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APPETITE, OBESITY AND METABOLISM
1Neurology Service, Department of Veterans Affairs Medical Center, East Orange 07018; and 2Department of Neurosciences, New Jersey Medical School, Newark, New Jersey 07103; and 3Division of Molecular Genetics, Department of Pediatrics, Columbia University, New York, New York 10032
Submitted 14 May 2003 ; accepted in final form 7 July 2003
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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diet-induced obesity; leptin; food intake; exercise; insulin resistance
One consequence of the selective breeding process is that, unlike the outbred rats, the selectively bred DIO rats become hyperphagic and begin to gain excess weight by 4-6 wk of life, even when fed chow from weaning (9, 13). Thus, by 10-12 wk of age, selectively bred chow-fed DIO rats become considerably heavier than comparable DR rats (8, 13). However, even though the selective breeding process produces DIO rats, which are somewhat different from the outbred parent strain, the intrinsic DIO phenotype is maintained when they are fed the HE diet (8, 13). The present studies were undertaken with the hope of using these selectively bred DIO rats to confirm that the DIO phenotype is, indeed, a heritable trait. Toward this end, we crossed selectively bred DIO rats with the relatively obesity-resistant inbred Fischer F344 strain (11). This was followed by successive cycles of inbreeding the resultant offspring. Using this strategy, we have developed a new strain of rats that gain weight on low-fat chow almost comparably to F344 rats, but maintain the DIO trait when fed a HE diet. In addition, these rats are glucose intolerant even on a low-fat diet. They are named F.DIO to reflect their parental backgrounds (Fischer F344 x DIO).
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METHODS |
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For production of the first (N1) generation, each of four selectively bred DIO males was mated with three Fischer F344 females (Charles River Laboratories, Kingston, NY). From the 12 potential breeding pairs, there were nine successful pregnancies, producing 20 male and 19 female N1 offspring. These offspring were placed with four of the original dams in litters of 9-10 pups each at a male-to-female pup ratio of 
1:1. Offspring of all subsequent breeding cycles were similarly handled with regard to litter size and sex ratio. At 10 wk of age, one-half of all N1 males and females were placed on HE diet. The other one-half were kept on chow for 3 wk. Ten randomly selected (without regard to body weight gain on either chow or HE diet) male N1 rats were paired with 12 F344 females. This produced 12 successful pregnancies that yielded 65 male and 33 female N2 offspring. The entire N2 generation was placed on HE diet for 3 wk at 10 wk of age for weight-gain phenotyping. Of these N2 rats, data from the 10 highest male and 10 highest female weight gainers after 3 wk of HE diet were used for comparison to other groups (Fig. 1). The 10 highest male N2 weight gainers were then crossed with one F344 female each. The N3 offspring of these matings (46 males, 58 females) were designated as F.DIO. Initial phenotyping of these F.DIO rats was carried out by either placing 10-wk-old, randomly selected F.DIO male (n = 8-9) or female (n = 10-11) and F344 male (n = 8) or female (n = 8) rats on HE diet or continuing them on chow for 3 wk. From this first generation of F.DIO rats, three successive additional generations of F.DIO rats were produced from pairings of 4-5 male and 8-10 female F.DIO breeders per generation. Selection of these breeders was made without regard to weight-gain phenotype. Offspring of these F.DIO x F.DIO crosses were used to assess running wheel activity (generation 2) and glucose tolerance (generation 3).
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Spontaneous wheel running. At 10 wk of age, seven chow-fed F.DIO generation 2 offspring and seven chow-fed F344 male rats were given continuous access to a running wheel in their home cage for 14 days. Animals were allowed ad libitum access to chow and water during this time. Data were collected remotely by a computerized system (Mini Mitter), and the data were expressed as the number of revolutions/12 h for the dark (1801-0600) and light (0601-1800) cycles, respectively.
Oral glucose tolerance test. Groups of eight male F.DIO (generation 3) and eight male F344 rats were fed chow from weaning until 10 wk of age. They were fasted overnight, and a baseline tail blood sample was taken for glucose, insulin, and leptin levels. They were then gavaged with glucose (0.5 g/kg body wt), and repeated tail blood samples of 0.25 ml were taken at 15, 30, 60, 90, and 120 min. They were then placed on the HE diet for 3 wk, and the glucose tolerance test was repeated (8).
Plasma glucose, insulin, and leptin levels. Glucose levels were measured by automated glucose oxidase method (Beckman). Insulin and leptin levels were measured by using radioimmunoassay kits (Linco).
Statistics. Parameters were compared among various groups [DIO, F344, N1, N2, N3 (F.DIO)] by one-way ANOVA with post hoc Scheffé comparisons. Data from each sex were analyzed separately. For the glucose tolerance test, areas under the curve for glucose and insulin were calculated as change from baseline by using the trapezoidal rule (Graph-Pad Prism 3.0 software). Glucose and insulin curves were also compared by using repeated-measures ANOVA. Feed efficiency was calculated by dividing the body weight gain by the calories of diet ingested during 3 wk on either chow or HE diet.
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RESULTS |
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Overall, caloric intake of the HE diet in both F344 rats and the first generation of F.DIO rats was greater than that of chow [Table 1; F(1,31) = 13.67; P = 0.001]. However, post hoc analysis showed that it was only the F.DIO rats that ate more on the HE diet than their chow-fed counterparts. Intake of HE diet led to a generally higher feed efficiency than when rats of both genotypes were fed chow [F(1,31) = 4.41; P = 0.044]. But again, post hoc analysis showed that only the F.DIO males on the HE diet had higher feed efficiency than their respective chow-fed counterparts.
Consumption of HE diet led to an overall increase in total fat pad weights (epididymal, retroperitoneal, perirenal, and mesenteric) for both male F.DIO and F344 rats [Table 1; F(1,20) = 9.21; P = 0.007]. However, whereas there was no statistically significant difference in fat pad weights between chow-fed F.DIO and F344 rats, 3 wk on the HE diet increased F.DIO fat pad weights by 85% and F344 fat pad weights by only 71% compared with their respective chow-fed controls. Thus F.DIO rats on HE diet had the heaviest fat pad weights of all groups by post hoc analysis (Table 1). Similarly, as a percentage of total body weight, total fat pad weights did not differ between chow-fed F.DIO and F344 males, but F.DIO rats on the HE diet had heavier fat pads as a percentage of body weight than comparable F344 rats [Table 1; F(1,20) = 8.91; P = 0.008]. The increase in fat pad weights associated with HE diet intake was reflected in higher plasma leptin levels in F.DIO (132%) and F344 (70%) rats compared with their respective chow-fed controls [F(1,20) = 71.16; P = 0.0001]. However, F.DIO males had higher leptin levels than F344 males, regardless of diet [F(1,20) = 0.030]. On the HE diet, F.DIO leptin levels were 98% higher than those of comparable F344 rats by post hoc analysis (Table 1). In addition to heavier adipose pads, F.DIO males on the HE diet had 20% heavier livers than their chow-fed counterparts, whereas there was no diet effect on liver weights in F344 males (Table 1).
Fasting (Fig. 3) and nonfasting (Table 1) basal glucose levels did not differ significantly between chow-fed male F.DIO and F344 males. Neither did they differ in their plasma glucose response (area under the curve) to an oral glucose load (Fig. 3; Table 1). On chow, fasting insulin levels did not differ between F.DIO and F344 males. But F.DIO rats had a 96% greater insulin response (area under the curve) than F344 rats after an oral glucose load. Intake of HE diet for 3 wk altered neither fasting (Fig. 3) nor nonfasting (Table 1) basal glucose levels in F.DIO or F344 rats. However, F.DIO fasting insulin levels rose by 168% (Fig. 3) and nonfasting levels by 176% (Table 1). Furthermore, their glucose response to oral glucose was increased by 60%, and their insulin response by 370%, compared with their own chow-fed responses (Table 1, Fig. 3). In fact, HE diet exposure was associated with a heightened insulin response to oral glucose in both F.DIO and F344 males [F(1,28) = 7.51; P = 0.011], although the increase in F344 rats (96%) over their chow-fed responses was less than that in F.DIO rats (Table 1, Fig. 3). Thus F.DIO rats had an abnormal insulin response to an oral glucose load, even when fed chow from weaning, and this response was markedly exaggerated and accompanied by an abnormal glucose response after 3 wk on the HE diet.
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As with the male rats, there was a progressive reduction in chow-fed body weights in female offspring with each subsequent backcross to F344 rats. At 10 wk of age, chow-fed female DIO rats weighed 59% more than F344 females (Fig. 4). The chow-fed N1 females weighed 37% more and the N2 females weighed only 15% more than F344 females. By the N3 generation, chow-fed F.DIO females weighed 10% more than comparable chow-fed F344 females [Table 2, Fig. 4; F(1,31) = 18.94; P = 0.001]. Intake of HE diet for 3 wk resulted in a significant genotype [F(1,32) = 13.21; P = 0.001] and diet [F(1,32) = 9.71; P = 0.004] effect (Table 2; Fig. 4). F.DIO females gained 152%, whereas F344 females gained only 89% more body weight than their respective chow-fed controls. As was seen in the male rats, there was a wider spread of body weight gains in F.DIO female rats on chow for 3 wk (4-29 g) than in F344 females (7-20 g; Fig. 5). However, unlike the males, intake of HE diet for 3 wk produced a wider spread of body weight gain in F.DIO (26-58 g) than F344 females (16-35 g; Fig. 5).
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Overall, F.DIO females consumed more calories than F344 females, regardless of diet [Table 2; F(1,32) = 30.42; P = 0.0001], and this was accentuated by intake of HE diet [F(1,32) = 17.05; P = 0.0001]. F.DIO females ate 16% more calories as chow and 22% more as HE diet than F344 females. Increased body weight gain on HE diet was associated with a 16% greater intake in F.DIO females and 11% more in F344 females compared with their respective chow-fed controls. F.DIO females also had higher overall feed efficiency than F344 females, independent of diet [Table 2; F(1,32) = 6.56; P = 0.014]. This difference was significant by post hoc comparison only on the HE diet, where only F.DIO females had higher feed efficiency (115%) than their respective chow-fed controls.
When considered across all groups, F.DIO females had heavier fat pad weights (ovarian, retroperitoneal, perirenal, and mesenteric) than F344 rats, regardless of diet [Table 2; F(1,20) = 15.14; P = 0.001]. There was no significant difference in total fat pad weights between chow-fed F.DIO and F344 females (Table 2). Whereas fat pad weights were increased in both strains by intake of the HE diet, this effect was 36% greater in F.DIO females. However, both F.DIO and F344 females increased the weight of their adipose pads as a percentage of body weight comparably on the HE diet (Table 2). In keeping with the development of DIO in both F344 and F.DIO females, HE diet produced a significant increase in plasma leptin levels in both strains [F(1,32) = 46.12; P = 0.0001]. However, this diet-induced leptin increase was much greater in F.DIO females. F344 females on HE diet had 142% higher leptin concentrations, but F.DIO females had 340% higher leptin levels than their comparable chow-fed controls. Nonfasting basal insulin levels were also higher in F.DIO than F344 females, regardless of diet [F(1,32) = 16.25; P = 0.0001]. This effect was due primarily to the fact that HE diet intake increased nonfasting insulin levels only in F.DIO rats. Nonfasting plasma glucose levels differed as a function of neither genotype nor diet. Finally, F.DIO females had heavier livers than F344 females, regardless of diet [Table 2; F(1,20) = 8.64; P = 0.0001]. HE diet exposure increased liver weight in F.DIO females by 13%, but had no effect on F344 females compared with chow-fed controls.
Running-wheel activity. Chow-fed male F.DIO and F344 rats were evaluated for their dark vs. light cycle activity across 14 days of continuous access to running wheels in their home cages (Fig. 6; Table 1). There was enormous interindividual variability in both groups across the 14 days. F.DIO males ranged from 2,554 to 45,199 revolutions and F344 males from 3,383 to 40,099 revolutions per 24 h across the 14-day period of observation. Although F.DIO males tended to run more, the large variability in running resulted in no statistically significant difference from F344 rats during the dark cycle. However, F.DIO rats ran 53% less during the light cycle than F344 rats (P = 0.032). Because the light-cycle running made up only 4% of total running in F.DIO rats and 10% in F344 rats, the two strains did not differ from each other in total 24-h running across the entire 14-day period.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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The repetitive backcrossing of offspring against the F344 strain led to a progressive reduction in chow-fed body weight from that of the DIO to that of the F344 parent strain. Somewhat surprisingly, both male and female F.DIO rats have a fairly wide range of body weight gains compared with respective F344 rats during a 3-wk period on chow. Because chow-fed F.DIO males have slightly higher leptin levels than comparable F344 males, it is likely that they also have greater carcass adiposity (6). Some of this increased body weight and adipose gain on chow can be attributed to increased food intake in female but not male F.DIO rats compared with the F344 parent strain. But feed efficiency on chow does not differ between the genotypes. Thus, whereas chow-fed F.DIO rats do become slightly more obese than the parent F344 strain, the differences are quite small and differ markedly from the development of the obesity, which was described in the DIO parent strain when they were fed chow (8).
During 3 wk on the HE diet, both male and female F.DIO rats become more obese than comparable F344 rats. Although we did not carry out full carcass analysis on these animals, the combination of elevated fat pad weights and leptin levels in F.DIO rats fed the HE diet strongly suggests that they truly develop DIO. Whereas both F344 males and females on the HE diet also have heavier fat pads and/or leptin levels than comparable chow-fed controls, these changes are small compared with comparable F.DIO rats. This is in keeping with our laboratory's prior report (11) in male F344 rats, showing that they are relatively obesity resistant compared with outbred DIO rats. Whereas F.DIO male rats have a narrow, and probably unimodal, range of weight gains on the HE diet, F344 males have a wider range of weight gains. On the other hand, female F.DIO rats have a wider, but still probably unimodal, pattern of weight gain on the HE diet. Female F344 rats also have a wide range of weight gains on the HE diet. Although the numbers of animals tested were too small to be certain, the tight clustering of male weight gains on the HE diet supports the contention that the DIO phenotype was passed on to F.DIO rats as an inherited, polygenic trait. The wide spread of weight gains in chow-fed F.DIO rats might suggest that the DIO phenotype could be due to nongenetic factors. For example, inbred C57BL/6J mice show a highly variable weight gain and insulin sensitivity response to high-fat diet (4). Maternal factors might explain such variability in F.DIO rats (12), were it not for the fact that both F.DIO and F344 offspring had F344 mothers. Thus the strongest arguments favoring a heritable DIO trait in F.DIO rats are the tight clustering of weight gains on the HE diet and the preservation of the DIO phenotype after three successive backcrosses against obesity-resistant F344 rats, followed by three successive generations of inbreeding F.DIO with F.DIO rats.
As with weight gains on chow or HE diet, there is also a large interindividual variability in spontaneous running rates in both F.DIO and F344 males. Despite this inherent variability, chow-fed male F.DIO and F344 rats have comparable 24-h activity levels. If these running rates are related in any way to spontaneous activity in the home cage, it is unlikely that reduced physical activity accounts for the slightly greater weight gain of F.DIO rats on chow. However, the large variations in running rates might contribute to the similarly large variations in weight gain on chow of both F.DIO and F344 males. Interestingly, obese selectively bred DIO rats are less active in a running wheel than selectively bred DR rats (2). This appears to be an inherent trait of the DIO parent strain, because they also run less than selectively bred DR rats at 4-5wkof age, before DIO rats become obese (unpublished observation). However, even if reduced wheel activity is characteristic of the selectively bred DIO parent strain, this trait is not required for the development of the DIO phenotype in F.DIO rats.
In summary, we have derived a new strain of obesity-prone rats that exhibit DIO only when challenged with a diet relatively high in fat and caloric density. A striking new feature of this strain is the development of glucose intolerance, even when fed chow from weaning. As with the parent DIO strain (8), the F.DIO rats have an enormously exaggerated hyperinsulinemic response after only 3 wk on the HE diet. The preservation of the DIO trait, as well as the exaggeration of the diet-induced glucose intolerance seen in both DIO and F344 parent strains, despite three successive backcrosses against the obesity-resistant inbred F344 strain, followed by three successive cycles of inbreeding, suggests that both the DIO and glucose-intolerant phenotypes are inherited as a polygenic trait in this model.
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DISCLOSURES |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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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REFERENCES |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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0.05 by post hoc Scheffé test after ANOVA showed significant intergroup differences.
