/db than Ldb/db. Leptin production-deficient mice (Fob/ob and Lob/ob) were also fatter than the wild-type mice. The difference in fat amount between the lines was preserved in females (Fob/ob 4.1 g higher than Lob/ob) but not in males (Fob/ob 0.8 g higher than Lob/ob). Heterozygous mice (Fob/+, Fdb/+, Lob/+, and Ldb/+) had body fat content not significantly different (P>0.05) from the wild-type mice; differences between the lines were preserved in each case. The differences between Fob/+ vs. Lob/+ were 4.9 g (females) and 5.8 g (males); differences between Fdb/+ vs. Ldb/+ were 5.4 g (females) and 4.5 g (males). Similar results using uncorrected fat amounts were obtained when fat-free body weight (ffBW) was used as a covariate (Fig. 1
). This analysis accounts for differences in body weight and avoids the automatic ‘part-whole regression’ of fat weight on total body weight.
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The above results strongly suggest that the causal genetic differences between the F and L lines are in genes that work independent of the leptin regulatory system. Of eight comparisons in both sexes between F and L genetic backgrounds with introgressed Lepob or Leprdb mutation, the difference in fat amount observed between F+/+ vs. L+/+ was preserved in seven cases. Regression of fat (g) on ffBW (g) was significantly (P<0.05) affected by line, being much higher for F than L, but not by degree of fatness (data not shown). Consequently, there is a clear difference between the lines in the relationship between the amount of fat and ffBW. Taken together, this suggests that body fatness in F and L lines is under the control of a regulation system that is independent of leptin production and reception.
3. The overall patterns of variation in leptin production occurred independent of the F and L lines, pointing to an independent regulatory system controlling the genetic line difference in fatness
If leptin values are expressed as circulating levels per gram of body fat tissue, the lowest leptin levels (0.05–0.09 ng/mL/g fat) were observed in the homozygous leptin-deficient mice (Fob/ob and Lob/ob) of both sexes, as expected. Wild-type mice from F and L lines produced leptin at a level 10- to 20-fold greater than ob/ob animals, 0.3–0.9 ng/mL/g fat. Absolute circulating levels were higher in the F than in L animals, but differences did not reach statistical significance (P>0.05). L males produced significantly more leptin per gram of body fat than F males. Very high levels of leptin production were observed in the Fdb/db and Ldb/db mice (0.44 to 1.3 ng/mL/g fat), again not significantly different between strains in females but higher in Ldb/db than Fdb/db males (0.7 vs. 0.4 ng/mL/g fat). In F and L lines, the heterozygote ob/+ animals consistently, but without reaching statistical significance (P>0.05), produced a lower amount of leptin/g of body fat than wild-type animals (49–60% of wild-type in F and 80% in L), although body fatness was similar (above). db/+ animals had levels of leptin production at the same level as db/db homozygotes, notwithstanding the normalization of body fatness. Despite these heterozygote effects, the overall patterns of variation in leptin production occurred independent of the F and L lines, again pointing to an independent regulatory system controlling fatness in these lines.
4. The calculation of energy budgets suggests that the major difference between the L and F lines resides in differences in the amount of energy devoted to physical activity rather than differences in food intake and resting metabolic rate and that these differences are preserved when leptin production and reception deficiencies are introgressed
Energy budgets for the different genetic groups of mice using the recorded food intakes and resting metabolic rates were constructed. Food intake of the wild-type and heterozygote mice did not differ between the F and L lines (averaging 5.5 g/day in the fat line and 5.3 g/day in the L line). When leptin production and receptor deficiencies were introgressed, daily food intake increased by 2.5–3 g and by 1.3–1.4 g, respectively, but there were no significant differences between L and F lines in the extent of increase after introgression. We converted these estimates of food intake into total daily energy demands using an estimated metabolizable energy content of 9.8 kJ/g for their diet. Resting metabolic rate (O2 consumption mL/min, RMR) in thermoneutrality was strongly dependent on body mass (F=13, P<0.05). We converted these RMRs to daily energy expended on RMR, assuming a respiratory quotient of 0.8. Mice from the F line were heavier on average in all conditions than mice from the L line, so total energy expenditure at rest was much higher in F vs. L line animals. Combining data obtained from females and males allows the preliminary calculation of a difference between the energy expended at rest and total daily energy expenditure, reflecting the combined energy expended on physical activity and thermoregulation. The ratio between the physical activity (PA) and thermoregulation is called the physical activity level (PAL). Total energy expenditure on PA and thermoregulation was much greater in the L line than the F line mice independent of the introgression; PAL was also much greater in the L (3.2 to 3.6) than the F line (1.6 to 2.5). These data on energetics strongly suggest that the major difference between the L and F lines resides in differences in their physical activity levels and expenditure on thermoregulation, rather than differences in food intake and resting metabolic rate, and that these differences are preserved when the leptin production and receptor deficiencies are introgressed. Consistent with this are the body temperature (Tb) data. For the L+/+ animals, Tb was higher than for the F+/+ mice, by 0.85°C (SE=0.87, P>0.05) in females and 1.32°C (SE=0.63, P<0.05) in males. When leptin production and reception were inactivated by introgression the Tb dropped in both lines by 2.5°C in females and by 0.9°C in males, but did not change significantly in heterozygotes. Differences between the L and F lines were preserved in all pairwise comparisons across genotype and sexes apart from Fdb/+ vs. Ldb/+, where the difference was negligible. Since introgression resulted in decreased Tb but the energy budgets were essentially similar, the indication is that the major difference between the F and L lines could be in physical activity rather than thermogenesis.
5. Polymorphisms in the Lepob and Leprdb genes with major phenotypic effects probably did not contribute to the difference between the F and L lines
Mutations with considerable effect in the Lepob and Leprdb genes seem not to segregate in the F or L line. 1) The outbred F line seems to approach a selection plateau with body fat% values of 20–23%. The introgression of defects in the leptin receptor and leptin production increased the body fatness to between 34 to 38%, indicating that introgression of both mutations brought in genetic variability that was not present before. 2) The effects of introgressing the leptin production and reception deficiencies were additive to the effects generated by the selection lines. 3) The four QTL found in an F2 cross between F and L did not map to regions of known single gene mutations for obesity, including Lepob and Lepr db.
CONCLUSIONS AND SIGNIFICANCE
The present and related experiments show that long-term selection can easily split a common base population into highly diverging lines, confirming that body fatness has a strong genetic basis. The F line derived here resembles in several respects typical westernized human populations where the genetic basis of obesity is predominantly polygenic; humans homozygous for loss-of-function mutation in genes identified as causal in monogenic rodent obesity are relatively rare. Major mutations in these loci do not segregate in the F and L lines.
Selection over > 60 generations has changed the frequency of ‘lean and fat’ alleles in both lines. Together with our previous studies of the effects of providing exogenous leptin to the F line, we have shown that this long-term selection response is independent of the leptin regulatory system. Energy budgets for the L and F lines show that major differences between the lines seem to reside in the energy devoted to physical activity, and not in food intake or resting energy demands. Since the dominant effect of leptin is on regulation of food intake, with secondary effects on resting energy expenditure, the independence of the line effects from the introgressions is consistent with the energy budgets.
These data support the notion that regulation of body fatness involves multiple regulatory systems that may act independently. The data point to a genetic basis underlying differences in physical activity energy expenditure that leads to differences in body fatness. Independent regulatory systems may serve as independent targets for intervention-based therapies (Fig. 2
).
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0111fje; to cite this article, use FASEB J. (November 1, 2002) 10.1096/fj.02-0111fje 
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) was maintained after introgression, leading to the acceptance of H0. Leptin production and receptor deficiencies had separate effects from long-term selection, indicating that the genes responsible for the line divergence must act independent of the leptin regulatory system. The construction of energy budgets indicated the major line differences were in level of energy expended on physical activity, and these differences were preserved independent of the introgressed mutations. These data strongly suggest that multiple pathways regulate fatness, which may be independently responsive to intervention.