Hierarchical clustering of differential gene expression in the hypothalamus of non-recombinant B6.CAST-17.1-derived F₂ mice.

<p>Legend: Gene expression was measured in hypothalamus on day 2 of macronutrient selection diet using tag-based transcriptome sequencing (n = 12 individual libraries per genotype). Animals used in this experiment were either <i>cast/cast</i> (carbohydrate-preferring) or <i>b6/b6</i> (fat-preferring) within the subcongenic segment, and <i>b6/b6</i> across the rest of the genome. Genes were filtered based on ≥1.5-fold change and <i>P</i> value≤0.01 using the DESeq method. The resulting 55 differentially expressed genes are displayed on the vertical axis; individual animals are clustered by genotype on the horizontal axis. Genes whose expression changed significantly in animals with the <i>cast/cast</i> genotype (higher carbohydrate consumption), relative to <i>b6/b6</i> (higher fat consumption), cluster together as increased (red) or decreased (blue) cells, with gray representing no change. Brighter shades of red and blue reflect higher degrees of up- and down-regulation.</p

High-Resolution Mapping of a Genetic Locus Regulating Preferential Carbohydrate Intake, Total Kilocalories, and Food Volume on Mouse Chromosome 17

<div><p>The specific genes regulating the quantitative variation in macronutrient preference and food intake are virtually unknown. We fine mapped a previously identified mouse chromosome 17 region harboring quantitative trait loci (QTL) with large effects on preferential macronutrient intake-carbohydrate (<i>Mnic1</i>), total kilcalories (<i>Kcal2</i>), and total food volume (<i>Tfv1</i>) using interval-specific strains. These loci were isolated in the [C57BL/6J.CAST/EiJ-17.1<i>-(D17Mit19</i>-<i>D17Mit50)</i>; B6.CAST-17.1] strain, possessing a ∼40.1 Mb region of CAST DNA on the B6 genome. In a macronutrient selection paradigm, the B6.CAST-17.1 subcongenic mice eat 30% more calories from the carbohydrate-rich diet, ∼10% more total calories, and ∼9% more total food volume per body weight. In the current study, a cross between carbohydrate-preferring B6.CAST-17.1 and fat-preferring, inbred B6 mice was used to generate a subcongenic-derived F₂ mapping population; genotypes were determined using a high-density, custom SNP panel. Genetic linkage analysis substantially reduced the 95% confidence interval for <i>Mnic1</i> (encompassing <i>Kcal2</i> and <i>Tfv1</i>) from 40.1 to 29.5 Mb and more precisely established its boundaries. Notably, no genetic linkage for self-selected fat intake was detected, underscoring the carbohydrate-specific effect of this locus. A second key finding was the separation of two energy balance QTLs: <i>Mnic1/Kcal2/Tfv1</i> for food intake and a newly discovered locus regulating short term body weight gain. The <i>Mnic1/Kcal2/Tfv1</i> QTL was further de-limited to 19.0 Mb, based on the absence of nutrient intake phenotypes in subcongenic HQ17IIa mice. Analyses of available sequence data and gene ontologies, along with comprehensive expression profiling in the hypothalamus of non-recombinant, <i>cast/cast</i> and <i>b6/b6</i> F₂ controls, focused our attention on candidates within the QTL interval. <i>Zfp811</i>, <i>Zfp870</i>, and <i>Btnl6</i> showed differential expression and also contain stop codons, but have no known biology related to food intake regulation. The genes <i>Decr2</i>, <i>Ppard</i> and <i>Agapt1</i> are more appealing candidates because of their involvement in lipid metabolism and down-regulation in carbohydrate-preferring animals.</p></div

Differentially expressed genes in the hypothalamus of homozygous <i>cast/cast</i> vs. <i>b6/b6</i> subcongenic-derived F₂ mice, located within the fine mapped QTL interval of 26.08–45.12 Mb.

<p>Positive fold change indicates increased expression, negative value indicates decreased expression in homozygous <i>cast/cast</i> (CC) subcongenic-derived F₂ mice (n = 12) relative to <i>b6/b6</i> (BB) subcongenic-derived F₂ (n = 12). Genes were selected based on a significance value of <i>P</i><0.01 and an expression change of ≥1.5-fold, based on sequence tag counts.</p><p>Differentially expressed genes in the hypothalamus of homozygous <i>cast/cast</i> vs. <i>b6/b6</i> subcongenic-derived F₂ mice, located within the fine mapped QTL interval of 26.08–45.12 Mb.</p

Phenotypic data for food intake, body weight, body composition and organ weights in non-recombinant B6.CAST-17.1-derived F₂ mice.

<p>Values indicate least squares means ± SE.</p>a<p>Difference between B/B and C/C; probability value indexed in B/B column.</p>b<p>Difference between the B/C population and the average of its two parental strains combined, adjusted for fat source, initial age, baseline body weight, and baseline lean mass; probability value indexed in B/C column.</p><p>*<i>P</i><0.05; **<i>P</i><0.005; ^§<i>P</i><0.0001. Carbohydrate/protein (C/P) and fat/protein (F/P) diets were presented simultaneously for 10 days. BW, body weight; NMR, nuclear magnetic resonance; EPI, epididymal.</p><p>Phenotypic data for food intake, body weight, body composition and organ weights in non-recombinant B6.CAST-17.1-derived F₂ mice.</p

The critical <i>Mnic1/Kcal1</i>/<i>Tfv1</i> QTL region on mouse chromosome 17 was reduced to 19.0 Mb.

<p>Legend: Congenic and subcongenic strains with CAST/EiJ alleles introgressed on the wild type C57BL/6J (B6) or mutant C57BL/6J-<i>^hg/hg</i> genome are illustrated. Solid bars indicate CAST donor regions, open bars indicate B6 genotype, and hatched bars designate intervals of undetermined genotype, as defined by SNP or Mit markers (top). The fine-mapped interval encompassing carbohydrate-specific macronutrient intake (<i>Mnic1</i>; peak at 32.49 Mb), total kilocalories (<i>Kcal1</i>; peak at 27.19 Mb) and total food volume (<i>Tfv1</i>; peak at 27.10) is specified by the bar outlined in red.</p

Non-recombinant B6.CAST-17.1 F₂ mice exhibit increased intake of carbohydrate kcal, total kcal and total food volume.

<p>Legend: Daily consumption of (A) carbohydrate/protein kcal (C/P) versus (B) fat/protein kcal (F/P), total kcal (C) and total food volume (D) in non-recombinant B6.CAST-17.1 F₂ mice. Values are mean ± SE. Relative to <i>b6/b6</i> F₂, both <i>cast/cast</i> (<i>P</i><0.005, Tukey-Kramer) and <i>b6/cast</i> (<i>P</i><0.001) F₂ mice consumed more C/P kcal (A), with the exception of <i>day 1</i> when <i>b6/cast</i>><i>b6/b6</i> F₂ mice = <i>cast/cast</i> F₂ [genotype by day: <i>F</i> (18, 1187) = 2.16, <i>P</i><0.003]. By contrast, the <i>b6/b6</i> F₂ ate more F/P kcal when compared to <i>cast/cast</i> F₂ (<i>P</i><0.008) but not <i>b6/cast</i> F₂ (<i>P</i> = 0.10) (B), during the 10 d study. With respect to total kcal, all genotypes displayed a pronounced hyperphagic response to the diets at the beginning of the study, which subsided gradually over time [day: <i>F</i> (9, 938) = 21.10, <i>P</i><0.0001], independent of genotype [genotype×day: <i>F</i> (18, 938) = 0.94, <i>P</i> = 0.53]. Relative to <i>b6/b6</i> F₂, both <i>cast/cast</i> (<i>P</i><0.005) and <i>b6/cast</i> (<i>P</i><0.0001) F₂ mice consumed more total g over 10 d.</p

Preferential carbohydrate and total food intake is controlled by <i>Mnic1</i> and <i>Kcal/Tfv1</i> loci, respectively.

<p>Legend: Interval plot of proximal Chr 17 illustrates fine-mapped QTL for carbohydrate intake (<i>Mnic1</i>), total calorie intake (<i>Kcal2</i>) and total food volume consumed (<i>Tfv1</i>) in B6.CAST-17.1 subcongenic F₂ mice (left) and uncorrected phenotypic means for the peak marker of each QTL (right). Significance threshold values established for α = 0.01 and α = 0.05 were LOD scores of 2.98 and 2.14, respectively, as determined by 1,000 permutation tests. On the interval map, locations of markers nearest the peak LOD scores are shown on the x-axis, e.g., <i>rs33797547</i>. On the right, genotype effect plots at marker <i>rs33797547</i> show the CAST (CC) allele is associated with a higher intake of g, total kcal, and carbohydrate/protein kcal compared to the B6 (BB) allele, and the F1 (BC) responses for all three phenotypes resembled those of the CC, indicating dominance of the CAST allele. Values are mean ± SEM. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110424#pone-0110424-t002" target="_blank">Table 2</a> for a list of all QTLs.</p

QTLs for nutrient intake, body weight, and body composition in B6.CAST-17.1-derived F₂ mice.

<p>Genome coordinates are based on the Genome Reference Consortium Mouse Build 38 (GRCm38). Mb = megabase. LOD = logarithm (base 10) of odds. Significant linkage (<i>P</i><0.01 and <i>P</i><0.05) was defined as LOD>2.98 and LOD>2.14 respectively, based on 1,000 permutations. “----”, un-named. CI = confidence interval. C/P = carbohydrate/protein; BW = body weight. NMR = nuclear magnetic resonance. F/P = fat/protein. EPI = epididymal fat. ***<i>P</i>≤0.0001; **<i>P</i>≤0.001; *<i>P</i>≤0.01; ^§<i>P</i>≤0.05.</p><p>QTLs for nutrient intake, body weight, and body composition in B6.CAST-17.1-derived F₂ mice.</p