An interval of the obesity QTL Nob3.38 within a QTL hotspot on chromosome 1 modulates behavioral phenotypes

Abstract

A region on mouse distal chromosome 1 (Chr. 1) that is highly enriched in quantitative trait loci (QTLs) controlling neural and behavioral phenotypes overlaps with the peak region of a major obesity QTL (Nob3.38), which we identified in an intercross of New Zealand Obese (NZO) mice with C57BL/6J (B6). By positional cloning we recently identified a microdeletion within this locus causing the disruption of Ifi202b that protects from adiposity by suppressing expression of 11β-Hsd1. Here we show that the Nob3.38 segment also corresponds with the QTL rich region (Qrr1) on Chr. 1 and associates with increased voluntary running wheel activity, Rota-rod performance, decreased grip strength, and anxiety-related traits. The characterization of a subcongenic line carrying 14.2 Mbp of Nob3.38 with a polymorphic region of 4.4 Mbp indicates that the microdeletion and/or other polymorphisms in its proximity alter body weight, voluntary activity, and exploration. Since 27 out of 32 QTL were identified in crosses with B6, we hypothesized that the microdeletion and or adjacent SNPs are unique for B6 mice and responsible for some of the complex Qrr1-mediated effects. Indeed, a phylogenic study of 28 mouse strains revealed a NZO-like genotype for 22 and a B6-like genotype for NZW/LacJ and 4 other C57BL strains. Thus, we suggest that a Nob3.38 interval (173.0-177.4 Mbp) does not only modify adiposity but also neurobehavioral traits by a haplotype segregating with C57BL strains.

Figure 1. The peak region of the obesity QTL Nob3 corresponds with the QTL hotspot Qrr1.

The map depicts the critical interval recently identified to be responsible for differences in body weight of NZO and B6 allele carriers . Genes located in the region on Chr. 1 and a selection of microsatellite markers used for genotyping of the subcongenic line RCS-IX are indicated. QTL rich region on Chr. 1 (Qrr1) extending from the Fcgr3 to Rgs7 gene is shown in blue (NCBI Build 37/mm9).

Figure 2. Characterization of voluntary activity and the metabolism of skeletal muscle of Nob3.38^B/B and Nob3.38^N/N mice.

(A) Voluntary activity of B/B and N/N mice detected with a running wheel over 24 h (n = 13). (B) Glucose uptake into isolated skeletal muscle under basal and insulin-stimulated conditions (upper panel) and palmitate oxidation (lower panel) in isolated skeletal muscle under basal and AICAR stimulated conditions (EDL, left panels; Soleus, right panels) of B/B and N/N mice. (C) Characterization of mitochondrial respiration by determination of oxygen consumption rate of isolated mitochondria from skeletal muscles of B/B and N/N mice (n = 4–6).

Figure 3. Characterization of the behavior of B6.NZO-Nob3.38^B/B and B6.NZO-Nob3.38^N/N.

(A) Grip strength (maximum and average of 5 measurements), (B) Rota-rod performance at 24 and 32 rpm constant speed for 10 min, (C) time spent in illuminated compartments and number of transitions during light-dark avoidance test, (D) avoidance of the open areas of the O-Maze (left panel) and the center of the open field during the initial 5 min (right panel). (E) Spatial learning was assessed by the Morris Water Maze test as described in Material and Methods (n = 12). For the behavioral analysis, sex- and age-matched (10–12 weeks) mice were used (12 females and 11 males for each B6.NZO-Nob3.38^N/N and B6.NZO-Nob3.38^B/B line). (#p<0.05; *p<0.001).

Figure 4. Characterization of the body weight and behavior of RCS-IX.

(A) Body weight, (B) running wheel activity, (C) grip strength, and (D) preference on the O-Maze were compared between B/B and N/N allele carriers. (E) Spatial learning was assessed by the Morris Water Maze test and escape latency (left panel) and activity (right panel) were determined during acquisition (day 1–3) and reversal (day 4–5). For testing the subcongenic line RCS-IX 5 females of B/B and 7 females of N/N genotype were used. (#p<0.05; *p<0.01).

Figure 5. Expression of Ifi202b in different inbred strains of mice.

(A) Strains in which Ifi202b is expressed as detected by microarray are marked in black, lack of Ifi202b expression is marked in red. The figure shows a phylogenetic tree of 28 strains. It was generated by the Mouse Phylogeny Viewer (http://msub.csbio.unc.edu/) based on SNP information of the segment 175,860,234–175,928,281 bp (Chr. 1, NCBI Build 37/mm9). Of these 28 strains, 22 express Ifi202b, whereas 6 do not. (B) Quantitative real time PCR performed on white adipose tissue of indicated mouse strains as described in the Method section. (C) PCR products received by amplification of Ifi202b cDNA (white adipose tissue) between exon 1 and exon 2 as indicated in the scheme. (D) The different size of the PCR product is the result of integration of 68 bp between the sequence of exon 1 and exon 2 as indicated in red. Sequence of SJL corresponds to EST clone M31418, sequence of NZO exhibits GenBank accession number BankIt1573081 JX945582.

Figure 6. Expression of Kcnk2 in cerebellum of B6.NZO-Nob3.38^B/B and B6.NZO-Nob3.38^N/N mice.

Expression of Kcnk2 was determined by qRT-PCR (B6.NZO-Nob3.38^B/B: n = 7; B6.NZO-Nob3.38^N/N: n = 8). (*p<0.005).