Measurements of the obesity effect arising from the high-fat meal challenge were done every two weeks to ascertain the CC lines' response to obesity after 12 weeks of the HFD challenge. Beginning at the age of eight weeks following weaning, body mass was assessed every two weeks using a traditional laboratory scale. Afterward, a marker for the indication of obesity was created using the entire time series data of body weight changes at various time points during the mice's age that came from distinct CC lines in response to an HFD (42 % Fat) dietary challenge. Utilizing the HAPPY software, the genotypic data of the CC lines were analyzed to determine any genetic correlation with the body weight features for both the male and female populations as well as for the entire population.
Significant body weight variation between CC lines
Figures 1A-C illustrate the significant differences in percent body weight means between the entire (overall) CC lines population (Figure 1A), as well as for males and females as shown in Figures 1B and 1C respectively separately. The mean body weight change for the entire population of CC lines (n = 525/55 CC lines) was observed in varying degrees from the highest mean values in IL-2131, with the mean values from IL-2668 representing the lowest value in the overall population. In the male-only group, the mean body weight change ranged from the highest mean values in IL-2131, with the mean values from IL-2462 representing the lowest value. The mean body weight change ranged from the highest mean values in IL-2146, with the mean values from IL-2668 representing the lowest value in the female-only group.
Identification of candidate genes within the mapped QTL intervals
A total of 62 QTLs were identified in our study and were designated as ObSL, for Obesity Specific Locus and its number. These mapped QTL, along with their genomic interval locations, peak interval sizes, chromosome specificity, and specific traits, were listed in Tables 1, 2, and 3, and Figures 2A-C, respectively, representing the overall population and males and females separately. 28 QTLs of the total were not previously mapped. The genomic interval of the remaining 34 QTL was previously reported, and their genomic intervals were fine-mapped in our study, as shown in Table 4. The genomic positions of 42 of the mapped QTL overlapped (clustered) on seven different peaks or close genomic positions, as shown in Table 5.
Mapping new QTL
Our results showed that 28 new QTLs were not mapped in previous reports that were associated with our phenotypic data. The genomic interval positions of 16 of these QTLs overlapped with previously mapped QTLs (Tables 4, 5), while 12 of them are unique QTLs. The first new QTL ObSL1 mapped during our study was on chromosome 14 at a position of 52.87-61.00 Mbp, peaking at 57.24 Mbp in the overall population at BW02. The other observed new QTLs of ΔBW02 in the overall population were ObSL2 and ObSL3, both on chromosome 15, on interval positions of 23.64-37.32 and 44.88-62.17 Mbp with their respective peak positions at 28.45 and 54.46 Mbp. In ΔBW04 phenotype in the overall population, three new QTL ObSL4, ObSL5, and ObSL6 were identified, the former on chromosome 11 and later two on chromosome 16. In ΔBW08 phenotype in the overall population, a single new QTL ObSL8 was identified on chromosome 15 with an interval position of 24.83-32.46 Mbp with its respective peak positions at 28.45 Mbp. In ΔBW012 phenotype in the overall population, a single new QTL ObSL13 was identified on chromosome 11 with an interval position of 14.71-20.08 Mbp reaching to its peak position at 18.24 Mbp. In the overall population with phenotype ∆BW2-4, three new QTL ObSL14, ObSL15, and ObSL16 were identified on two different chromosomes with their respective interval positions of 118.83-122.67, 39.69-59.10, 66.36-84.97Mbp. In the overall population with phenotypes of ∆BW810 and ∆BW1012, two single new QTL ObSL21 and ObSL22 were identified respectively on chromosomes 10 and 13 in each phenotype. In the overall population with phenotype ∆BW612, a single new QTL ObSL27 was identified on chromosome 6 with an interval position of 85.56-104.52Mbp having a peak position at 94.57 Mbp.
The other major group of mapped QTLs were from the male population. Here in males on chromosome 3, the identified QTL at ΔBW02 was ObSL29, on interval position of 77.92-89.1Mbp with its peak position at 81.65 Mbp. In ΔBW04 two new QTL ObSL39 and ObSL40 on chromosomes 11 and 15 respectively with the respective interval positions of 16.92-20.24 and 24.70-31.64Mbp. For ΔBW06 phenotype a new QTL ObSL41 with the interval position of 24.86-31.75 Mbp peaked at 28.04 Mbp was identified on chromosome 15. With ΔBW08 phenotype, a novel QTL ObSL43 with its respective interval position of 25.38-30.97Mbp was identified on chromosome 15. In males with phenotype ∆BW24, two new QTL ObSL45 and ObSL46 were identified on chromosome 2 with the respective interval positions of 100.99-117.72Mbp reaching their peaks at 109.23 and 115.19Mbp. In males ∆BW10-12 phenotype, a new QTL ObSL51 was identified on chromosome 4 with the interval position of 95.56-114.02Mbp, reaching its peak at 104.57. In females with phenotypes of ΔBW08 and ∆BW68, two new QTL ObSL56 and ObSL60 were identified respectively on chromosomes 16 and 13 with the respective interval positions of 44.01-56.03 and 32.12-46.94Mbp reaching their peaks at 49.24 and 8.75Mbp.
Fine mapping of previously identified QTL
The other 34 QTLs that were observed during our study were also new but had already been identified by earlier researchers, although our study fine mapped their interval position. In the overall population, 14 fine-mapped QTLs were ObSL7, ObSL9, ObSL10, ObSL11, ObSL12, ObSL17, ObSL18, ObSL19, ObSL20, ObSL23, ObSL24, ObSL25, ObSL26, and ObSL28 on different chromosomes (Table 2). Similarly, in males-only group, 12 fine-mapped QTL were observed on five different chromosomes throughout the genome (Table 2). In the only female population, eight fine-mapped QTLs observed were ObSL53, ObSL54, ObSL55, ObSL57, ObSL58, ObSL59, ObSL61, and ObSL62, on chromosomes 1, 3, and 13 (Table 2).
As far as the genome-wide significance threshold is concerned, three sets of QTLs were identified during our entire study (Figures 2A-C). The first group is the one which had a 50% of genome-wide significance threshold. The second group is formed having a 90% of genome-wide significance threshold while the third group is consisting of a 95% of genome-wide significance threshold.
Founder effect
The results of this analysis were presented for the QTL in Figures 3A-C respectively for the overall population, only males and only females. We evaluated the effect of each founder genotype on the overall population, males and females ΔBW mapped QTL interval, and estimated the assessed trait. The wild-derived strains, primarily PWK, were a major factor in the rise in ΔBW values, as demonstrated by the loci complicated pattern of founder haplotype effects except in females where NOD plays the same role instead. Although other strains also contributed (positively or negatively) to the overall QTL effect, the NZO and NOD genotypes decrease this characteristic.
Gene prioritization and functional analysis
The results of the overall population have been presented in Table 1 and Figure 2a.
For the DBW0-2 phenotype three QTLs (ObSL1, ObSL2, ObSL3) one on chromosome 14 and two on chromosome 15 containing 414, 145, and 158 genes in the 95% CI, respectively, were reported.
Interestingly, for this early timepoint, one of the systemic processes highly enriched in the MGI Mammalian Phenotype is abnormal prenatal growth/weight/body size and from Gene Ontology lipid storage. Exostosin glycosyltransferase 1 (Ext1) is the highest prioritized gene. Ext1 is down-regulated by a high-fat diet and this downregulation is considered to exacerbate ER stress and NAFLD progression [31]. Other prioritized genes include fat storage-inducing transmembrane protein 1 (Fitm1) and hyaluronan synthase 2 (Has2). Has2 overexpression in mice has been shown to reduce fat accumulation and improve glucose tolerance [32].
For the DBW0-4 phenotype three QTLs (ObSL4, ObSL5, ObSL6) one on chromosome 11 and two on chromosome 16 containing 72, 211, and 140 genes in the 95% CI, respectively, were reported. Relevant systemic processes highly enriched in the MGI Mammalian Phenotype have enhanced lipolysis and abnormal circulating leptin level with genes coiled-coil domain containing 80 (Ccdc80) and SERTA domain containing 2 (Sertad2) being involved in both processes. Ccdc80 is an adipocyte-secreted protein that modulates glucose homeostasis in response to diet-induced obesity in mice and its levels have been associated with chronic obesity complications [33]. Ablation of Sertad2 has been shown to protect mice from obesity and associated metabolic dysfunction [34]. Another prioritized gene was microRNA 155 (Mir155), deletion of which has been shown to prevent diet-induced obesity in female mice [35] but aggravate high‐fat diet‐induced adipose tissue fibrosis in male mice [36].
For the DBW0-8 phenotype two QTLs (ObSL7, ObSL8) on chromosome 5 and chromosome 15 containing 147 and 68 genes in the 95% CI, respectively were reported. A systemic process enriched in the MGI Mammalian Phenotype was abnormal retroperitoneal fat pad morphology involving genes peroxisome proliferative activated receptor, gamma, coactivator 1 alpha (Ppargc1a), and superoxide dismutase 3 (Sod3). Over-expression of Sod3 in high-fat diet-fed mice has been shown to block diet-induced obesity[37].
For the DBW0-10 phenotype two QTL (ObSL9, ObSL10) both on chromosome 5, containing 129 and 88 genes in the 95% CI, respectively, were reported. One highlighted systemic process enriched for this phenotype is a response to dietary excess, involving again genes Ppargc1a and Sod3, as well as cholecystokinin A receptor (Cckar) and glucose-6-phosphate dehydrogenase 2 (G6pd2). Rats with naturally mutated Cckar gene (Otsuka Long-Evans Tokushima Fatty (OLETF) have been shown to develop both diabetes and obesity [38].
For the DBW0-12 phenotype three QTLs (ObSL11, ObSL12, ObSL13) on chromosome 1, chromosome 5, and chromosome 11 containing 76, 117, and 61 genes in the 95% CI, respectively, were reported. For this phenotype, again Ppargc1a, Cckar, and Sod3 are highly prioritized, with response to dietary excess being a highly enriched systemic process.
For the DBW2-4 phenotype three QTLs (ObSL14, ObSL15, ObSL16) on chromosome 14, chromosome 15, and chromosome 16 containing 273, 248, and 132 genes in the 95% CI, respectively, were reported. Systemic processes highly enriched in the MGI Mammalian Phenotype are increased glycogen level and abnormal pancreatic islet size, with notable genes prioritized thrombospondin 1 (Thbs1) and secretogranin V (Scg5). Thbs1 has been suggested as a marker of obesity and metabolic syndrome [39], and targeted disruption of Thbs1 in male mice ameliorated high-fat diet-induced insulin resistance and adipose tissue inflammation [40]. Scg5 overexpression had been inversely correlated with body weight in congenic mice[41].
For the DBW6-8 phenotype four QTLs (ObSL17, ObSL18, ObSL19, ObSL20) two on chromosome 1, one on chromosome 12, and one on chromosome 19 containing 106, 120, 255, and 229 genes in the 95% CI, respectively, were reported. A highly enriched systemic process in Gene Ontology is the regulation of lipid metabolic process, with notable genes prioritized being insulin 1 (Ins1), twist basic helix-loop-helix transcription factor 1 (Twist1), and phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit gamma (Pik3cg).
For the DBW6-12 phenotype six QTLs (ObSL23, ObSL24, ObSL25, ObSL26, ObSL27, ObSL28) two on chromosome 1, one on chromosome 2, one on chromosome 5, one on chromosome 6, and one on chromosome 12, containing 165, 198, 127, 177, 288, and 218 genes in the 95% CI, respectively, were reported. Enriched systemic processes include increased body size and abnormal pituitary hormone level with notable genes prioritized being gonadotropin-releasing hormone receptor (Gnrhr), ATPase, aminophospholipid transporter (APLT), class I, type 8A, member 1 (Atp8a1), phosphoribosyl pyrophosphate amidotransferase (Ppat), and neuromedin U (NmU). Nmu−/− mice are more susceptible to obesity [42] and homozygous carriers of NMU gene mutations have increased the prevalence of overweight and obesity [43].
Finally, for the DBW8-10 and DBW10-12 phenotypes, one QTL was reported for each. ObSL21 on chromosome 10 for DBW8-10 and ObSL22 on chromosome 13 for DBW10-12 containing 25, and 217 genes in the 95% CI, respectively. One systemic process highly enriched in our analysis was abnormal fasting circulating glucose level, with notable prioritized genes being follistatin (Fst) and ubiquitin-specific peptidase 15 (Usp15). Knockdown of Fst has been shown to improve glucose tolerance in diet-induced obese mice[44]. Usp15 has been suggested to modify obesity via the control of adipocyte differentiation [45].
The results for the male population have been presented in Table 2 and Figure 2b.
For the DBW0-2 phenotype eight QTLs (ObSL29, ObSL30, ObSL31, ObSL32, ObSL33, ObSL34, ObSL35, ObSL36) one on chromosome 3, two on chromosome 5, two on chromosome 7, one on chromosome 11, and two on chromosome 15, containing 246, 343, 126, 213, 142, 54, 91 and 118 genes in the 95% CI, respectively, were reported. The enriched systemic processes include impaired glucose tolerance and abnormal adipose tissue physiology, these include highly prioritized genes in common with the overall population QTL, such as Ppargc1a. Additionally, interesting, prioritized genes are (Pepd), a gene suggested to play a role in obesity-induced insulin resistance[46], and neuropeptide Y receptor Y2 (Npy2r). Npy2r knockout mice are susceptible to weight gain and obesity and NPY2R is a target for obesity drugs currently in phase I clinical trial (NCT05751226).
For the DBW0-4 phenotype four QTLs (ObSL37, ObSL38, ObSL39, ObSL40) two on chromosome 5, one on chromosome 11, and one on chromosome 15, containing 351, 127, 50, and 63 genes in the 95% CI, respectively, were reported. For the DBW0-6 phenotype a single QTL (ObSL41) on chromosome 15, containing 63 genes in the 95% CI was reported. This single QTL overlaps the same locus as QTL mapped for phenotypes DBW0-2, DBW0-4, DBW0-6, and DBW0-8.
Systemic processes enriched for these two phenotypes include abnormal retroperitoneal fat pad morphology and slow postnatal weight gain. Ppargc1a is again very highly prioritized, along with calcineurin B, type I (Ppp3r1). In mice, tissue-specific ablation of Ppp3r1 from skeletal muscle leads to protection from high-fat-diet-induced obesity and relevant comorbidities[47].
For the DBW0-8 phenotype two QTL (ObSL42, ObSL43) one on chromosome 5, and one on chromosome 15, containing 94 and 45 genes in the 95% CI, respectively, were reported. For the DBW0-12 phenotype a single QTL (ObSL44) on chromosome 5, containing 120 genes in the 95% CI was reported. For these two phenotypes again genes Ppargc1a and Cckar are highly prioritized, showing a strong association with all time points.
For the DBW2-4 phenotype two QTL (ObSL45, ObSL46) both on chromosome 2, containing 304 and 393 genes in the 95% CI, respectively, were reported. Important enriched systemic processes are cellular lipid catabolic process, glycerolipid metabolic process & phospholipid metabolic process. Prioritized genes include catalase (Cat), glycine amidinotransferase (Gatm), and Thbs1. Cat knockout mice are prone to obesity development, most likely due to decreased energy expenditure[48]. Similarly, Gatm knockout mice are prone to diet-induced obesity due to decreased energy expenditure [49].
For the DBW6-8 phenotype three QTLs (ObSL47, ObSL48, ObSL49) one on chromosome 1, one on chromosome 7, and one on chromosome 12 containing 71, 419, and 240 genes in the 95% CI, respectively, were reported. Enriched systemic processes include decreased pancreatic islet number and increased body size. Prioritized genes include aryl-hydrocarbon receptor (Ahr) and insulin-like growth factor I receptor (Igf1r). There is evidence linking Ahr signaling activity, especially to diet-induced obesity[50].
For the DBW8-10 phenotype a single QTL (ObSL50) on chromosome 5, containing 168 genes in the 95% CI was reported. For the DBW6-12 phenotype a single QTL (ObSL52) on chromosome 5, containing 201 genes in the 95% CI was reported. For both phenotypes, abnormal pituitary hormone level is an enriched systemic process and the Nmu gene is highly prioritized, same as the DBW6-12 phenotype for the overall population.
For the DBW10-12 phenotype a single QTL (ObSL51) on chromosome 4, containing 322 genes in the 95% CI was reported. A very highly enriched systemic process is abnormal lipid homeostasis & hyperglycemia, as well as energy homeostasis. One highly prioritized gene is the leptin receptor (Lepr), which is involved in the regulation of the feeding behavior-enriched process. Mutations in LEPR/Lepr cause a monogenic form of obesity both in humans and mice [51].
The results of the female population have been presented in Table 3 and Figure 2c.
For female mice we observed QTL on chromosome 3 for phenotypes DBW0-4, DBW0-6, DBW0-8, DBW0-10, and DBW0-12 (ObSL53, ObSL54, ObSL55, ObSL57, ObSL58), showing strong involvement of the locus in many different timepoints. ObSL58 has been previously described by our lab as ObFL in Binenbaum et al. [12]. Of the 58 genes in total on this locus, ontologies such as increased circulating free fatty acids levels, decreased susceptibility to diet-induced obesity, decreased circulating ketone body level, decreased susceptibility to weight gain, and increased energy expenditure were observed to be enriched. Of these hydroxyacyl-Coenzyme A dehydrogenase (Hadh) is an enzyme involved in fatty acid oxidation and has been previously identified as a putative obesity gene. Mice lacking the Hadh gene are protected from diet-induced obesity [52]. Sphingomyelin synthase 2 (Sgms2) is as well a proposed therapeutic target for obesity and Sgms2 deficiency has been found to prevent high-fat diet-induced obesity and insulin resistance [53].
For the DBW6-8 phenotype two QTLs (ObSL59, ObSL60) on chromosome 13, and chromosome 1 containing 34 genes and 112 genes in the 95% CI, respectively, were reported. This gene list is enriched with genes related to cholesterol biosynthesis and metabolism of steroids, such as Idi1 and Idi2.
For the DBW6-12 phenotype two QTL (ObSL61, ObSL62) on chromosome 1 and chromosome 18 containing 230 genes and 49 genes in the 95% CI, respectively, were reported. This gene list is enriched with genes related to abnormal digestion, abnormal digestive system physiology, and chronic inflammation such as solute carrier family 9 members Slc9a2 and Slc9a4.