Impact of Chromosome 2 Obesity Loci on Cardiovascular Complications of Insulin Resistance in LDL Receptor–Deficient C57BL/6 Mice
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《糖尿病学杂志》
1 Department of Human Genetics, University of California Los Angeles, Los Angeles, California
2 Department of Medicine/Division of Endocrinology, Diabetes and Hypertension, University of California Los Angeles, Los Angeles, California
3 Department of Medicine, Division of Cardiology, University of California Los Angeles, Los Angeles, California
AUC, area under the curve; NMR, nuclear magnetic resonance
ABSTRACT
Previous characterization of mouse chromosome 2 identified genomic intervals that influence obesity, insulin resistance, and dyslipidemia. For this, resistant CAST/Ei (CAST) alleles were introgressed onto a susceptible C57BL/6J background to generate congenic strains with CAST alleles encompassing 67–162 Mb (multigenic obesity 6 [MOB6]) and 84–180 Mb (MOB5) from mouse chromosome 2. To examine the effects of each congenic locus on atherosclerosis and glucose disposal, we bred each strain onto a sensitizing LDL receptor–null (LDLR–/–) C57BL/6J background to predispose them to hypercholesterolemia and insulin resistance. LDLR–/– congenics and controls were characterized for measures of atherogenesis, insulin sensitivity, and obesity. We identified a genomic interval unique to the MOB6 congenic (72–84 Mb) that dramatically decreased atherosclerosis by approximately threefold and decreased insulin resistance. This region also reduced adiposity twofold. Conversely, the congenic region unique to MOB5 (162–180 Mb) increased insulin resistance but had little effect on atherosclerosis and adiposity. The MOB congenic intervals are concordant to human and rat quantitative trait loci influencing diabetes and atherosclerosis traits. Thus, our results define a strategy for studying the poorly understood interactions between diabetes and atherosclerosis and for identifying genes underlying the cardiovascular complications of insulin resistance.
Insulin resistance increases the risk of hypertension, obesity, and dyslipidemia and is thereby a principal risk factor for cardiovascular disease (1–4), but the mechanisms involved are largely unknown (3–5). Loci on mouse chromosome 2 are concordant (6) for insulin resistance and atherosclerosis quantitative trait loci from homologous genomic intervals in rats, mice, and humans (supplemental data, which is detailed in the online appendix [available at http://diabetes.diabetesjournals.org]). Previously, we characterized overlapping multigenic obesity (MOB)5 and -6 congenic mice (7) for these traits to better understand their impact. Here, chromosome 2 alleles from CAST/Ei (CAST) mice that are resistant to obesity were introgressed onto an obesity-susceptible C57BL/6J background (available online from http://phenome.jax.org/pub-cgi/phenome). We showed that MOB5 congenics became insulin resistant, suggesting that genes underlying this trait resided in the congenic interval unique to this strain (CAST alleles from 162 to 180 Mb). By contrast, MOB6 congenics displayed an atherosclerosis-susceptible cholesterol profile when fed a diet high in fat, cholesterol, and cholic acid, suggesting that genes in the interval unique to this strain (CAST alleles from 72 to 84 Mb) underlie the atherogenic cholesterol profile (7). However, the experimental diet used in that study, though enriched in lipid and cholesterol, did not trigger atherogenesis or obesity in these strains.
C57BL/6J (B6) mice with homozygous null alleles for the LDL receptor (LDLR–/–) become obese and develop atherosclerosis associated with hypertriglyceridemia, hyperglycemia, hyperinsulinemia, and decreased plasma HDL (8,9) when fed a Western diet for 16–18 weeks. To determine how CAST alleles from the MOB chromosome 2 loci modify the dyslipidemia, insulin resistance, and atherosclerosis of the LDLR–/– strain, we bred each congenic strain onto the LDLR–/– background. LDLR–/– MOB5 and LDLR–/– MOB6 congenic strains possess genetic variations that mimic the composite interactions between human syntenic loci that underlie multiple insulin resistance risk factors for cardiovascular disease. Introgressing the MOB loci onto the LDLR–/– background susceptible to the cardiovascular vascular complications of insulin resistance greatly enabled the dissection of genetic interactions that characterize these multigenic disorders.
RESEARCH DESIGN AND METHODS
Animals were housed in an Association for Assessment and Accreditation of Laboratory Animal Care–accredited, specific pathogen-free facility under a 12-h light/dark cycle with free access to food and water. Male MOB5 (B6.CAST-D2mit 9 to D2Mit 50) and MOB6 (B6.CAST-D2Mit 14 to D2Mit457) congenic mice (7) were bred to female LDLR–/– (Ldlrtm1Her/Ldlrtm1Her) mice (10) from The Jackson Laboratory (stock no. 2207; Bar Harbor, ME). Male N1 mice were backcrossed to LDLR–/– females, and N2 offspring homozygous for LDLR null allele and heterozygous for the congenic region were intercrossed to obtain mice homozygous for LDLR null allele and the congene. To maintain the integrity of the MOB congenic loci, internal markers (D2Mit19, 133 Mb; and D2Mit92, 71 Mb) were also genotyped. Positions for microsatellite markers were determined using the Mouse Genome Browser Gateway from Genome Bioinformatics of the University of California Santa Cruz (available from http://genome.ucsc.edu/cgi-bin/hgGateway, mm6 assembly).
At 21 days of age, mice were weaned to chow containing 6% fat by weight (no. 7013; Harlan Teklad, Madison WI). At 12 weeks, males were randomly assigned to chow or Western diet (40% of calories from fat, 17% from protein, and 43% from carbohydrate; no. R12079B; Research Diets, New Brunswick, NJ). At weekly intervals mice were weighed and characterized for fat and lean mass by nuclear magnetic resonance (NMR) during the diet period (from 12 to 30 weeks of age). Selected mice from each strain were characterized for glucose tolerance and insulin sensitivity. Mice were killed at 28–30 weeks of age. Randomly selected LDLR–/– and LDLR–/– MOB congenics were characterized for atherosclerosis.
Atherosclerotic lesion analysis.
Nonfasted 28- to 30-week-old male mice were killed by Isoflurane vapor (Abbott Laboratories, Chicago, IL) after 16–18 weeks on Western diet. Analysis of 30 sequential hematoxilin and eosin–stained aortic root sections was used to assay for morphology of the aortic root. Aortic roots in 12–13 males from each strain were scored for foam cell content and presence of a fibrous cap (11). In addition, whole aortas from 10 mice per strain were analyzed for total fatty streak lesion area by Sudan V staining, using the en face technique (8).
Plasma lipid, insulin, and glucose assays.
Plasma samples were collected at death after a fast of 9 h beginning between 5:00 and 7:00 A.M. Blood plasma drawn from the retro-orbital sinus under isoflurane anesthesia was assayed for lipids (free fatty acids and triglycerides), cholesterol (total, HDL, and unesterified), insulin, and glucose (12). VLDL/LDL concentrations were determined by subtracting HDL from total cholesterol (7).
Intraperitoneal glucose tolerance test and insulin sensitivity index.
Intraperitoneal glucose tolerance tests and insulin sensitivity index (Si) were performed as previously described (13). For intraperitoneal glucose tolerance testing, 5–16 male mice from each strain were fasted for 9 h and injected with glucose (2 g/kg body wt i.p.) in saline (American Pharmaceutical Partners, Schaumburg, IL). Glucose was measured from the blood of a saphenous vein puncture at 0, 30, 60, and 120 min, using a OneTouch Ultra Glucometer (Lifescan, Milpitas, CA). Differences in glucose clearance were measured as changes in the area under the curve (AUC) determined using the composite trapezoid rule:
The Si was evaluated in five to six male congenics and LDLR–/– controls. After 16–17 weeks on a Western diet, mice were fasted for 4 h and injected with porcine pancreatic insulin (Sigma-Aldrich, St. Louis, MO) at 2 IU/kg body wt i.p. in 200 μl saline (American Pharmaceutical Partners, Schaumburg, IL). Blood glucose was measured at 0, 15, 30, and 90 min postinjection. Si (13) was measured by the slope of the glucose concentration curve (Ki) over the first 15 min: Ki = [(glucose)t = 15 – (glucose)t = 0]/15 min.
NMR analysis.
Fat mass and lean mass were monitored weekly with a mouse Minispec (Bruker Woodlands, TX), using software from Echo Medical Systems (Houston, TX). The NMR spectroscopy apparatus (14) gave fat and lean mass measurements with coefficients of variation of <3%. Correlation between NMR and gravimetric measurements is better than 0.99.
Food efficiency and food intake.
Food efficiency (food required to produce 1 g of body mass) was measured as previously described (15). Weekly, mice and the food from each cage were weighed. Bedding was checked to ensure that no food was present. Cages that consistently contained food in the bedding were dropped from the analysis. Total amount of food consumed was divided by the change in body weight over the diet period to calculate food efficiency for each strain. To determine differences in food consumption between the strains, four mice per strain were singly housed and food weighed weekly, as above.
Statistical analysis.
The differences between individual groups were determined by ANOVA, using StatView 5.0 (SAS Institute).
RESULTS
CAST alleles from the MOB6 locus were associated with decreased atherosclerotic lesion formation and improved insulin sensitivity.
LDLR–/– mice develop extensive atherosclerotic lesions following a Western diet. However, when compared with LDLR–/– controls, LDLR–/– MOB6 congenics showed a two- to threefold decrease in both aortic root lesion area (Fig. 1A) and total aortic lesion area measured by en face analysis (Fig. 1B). By contrast, lesion areas in LDLR–/– MOB5 congenics were comparable to controls. Although there were differences in lesion area, there were no differences in morphology between strains. These data were especially striking because both LDLR–/– MOB congenic strains displayed decreased plasma cholesterol concentrations (Fig. 4).
Chow-fed LDLR–/– and LDLR–/– congenics developed 9- to 15-fold less aortic root lesion area than the same mice fed a Western diet. Compared with controls, both congenic strains now demonstrated a two- to threefold decrease in aortic lesion area, whereas plasma cholesterol concentrations were not significantly different between the strains (supplemental Figs. 2 and 4, which are detailed in the online appendix). No differences were observed in lesion morphology between controls and LDLR–/– MOB congenic strains.
Compared with LDLR–/–, LDLR–/– MOB6 congenics fed a Western diet had markedly improved glucose tolerance when challenged with an intraperitoneal glucose load (2 g/kg body wt i.p.). Here, we compared strains for areas under the plasma glucose curve (AUCs) as a function of time (Fig. 2A) after the glucose bolus (see RESEARCH DESIGN AND METHODS). The AUCs for LDLR–/– MOB6 and parental MOB6 congenics (37,500 and 35,100, respectively) were significantly lower than LDLR–/– (44,700). LDLR–/– MOB5 congenics showed no difference in response to glucose challenge compared with LDLR–/– controls. Unexpectedly, the parental MOB5 congenics had significantly impaired glucose clearance (AUC = 53,500) compared with LDLR–/–. These results suggested that sequences unique to the MOB5 congenic interval (162–180 Mb) carry allelic variations that strongly impact glucose clearance. Consistent with this hypothesis, we observed similarly decreased glucose clearance in preliminary experiments with a subcongenic carrying CAST alleles only from the region 172–180 Mb (supplemental data, which is detailed in the online appendix).
Si was used to determine sensitivity to insulin-stimulated glucose uptake (Fig. 2B). LDLR–/– MOB5 and parental MOB5 strains were significantly more resistant to insulin-stimulated glucose uptake (Ki = –1.52 and Ki = –0.72, respectively) than LDLR–/– controls (Ki = –3.44). By contrast, we observed increased sensitivity to insulin (Ki = –7.722) in parental MOB6 congenics. When fed a chow diet, all strains had markedly improved glucose clearance, but no significant differences were observed between the strains (supplemental Fig. 2, which is detailed in the online appendix).
CAST alleles in the MOB6 locus diminished the obesity of LDLR–/– mice.
LDLR–/– MOB6 congenics fed a Western diet were markedly less obese than controls, as measured by a dramatic drop in weight gain and NMR assessment of fat mass–to–lean mass ratio (Figs. 3 and Table 1). We observed a significant decline in lean mass for both of the LDLR–/– MOB5 and -6 congenics compared with controls (Figs. 3C and Table 1). However, the decreased adiposity of the LDLR–/– MOB6 congenics was attributable to a highly significant twofold reduction in fat mass (Figs. 3D and Table 1) compared with LDLR–/– controls. A small but significant decrease in final mean weight was observed in the LDLR–/– MOB5 congenics, with no significant difference in fat mass–to–lean mass ratio.
LDLR–/– MOB6 congenics fed a Western diet showed no significant difference in the amount of food consumed over the 16-week diet period compared with LDLR–/– controls, whereas LDLR–/– MOB5 congenics consumed significantly more (Figs. 3E and Table 1). LDLR–/– MOB6 congenics required two times more food to amass 1 g of body weight (food efficiency) than LDLR–/– controls (Figs. 3F and Table 1). This difference in food efficiency may arise from differences in energy expenditure or nutrient absorption. Further studies are required to determine the underlying mechanism and to identify the responsible genes. There was no significant difference in food efficiency between LDLR–/– MOB5 congenics and LDLR–/– controls.
On a chow diet, LDLR–/– control and LDLR–/– MOB5 strains weighed 10 g less than Western diet–fed mice, whereas LDLR–/– MOB6 congenics weighed 5 g less than the same strain fed a Western diet (supplemental data, which is detailed in the online appendix). Similar differences were observed for NMR traits (fat mass–to–lean mass ratios, fat mass, and lean mass), food intake, and food efficiency (supplemental Fig. 3, which is detailed in the online appendix).
CAST alleles from both MOB5 and MOB6 loci improved plasma cholesterol profiles, but only CAST alleles from the MOB5 locus reduced the dyslipidemia and hyperglycemia of LDLR–/– mice.
We observed a modest but significant decrease in plasma total cholesterol of both LDLR–/– MOB6 and LDLR–/– MOB5 compared with LDLR–/– controls. A similar drop in plasma HDL, LDL plus VLDL, and unesterified cholesterol was seen in both LDLR–/– congenics compared with controls (Fig. 4). In the absence of LDL receptors, it is clear that other mechanisms are required to explain the lower LDL levels in the congenic strains compared with background LDLR–/– mice. This may simply result from a decline in overall cholesterol pools because of decreased synthesis, or it may result from activation of an alternate clearance pathway.
Compared with LDLR–/–, LDLR–/– MOB5 congenics developed markedly lower plasma triglycerides, free fatty acids, and glucose when fed a Western diet (Fig. 5). By contrast, LDLR–/– MOB6 congenics were quite similar to LDLR–/– controls, although both LDLR–/– MOB congenic strains displayed lower plasma insulin compared with LDLR–/–. We hypothesize that the lack of weight gain, given the dyslipidemia and hyperglycemia of the LDLR–/– MOB6 congenics, suggests a defect in lipid storage in this strain.
When fed a chow diet, plasma cholesterol concentrations (with the exception of HDL) declined in all strains. Interestingly, LDLR–/– MOB6 animals showed a significant decline in HDL comparable to Western diet–fed mice. No difference in HDL was observed between LDLR–/– MOB5 congenics and controls (supplemental data, which is detailed in the online appendix). Chow-fed LDLR–/– mice and LDLR–/– MOB congenics also had lower plasma triglycerides and free fatty acids compared with the same mice fed a Western diet, but no significant differences were seen in these traits between congenics and controls (supplemental Fig. 2, which is detailed in the online appendix).
DISCUSSION
Robust (50–70%) reductions in atherosclerotic lesion area observed in MOB6 congenics suggest an atheroprotective locus in the genomic interval unique to that strain, 72–84 Mb (Fig. 1). Similar to data reported by Merat et al. (9), LDLR–/– controls showed profound insulin resistance as assayed by glucose tolerance testing after long-term exposure to high caloric intake. CAST alleles from each MOB genomic interval effectively modified the insulin resistance–related phenotypes observed in the LDLR–/– controls (Fig. 2). When compared by AUC, the LDLR–/– MOB6 congenic mice were able to clear glucose significantly (P = 0.008) faster than controls. Notably, although LDLR–/– MOB5 showed glucose clearance similar to that of LDLR–/– controls, clearance in parental MOB5 congenics was significantly (P = 0.004) impaired compared with LDLR–/– controls (Fig. 2). Although it cannot be generally assumed that impact of the congenic loci is the same in the presence or absence of the LDL receptor, the glucose clearance data suggest that the decline in glucose tolerance in the MOB5-derived congenics was independent of the LDLR–/– mutation. Similarly, insulin sensitivity, as measured by Ki, was significantly decreased in both LDLR–/– MOB5 (P = 0.02) and parental MOB5 congenics (P = 0.009), but Ki was significantly increased (P = 0.0002) in parental MOB6 congenics compared with controls. Thus, we hypothesize that CAST alleles from a locus unique to the MOB5 congenic interval (162–180 Mb) influenced the decrease in insulin sensitivity and glucose clearance, whereas a second CAST locus unique to the MOB6 genomic interval (72–84 Mb) influenced an increase in these measures (Fig. 2). Initial experiments with subcongenics specific to these regions support this hypothesis, but, given the size and complex genetic architecture of the MOB5 and -6 congenic intervals, genetic interactions between the MOB unique intervals and the shared genomic region cannot be ruled out.
Similar to previous results observed in the parental MOB congenics (7), the LDLR–/– MOB6 congenics gained weight at a significantly decreased rate when compared with control and LDLR–/– MOB5 strains. Additionally, although there was significantly less lean mass in both congenics compared with in controls, the decline in weight gain in the LDLR–/– MOB6 congenics was attributed to a dramatic drop in fat mass accretion, even though they consumed as much food as the obese LDLR–/– controls (Fig. 3). The decline in fat accumulation is suggestive of poor caloric utilization because LDLR–/– MOB6 congenics require more energy input to produce a gram of body mass compared with LDLR–/– MOB5 and LDLR–/– strains. However, the possibility of malabsorption of nutrients by the intestine cannot be ruled out.
Plasma lipid and glucose levels were greatly increased, despite decreased adipose storage in the LDLR–/– MOB6 mice. In this regard, the dyslipidemic, hyperglycemic condition in the LDLR–/– MOB6 congenics (Fig. 4) is similar to that seen in lipodystrophy (16). However, unlike lipodystrophy, glucose tolerance was increased in LDLR–/– MOB6 congenics compared with obese LDLR–/– controls. Therefore, although the lack of adipose stores predisposed the LDLR-MOB6 strain to elevated plasma lipids and glucose, the responses to glucose challenge and plasma insulin concentrations remained intact. We hypothesized that during times of food deprivation, all of the energy stores must be readily available in the plasma because the LDLR–/– MOB6 mice did not maintain enough reserves in the form of adipose. However, the degree of systemic steatosis caused by increased plasma triglycerides, free fatty acids, and hyperglycemia was not enough to trigger the insulin resistance associated with lipodystrophy (16).
Conversely, plasma lipid and glucose concentrations were decreased in LDLR–/– MOB5 congenics, but these mice cleared glucose no faster than LDLR–/– controls and showed decreased insulin sensitivity (Figs. 2 and 5). These data indicated a detachment between the dyslipidemia and hyperglycemia typically associated with insulin resistance. However, differences in non–insulin-mediated glucose uptake (17,18) may contribute to the discrepancy between insulin metabolic studies and plasma insulin and glucose assays.
Curiously, an improved cholesterol profile failed to protect the LDLR–/– MOB5 congenics from increased atherogenesis (Figs. 1 and 4), suggesting that genetic regulation of other atherosclerosis risk factors, such as an altered inflammatory response (19) or differences in adiposity (20) unique to the MOB5 locus (162–180 Mb), were responsible. Because both congenics showed decreases in total HDL, LDL, and unesterified cholesterol (25–40%) and insulin (nearly 50%), we hypothesized that the responsible genes lie within the genomic interval shared by both strains, 87–162 Mb.
Care must be taken in generalizing results from mice to humans. For instance, LDLR–/– controls exhibited cholesterol levels far in excess of those normally observed in humans with insulin resistance. However, given the concordance between mice, humans, and rats for traits associated with cardiovascular complications of insulin resistance (supplemental data, which is detailed in the online appendix), it appears that genetic perturbations in MOB5 and -6 loci on mouse chromosome 2 contribute importantly to several complex metabolic disorders relevant to humans.
We used overlapping congenic strains to better localize genetic influences underlying atherogenesis, obesity, and insulin resistance. Although these congenic intervals harbor hundreds of genes, we hypothesize that the MOB6-specific interval (72–84 Mb) is involved in the increased insulin sensitivity observed in this strain. This locus contains 283 genes, including Epac2, which is involved in -cell exocytosis (21), and NeuroD1, which is implicated in -cell neogenesis in obesity (22), as well as a maturity-onset diabetes of the young (MODY) candidate gene (23). Similarly, we hypothesize that the MOB5-specific interval (162–180 Mb) underlies the insulin resistance seen in this strain. This region of 225 genes includes hepatocyte nuclear factor 4 (HNF4), which was recently implicated in type 2 diabetes in human populations (24), as well as MODY (25). It also includes CCAAT/enhancer-binding protein (C/EBP), (CEBPB), which is involved in the regulation of adipogenesis (26) and -interferon inflammatory response (27). The increasing availability of dense single-nucleotide polymorphism maps (28–30) and strain-specific sequence information will greatly speed the identification and validation of candidate genes for these complex traits.
The fact that quantitative trait loci are concordant in humans and rats suggests that a common set of genes and pathways underlie these traits. Thus, we propose that characterizing concordant genetic models will greatly assist our understanding of the syntenic loci for obesity and insulin resistance in humans. Congenic mouse strains are complex genetic systems that model interactions between multiple human syntenic loci linked to many common diseases. Thus, they provide powerful tools for dissecting the interactions between the multiple genes and metabolic pathways that underlie these complex disorders.
ACKNOWLEDGMENTS
This work was supported by an National Science Foundation fellowship (to D.E.-S.) from 1999 to 2002 and by National Institutes of Health Fellowship 1F31DK63928 (to D.E.-S.) and National Institutes of Health Grants HL30568, HL70526, and HL60030 (to A.J.L. and R.C.D.).
FOOTNOTES
Additional information can be found in an online appendix at http://diabetes.diabetesjournals.org.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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2 Department of Medicine/Division of Endocrinology, Diabetes and Hypertension, University of California Los Angeles, Los Angeles, California
3 Department of Medicine, Division of Cardiology, University of California Los Angeles, Los Angeles, California
AUC, area under the curve; NMR, nuclear magnetic resonance
ABSTRACT
Previous characterization of mouse chromosome 2 identified genomic intervals that influence obesity, insulin resistance, and dyslipidemia. For this, resistant CAST/Ei (CAST) alleles were introgressed onto a susceptible C57BL/6J background to generate congenic strains with CAST alleles encompassing 67–162 Mb (multigenic obesity 6 [MOB6]) and 84–180 Mb (MOB5) from mouse chromosome 2. To examine the effects of each congenic locus on atherosclerosis and glucose disposal, we bred each strain onto a sensitizing LDL receptor–null (LDLR–/–) C57BL/6J background to predispose them to hypercholesterolemia and insulin resistance. LDLR–/– congenics and controls were characterized for measures of atherogenesis, insulin sensitivity, and obesity. We identified a genomic interval unique to the MOB6 congenic (72–84 Mb) that dramatically decreased atherosclerosis by approximately threefold and decreased insulin resistance. This region also reduced adiposity twofold. Conversely, the congenic region unique to MOB5 (162–180 Mb) increased insulin resistance but had little effect on atherosclerosis and adiposity. The MOB congenic intervals are concordant to human and rat quantitative trait loci influencing diabetes and atherosclerosis traits. Thus, our results define a strategy for studying the poorly understood interactions between diabetes and atherosclerosis and for identifying genes underlying the cardiovascular complications of insulin resistance.
Insulin resistance increases the risk of hypertension, obesity, and dyslipidemia and is thereby a principal risk factor for cardiovascular disease (1–4), but the mechanisms involved are largely unknown (3–5). Loci on mouse chromosome 2 are concordant (6) for insulin resistance and atherosclerosis quantitative trait loci from homologous genomic intervals in rats, mice, and humans (supplemental data, which is detailed in the online appendix [available at http://diabetes.diabetesjournals.org]). Previously, we characterized overlapping multigenic obesity (MOB)5 and -6 congenic mice (7) for these traits to better understand their impact. Here, chromosome 2 alleles from CAST/Ei (CAST) mice that are resistant to obesity were introgressed onto an obesity-susceptible C57BL/6J background (available online from http://phenome.jax.org/pub-cgi/phenome). We showed that MOB5 congenics became insulin resistant, suggesting that genes underlying this trait resided in the congenic interval unique to this strain (CAST alleles from 162 to 180 Mb). By contrast, MOB6 congenics displayed an atherosclerosis-susceptible cholesterol profile when fed a diet high in fat, cholesterol, and cholic acid, suggesting that genes in the interval unique to this strain (CAST alleles from 72 to 84 Mb) underlie the atherogenic cholesterol profile (7). However, the experimental diet used in that study, though enriched in lipid and cholesterol, did not trigger atherogenesis or obesity in these strains.
C57BL/6J (B6) mice with homozygous null alleles for the LDL receptor (LDLR–/–) become obese and develop atherosclerosis associated with hypertriglyceridemia, hyperglycemia, hyperinsulinemia, and decreased plasma HDL (8,9) when fed a Western diet for 16–18 weeks. To determine how CAST alleles from the MOB chromosome 2 loci modify the dyslipidemia, insulin resistance, and atherosclerosis of the LDLR–/– strain, we bred each congenic strain onto the LDLR–/– background. LDLR–/– MOB5 and LDLR–/– MOB6 congenic strains possess genetic variations that mimic the composite interactions between human syntenic loci that underlie multiple insulin resistance risk factors for cardiovascular disease. Introgressing the MOB loci onto the LDLR–/– background susceptible to the cardiovascular vascular complications of insulin resistance greatly enabled the dissection of genetic interactions that characterize these multigenic disorders.
RESEARCH DESIGN AND METHODS
Animals were housed in an Association for Assessment and Accreditation of Laboratory Animal Care–accredited, specific pathogen-free facility under a 12-h light/dark cycle with free access to food and water. Male MOB5 (B6.CAST-D2mit 9 to D2Mit 50) and MOB6 (B6.CAST-D2Mit 14 to D2Mit457) congenic mice (7) were bred to female LDLR–/– (Ldlrtm1Her/Ldlrtm1Her) mice (10) from The Jackson Laboratory (stock no. 2207; Bar Harbor, ME). Male N1 mice were backcrossed to LDLR–/– females, and N2 offspring homozygous for LDLR null allele and heterozygous for the congenic region were intercrossed to obtain mice homozygous for LDLR null allele and the congene. To maintain the integrity of the MOB congenic loci, internal markers (D2Mit19, 133 Mb; and D2Mit92, 71 Mb) were also genotyped. Positions for microsatellite markers were determined using the Mouse Genome Browser Gateway from Genome Bioinformatics of the University of California Santa Cruz (available from http://genome.ucsc.edu/cgi-bin/hgGateway, mm6 assembly).
At 21 days of age, mice were weaned to chow containing 6% fat by weight (no. 7013; Harlan Teklad, Madison WI). At 12 weeks, males were randomly assigned to chow or Western diet (40% of calories from fat, 17% from protein, and 43% from carbohydrate; no. R12079B; Research Diets, New Brunswick, NJ). At weekly intervals mice were weighed and characterized for fat and lean mass by nuclear magnetic resonance (NMR) during the diet period (from 12 to 30 weeks of age). Selected mice from each strain were characterized for glucose tolerance and insulin sensitivity. Mice were killed at 28–30 weeks of age. Randomly selected LDLR–/– and LDLR–/– MOB congenics were characterized for atherosclerosis.
Atherosclerotic lesion analysis.
Nonfasted 28- to 30-week-old male mice were killed by Isoflurane vapor (Abbott Laboratories, Chicago, IL) after 16–18 weeks on Western diet. Analysis of 30 sequential hematoxilin and eosin–stained aortic root sections was used to assay for morphology of the aortic root. Aortic roots in 12–13 males from each strain were scored for foam cell content and presence of a fibrous cap (11). In addition, whole aortas from 10 mice per strain were analyzed for total fatty streak lesion area by Sudan V staining, using the en face technique (8).
Plasma lipid, insulin, and glucose assays.
Plasma samples were collected at death after a fast of 9 h beginning between 5:00 and 7:00 A.M. Blood plasma drawn from the retro-orbital sinus under isoflurane anesthesia was assayed for lipids (free fatty acids and triglycerides), cholesterol (total, HDL, and unesterified), insulin, and glucose (12). VLDL/LDL concentrations were determined by subtracting HDL from total cholesterol (7).
Intraperitoneal glucose tolerance test and insulin sensitivity index.
Intraperitoneal glucose tolerance tests and insulin sensitivity index (Si) were performed as previously described (13). For intraperitoneal glucose tolerance testing, 5–16 male mice from each strain were fasted for 9 h and injected with glucose (2 g/kg body wt i.p.) in saline (American Pharmaceutical Partners, Schaumburg, IL). Glucose was measured from the blood of a saphenous vein puncture at 0, 30, 60, and 120 min, using a OneTouch Ultra Glucometer (Lifescan, Milpitas, CA). Differences in glucose clearance were measured as changes in the area under the curve (AUC) determined using the composite trapezoid rule:
The Si was evaluated in five to six male congenics and LDLR–/– controls. After 16–17 weeks on a Western diet, mice were fasted for 4 h and injected with porcine pancreatic insulin (Sigma-Aldrich, St. Louis, MO) at 2 IU/kg body wt i.p. in 200 μl saline (American Pharmaceutical Partners, Schaumburg, IL). Blood glucose was measured at 0, 15, 30, and 90 min postinjection. Si (13) was measured by the slope of the glucose concentration curve (Ki) over the first 15 min: Ki = [(glucose)t = 15 – (glucose)t = 0]/15 min.
NMR analysis.
Fat mass and lean mass were monitored weekly with a mouse Minispec (Bruker Woodlands, TX), using software from Echo Medical Systems (Houston, TX). The NMR spectroscopy apparatus (14) gave fat and lean mass measurements with coefficients of variation of <3%. Correlation between NMR and gravimetric measurements is better than 0.99.
Food efficiency and food intake.
Food efficiency (food required to produce 1 g of body mass) was measured as previously described (15). Weekly, mice and the food from each cage were weighed. Bedding was checked to ensure that no food was present. Cages that consistently contained food in the bedding were dropped from the analysis. Total amount of food consumed was divided by the change in body weight over the diet period to calculate food efficiency for each strain. To determine differences in food consumption between the strains, four mice per strain were singly housed and food weighed weekly, as above.
Statistical analysis.
The differences between individual groups were determined by ANOVA, using StatView 5.0 (SAS Institute).
RESULTS
CAST alleles from the MOB6 locus were associated with decreased atherosclerotic lesion formation and improved insulin sensitivity.
LDLR–/– mice develop extensive atherosclerotic lesions following a Western diet. However, when compared with LDLR–/– controls, LDLR–/– MOB6 congenics showed a two- to threefold decrease in both aortic root lesion area (Fig. 1A) and total aortic lesion area measured by en face analysis (Fig. 1B). By contrast, lesion areas in LDLR–/– MOB5 congenics were comparable to controls. Although there were differences in lesion area, there were no differences in morphology between strains. These data were especially striking because both LDLR–/– MOB congenic strains displayed decreased plasma cholesterol concentrations (Fig. 4).
Chow-fed LDLR–/– and LDLR–/– congenics developed 9- to 15-fold less aortic root lesion area than the same mice fed a Western diet. Compared with controls, both congenic strains now demonstrated a two- to threefold decrease in aortic lesion area, whereas plasma cholesterol concentrations were not significantly different between the strains (supplemental Figs. 2 and 4, which are detailed in the online appendix). No differences were observed in lesion morphology between controls and LDLR–/– MOB congenic strains.
Compared with LDLR–/–, LDLR–/– MOB6 congenics fed a Western diet had markedly improved glucose tolerance when challenged with an intraperitoneal glucose load (2 g/kg body wt i.p.). Here, we compared strains for areas under the plasma glucose curve (AUCs) as a function of time (Fig. 2A) after the glucose bolus (see RESEARCH DESIGN AND METHODS). The AUCs for LDLR–/– MOB6 and parental MOB6 congenics (37,500 and 35,100, respectively) were significantly lower than LDLR–/– (44,700). LDLR–/– MOB5 congenics showed no difference in response to glucose challenge compared with LDLR–/– controls. Unexpectedly, the parental MOB5 congenics had significantly impaired glucose clearance (AUC = 53,500) compared with LDLR–/–. These results suggested that sequences unique to the MOB5 congenic interval (162–180 Mb) carry allelic variations that strongly impact glucose clearance. Consistent with this hypothesis, we observed similarly decreased glucose clearance in preliminary experiments with a subcongenic carrying CAST alleles only from the region 172–180 Mb (supplemental data, which is detailed in the online appendix).
Si was used to determine sensitivity to insulin-stimulated glucose uptake (Fig. 2B). LDLR–/– MOB5 and parental MOB5 strains were significantly more resistant to insulin-stimulated glucose uptake (Ki = –1.52 and Ki = –0.72, respectively) than LDLR–/– controls (Ki = –3.44). By contrast, we observed increased sensitivity to insulin (Ki = –7.722) in parental MOB6 congenics. When fed a chow diet, all strains had markedly improved glucose clearance, but no significant differences were observed between the strains (supplemental Fig. 2, which is detailed in the online appendix).
CAST alleles in the MOB6 locus diminished the obesity of LDLR–/– mice.
LDLR–/– MOB6 congenics fed a Western diet were markedly less obese than controls, as measured by a dramatic drop in weight gain and NMR assessment of fat mass–to–lean mass ratio (Figs. 3 and Table 1). We observed a significant decline in lean mass for both of the LDLR–/– MOB5 and -6 congenics compared with controls (Figs. 3C and Table 1). However, the decreased adiposity of the LDLR–/– MOB6 congenics was attributable to a highly significant twofold reduction in fat mass (Figs. 3D and Table 1) compared with LDLR–/– controls. A small but significant decrease in final mean weight was observed in the LDLR–/– MOB5 congenics, with no significant difference in fat mass–to–lean mass ratio.
LDLR–/– MOB6 congenics fed a Western diet showed no significant difference in the amount of food consumed over the 16-week diet period compared with LDLR–/– controls, whereas LDLR–/– MOB5 congenics consumed significantly more (Figs. 3E and Table 1). LDLR–/– MOB6 congenics required two times more food to amass 1 g of body weight (food efficiency) than LDLR–/– controls (Figs. 3F and Table 1). This difference in food efficiency may arise from differences in energy expenditure or nutrient absorption. Further studies are required to determine the underlying mechanism and to identify the responsible genes. There was no significant difference in food efficiency between LDLR–/– MOB5 congenics and LDLR–/– controls.
On a chow diet, LDLR–/– control and LDLR–/– MOB5 strains weighed 10 g less than Western diet–fed mice, whereas LDLR–/– MOB6 congenics weighed 5 g less than the same strain fed a Western diet (supplemental data, which is detailed in the online appendix). Similar differences were observed for NMR traits (fat mass–to–lean mass ratios, fat mass, and lean mass), food intake, and food efficiency (supplemental Fig. 3, which is detailed in the online appendix).
CAST alleles from both MOB5 and MOB6 loci improved plasma cholesterol profiles, but only CAST alleles from the MOB5 locus reduced the dyslipidemia and hyperglycemia of LDLR–/– mice.
We observed a modest but significant decrease in plasma total cholesterol of both LDLR–/– MOB6 and LDLR–/– MOB5 compared with LDLR–/– controls. A similar drop in plasma HDL, LDL plus VLDL, and unesterified cholesterol was seen in both LDLR–/– congenics compared with controls (Fig. 4). In the absence of LDL receptors, it is clear that other mechanisms are required to explain the lower LDL levels in the congenic strains compared with background LDLR–/– mice. This may simply result from a decline in overall cholesterol pools because of decreased synthesis, or it may result from activation of an alternate clearance pathway.
Compared with LDLR–/–, LDLR–/– MOB5 congenics developed markedly lower plasma triglycerides, free fatty acids, and glucose when fed a Western diet (Fig. 5). By contrast, LDLR–/– MOB6 congenics were quite similar to LDLR–/– controls, although both LDLR–/– MOB congenic strains displayed lower plasma insulin compared with LDLR–/–. We hypothesize that the lack of weight gain, given the dyslipidemia and hyperglycemia of the LDLR–/– MOB6 congenics, suggests a defect in lipid storage in this strain.
When fed a chow diet, plasma cholesterol concentrations (with the exception of HDL) declined in all strains. Interestingly, LDLR–/– MOB6 animals showed a significant decline in HDL comparable to Western diet–fed mice. No difference in HDL was observed between LDLR–/– MOB5 congenics and controls (supplemental data, which is detailed in the online appendix). Chow-fed LDLR–/– mice and LDLR–/– MOB congenics also had lower plasma triglycerides and free fatty acids compared with the same mice fed a Western diet, but no significant differences were seen in these traits between congenics and controls (supplemental Fig. 2, which is detailed in the online appendix).
DISCUSSION
Robust (50–70%) reductions in atherosclerotic lesion area observed in MOB6 congenics suggest an atheroprotective locus in the genomic interval unique to that strain, 72–84 Mb (Fig. 1). Similar to data reported by Merat et al. (9), LDLR–/– controls showed profound insulin resistance as assayed by glucose tolerance testing after long-term exposure to high caloric intake. CAST alleles from each MOB genomic interval effectively modified the insulin resistance–related phenotypes observed in the LDLR–/– controls (Fig. 2). When compared by AUC, the LDLR–/– MOB6 congenic mice were able to clear glucose significantly (P = 0.008) faster than controls. Notably, although LDLR–/– MOB5 showed glucose clearance similar to that of LDLR–/– controls, clearance in parental MOB5 congenics was significantly (P = 0.004) impaired compared with LDLR–/– controls (Fig. 2). Although it cannot be generally assumed that impact of the congenic loci is the same in the presence or absence of the LDL receptor, the glucose clearance data suggest that the decline in glucose tolerance in the MOB5-derived congenics was independent of the LDLR–/– mutation. Similarly, insulin sensitivity, as measured by Ki, was significantly decreased in both LDLR–/– MOB5 (P = 0.02) and parental MOB5 congenics (P = 0.009), but Ki was significantly increased (P = 0.0002) in parental MOB6 congenics compared with controls. Thus, we hypothesize that CAST alleles from a locus unique to the MOB5 congenic interval (162–180 Mb) influenced the decrease in insulin sensitivity and glucose clearance, whereas a second CAST locus unique to the MOB6 genomic interval (72–84 Mb) influenced an increase in these measures (Fig. 2). Initial experiments with subcongenics specific to these regions support this hypothesis, but, given the size and complex genetic architecture of the MOB5 and -6 congenic intervals, genetic interactions between the MOB unique intervals and the shared genomic region cannot be ruled out.
Similar to previous results observed in the parental MOB congenics (7), the LDLR–/– MOB6 congenics gained weight at a significantly decreased rate when compared with control and LDLR–/– MOB5 strains. Additionally, although there was significantly less lean mass in both congenics compared with in controls, the decline in weight gain in the LDLR–/– MOB6 congenics was attributed to a dramatic drop in fat mass accretion, even though they consumed as much food as the obese LDLR–/– controls (Fig. 3). The decline in fat accumulation is suggestive of poor caloric utilization because LDLR–/– MOB6 congenics require more energy input to produce a gram of body mass compared with LDLR–/– MOB5 and LDLR–/– strains. However, the possibility of malabsorption of nutrients by the intestine cannot be ruled out.
Plasma lipid and glucose levels were greatly increased, despite decreased adipose storage in the LDLR–/– MOB6 mice. In this regard, the dyslipidemic, hyperglycemic condition in the LDLR–/– MOB6 congenics (Fig. 4) is similar to that seen in lipodystrophy (16). However, unlike lipodystrophy, glucose tolerance was increased in LDLR–/– MOB6 congenics compared with obese LDLR–/– controls. Therefore, although the lack of adipose stores predisposed the LDLR-MOB6 strain to elevated plasma lipids and glucose, the responses to glucose challenge and plasma insulin concentrations remained intact. We hypothesized that during times of food deprivation, all of the energy stores must be readily available in the plasma because the LDLR–/– MOB6 mice did not maintain enough reserves in the form of adipose. However, the degree of systemic steatosis caused by increased plasma triglycerides, free fatty acids, and hyperglycemia was not enough to trigger the insulin resistance associated with lipodystrophy (16).
Conversely, plasma lipid and glucose concentrations were decreased in LDLR–/– MOB5 congenics, but these mice cleared glucose no faster than LDLR–/– controls and showed decreased insulin sensitivity (Figs. 2 and 5). These data indicated a detachment between the dyslipidemia and hyperglycemia typically associated with insulin resistance. However, differences in non–insulin-mediated glucose uptake (17,18) may contribute to the discrepancy between insulin metabolic studies and plasma insulin and glucose assays.
Curiously, an improved cholesterol profile failed to protect the LDLR–/– MOB5 congenics from increased atherogenesis (Figs. 1 and 4), suggesting that genetic regulation of other atherosclerosis risk factors, such as an altered inflammatory response (19) or differences in adiposity (20) unique to the MOB5 locus (162–180 Mb), were responsible. Because both congenics showed decreases in total HDL, LDL, and unesterified cholesterol (25–40%) and insulin (nearly 50%), we hypothesized that the responsible genes lie within the genomic interval shared by both strains, 87–162 Mb.
Care must be taken in generalizing results from mice to humans. For instance, LDLR–/– controls exhibited cholesterol levels far in excess of those normally observed in humans with insulin resistance. However, given the concordance between mice, humans, and rats for traits associated with cardiovascular complications of insulin resistance (supplemental data, which is detailed in the online appendix), it appears that genetic perturbations in MOB5 and -6 loci on mouse chromosome 2 contribute importantly to several complex metabolic disorders relevant to humans.
We used overlapping congenic strains to better localize genetic influences underlying atherogenesis, obesity, and insulin resistance. Although these congenic intervals harbor hundreds of genes, we hypothesize that the MOB6-specific interval (72–84 Mb) is involved in the increased insulin sensitivity observed in this strain. This locus contains 283 genes, including Epac2, which is involved in -cell exocytosis (21), and NeuroD1, which is implicated in -cell neogenesis in obesity (22), as well as a maturity-onset diabetes of the young (MODY) candidate gene (23). Similarly, we hypothesize that the MOB5-specific interval (162–180 Mb) underlies the insulin resistance seen in this strain. This region of 225 genes includes hepatocyte nuclear factor 4 (HNF4), which was recently implicated in type 2 diabetes in human populations (24), as well as MODY (25). It also includes CCAAT/enhancer-binding protein (C/EBP), (CEBPB), which is involved in the regulation of adipogenesis (26) and -interferon inflammatory response (27). The increasing availability of dense single-nucleotide polymorphism maps (28–30) and strain-specific sequence information will greatly speed the identification and validation of candidate genes for these complex traits.
The fact that quantitative trait loci are concordant in humans and rats suggests that a common set of genes and pathways underlie these traits. Thus, we propose that characterizing concordant genetic models will greatly assist our understanding of the syntenic loci for obesity and insulin resistance in humans. Congenic mouse strains are complex genetic systems that model interactions between multiple human syntenic loci linked to many common diseases. Thus, they provide powerful tools for dissecting the interactions between the multiple genes and metabolic pathways that underlie these complex disorders.
ACKNOWLEDGMENTS
This work was supported by an National Science Foundation fellowship (to D.E.-S.) from 1999 to 2002 and by National Institutes of Health Fellowship 1F31DK63928 (to D.E.-S.) and National Institutes of Health Grants HL30568, HL70526, and HL60030 (to A.J.L. and R.C.D.).
FOOTNOTES
Additional information can be found in an online appendix at http://diabetes.diabetesjournals.org.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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