Brief Genetics Report Quantitative Trait Loci for Obesity- and Diabetes-Related Traits and Their Dietary Responses to High-Fat Feeding in LGXSM Recombinant Inbred Mouse Strains James M. Cheverud,1 Thomas H. Ehrich,1 Tomas Hrbek,1 Jane P. Kenney,1 L. Susan Pletscher,1 and Clay F. Semenkovich2 Genetic variation in response to high-fat diets is impor- tant in understanding the recent secular trends that have led to increases in obesity and type 2 diabetes. The he prevalence of obesity and its correlates, such examination of quantitative trait loci (QTLs) for both as type 2 diabetes, has increased greatly over the obesity- and diabetes-related traits and their responses last 20 years (1). There has also been a concom- to a high-fat diet can be effectively addressed in mouse Titant change in the age of onset for type 2 model systems, including LGXSM recombinant inbred diabetes, with increasing diagnoses of this disease in (RI) mouse strains. A wide range of obesity- and diabe- children, adolescents, and young adults (2). These secular tes-related traits were measured in animals from 16 RI changes in obesity and diabetes are not due to genetic strains with 8 animals of each sex fed a high- or low-fat changes in populations (3), rather they are due to environ- diet from each strain. Marker associations were mea- mental changes in nutrition and activity over time. Even sured at 506 microsatellite markers spread throughout so, there is genetic variation in humans in the response to the mouse genome using a nested ANOVA. Locations with significant effects on the traits themselves and/or dietary and activity factors. Some individuals respond trait dietary responses were identified after correction strongly to an obesogenic environment by becoming for multiple comparisons by limiting the false detection obese, while other individuals remain lean when chal- rate. Nonsyntenic associations of marker genotypes lenged by the same environment (4). These variations in were common at QTL locations so that the significant response to environmental stimuli are, in part, due to results were limited to loci still significant in multiple genetic variations between people. QTL models. We discovered 91 QTLs at 39 locations. Study of differential response to dietary factors in also showed genetic human populations can be challenging because of the (31 ؍ Many of these locations (n effects on dietary response, typically because the loci difficulty in controlling or accurately recording diet over produced significantly larger effects on the high-fat diet. an extended period of time. Animal studies of the genetic Fat depot weights, leptin levels, and body weight at basis for differential response to dietary factors can play necropsy tended to map to the same locations and were an important role in attempts to understand the genetics responsible for a majority of the dietary response QTLs. involved in recent increases in obesity and type 2 diabetes. Basal glucose levels and the response to glucose chal- We have used the cross of inbred mouse strains LG/J and lenge mapped together in locations distinct from those affecting obesity. These QTL locations form a panel for SM/J over the past years to study the genetic architecture further research and fine mapping of loci affecting underlying complex traits (5), including growth (6,7), obesity- and diabetes-related traits and their responses obesity (8), and skeletal morphology (5). In continuing to high-fat feeding. Diabetes 53:3328–3336, 2004 studies we have shown that these parental strains vary in obesity- and diabetes-related traits and in responses to dietary fat (9,10). Recently, we documented significant genetic variation in dietary response for fat depot weights, From the 1Department of Anatomy and Neurobiology, Washington University School of Medicine, St. Louis, Missouri; and the 2Department of Medicine, liver weight, leptin levels, fasting glucose levels, and area Washington University School of Medicine, St. Louis, Missouri. under the glucose response curve in the LGXSM recombi- Address correspondence and reprint requests to James M. Cheverud, nant inbred (RI) strain set (11). Additionally, genetic Department of Anatomy and Neurobiology, Washington University School of Medicine, 660 S. Euclid Ave., St. Louis, MO, 63110. E-mail: cheverud@ variation in dietary response for obesity- and diabetes- pcg.wustl.edu. related traits was detected in the F16 generation of an Received for publication 23 July 2004 and accepted in revised form 25 August 2004. advanced intercross (AI) line of LG/J and SM/J formed Additional information for this article can be found in an online appendix at from the same F2 intercross source population as the RI http:// diabetes.diabetesjournals.org. strains used here (WUSTL:LG,SM-G14), where litters were FDR, false discovery rate; FPR, false-positive rate; QTL, quantitative trait locus. divided into high- and low-fat diet treatments (12). An AI © 2004 by the American Diabetes Association. line is a randomly bred population formed from the F2 3328 DIABETES, VOL. 53, DECEMBER 2004 J.M. CHEVERUD AND ASSOCIATES TABLE 1 solution for every gram of body weight. Additional glucose readings were Composition of high- and low-fat diets obtained 15, 30, 60, and 120 min after the initial injection. The graph of the glucose levels over the period of the test are summarized by the area under the Component High fat Low fat curve, which is used as a general measure of an animal’s response to a glucose challenge. Lower values indicate a more robust response to glucose. Strain- Energy from fat 42% 15% and sex-specific glucose tolerance curves were presented by Cheverud et al. Casein (g/kg) 195 197 (11). Sugars (g/kg) 341 307 At a later time, animals were again fasted for 4 h and anesthetized with Corn starch (g/kg) 150 313 sodium pentobarbital, and a terminal blood sample was obtained via cardiac Cellulose (g/kg) 50 30 puncture. Blood plasma was separated through centrifugation and analyzed Corn oil (g/kg) 58 for free fatty acids, cholesterol, triglycerides, leptin, and insulin. Internal organs (liver, spleen, heart, and kidneys) and fat depots (reproductive, renal, Hydrogenated coconut oil (g/kg) 7 mesenteric, and inguinal) were removed and weighed. Anhydrous milk fat (g/kg) 210 Genotypes are available for the RI strains at 506 microsatellite loci spread Cholesterol (g/kg) 1.5 across the mouse chromosomes with a marker every 2 or 3 cM (T.H., L.S.P., Kilojoules per gram 18.95 16.99 R. Alves de Brito, J.M.C., unpublished observations). Genotypes were scored as corresponding to the LG/J or SM/J parental alleles. Loci showing residual heterozygosity were scored as missing data. A list of the loci scored and their intercross of a pair of inbred strains and maintained at a genomic positions is provided in online appendix A (available from http:// reasonably large census size (N Ͼ 100). It is designed to diabetes.diabetesjournals.org). Genotypes for specific strains can be obtained allow fine scale quantitative trait loci (QTLs) mapping from our laboratory web site (http://thalamus.wustl.edu/cheverudlab/ using the additional recombinations accumulated over the projects.html). Statistical analysis. The data were first analyzed for QTL effects at each generations. These earlier results promise success in map- marker scored along the mouse chromosomes using the following nested ping QTLs in crosses of the LG/J and SM/J inbred mouse ANOVA model (17), lines. Here, we present QTL mapping of obesity- and ϭ␮ϩ ϩ ϩ ϩ ϫ ϩ ϫ ϩ diabetes-related traits and their responses to increased Yijklm sexi dietj genotypek sexi dietj sexi genotypek dietj ϫ ϩ ϫ ϫ ϩ dietary fat in the LGXSM RI strain set. While a few of these genotypek sexi dietj genotypek strainl(sexi ϫ ϫ ϩ traits have been subject to previous genome-wide QTL dietj genotypek) eijklm scans, many novel traits and dietary responses have not th where Yijklm is the phenotype of interest for the m animal of sex (i), diet (j), been mapped previously in our cross, including regional and genotype (k) in strain l, and ␮ is the constant. Strain is nested within the fat depots, leptin, cholesterol, free fatty acid, triglycerides, sex-by-diet-by-genotype interaction. The effects of sex, diet, strain, and their insulin, fasting glucose levels, and response to an intra- interactions were reported by Cheverud et al. (11) and do not vary substan- tially across the genome scan. The effects of interest here are those due to peritoneal glucose tolerance test. Regions identified here genotype and its interactions. A significant test for the genotype effect as carrying QTLs will be subject to further research and indicates that a QTL affecting the trait of interest resides near the marker. A fine mapping in the WUSTL:LG,SM-G14 AI line. significant diet-by-genotype interaction indicates that the effect of the QTL varies depending on the diet. Thus, this factor identifies QTLs affecting dietary response. A significant sex-by-genotype interaction identifies a QTL with RESEARCH DESIGN AND METHODS different effects in males and females. A significant sex-by-diet-by-genotype The LGXSM recombinant inbred line set was produced from the F2 intercross interaction indicates that a QTL with different effects on male and female of LG/J females with SM/J males obtained from Jackson Laboratories. Details dietary responses is present in the region. of the line formation and history are presented by T.H., L.S.P., R. Alves de Given interactions between genotype and other factors, we also performed Brito, and J.M.C. (unpublished observations). The parental strains were genome-wide mapping analyses on each sex and diet cohort separately using derived from separate selection experiments for high (LG/J) (14) and low the following nested ANOVA model, (SM/J) (15) adult body weight, respectively.