A new obesity-prone, glucose intolerant rat strain (F.DIO)

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1 A new obesity-prone, glucose intolerant rat strain (F.DIO) Barry E. Levin 1,2, Ambrose A. Dunn-Meynell 1,2, Julie E. McMinn 3, Michael Alperovich 3, Amy Cunningham-Bussel 3, Streamson C. Chua, Jr. 3 Neurology Service, Department of Veterans Affairs Medical Center, E. Orange, NJ 718 1, Department of Neurosciences, New Jersey Medical School, Newark, NJ, 713 2, Division of Molecular Genetics, Department of Pediatrics, 115 St. Nicholas Avenue, Columbia University, New York, NY Running title: A new strain of diet-induced obese rats Key Words: diet-induced obesity, leptin, food intake, exercise, insulin resistance Corresponding Author: Barry E. Levin, MD Neurology Service (127C) VA Medical Center 385 Tremont Ave. E. Orange, NJ Tel: , x1442 Fax: levin@umdnj.edu 1

2 ABSTRACT Previous breeding for the diet-induced obese (DIO) trait from outbred Sprague-Dawley rats produced a substrain with selection characteristics suggesting a polygenic mode of inheritance. To assess this issue further, selectively bred DIO male rats were crossed with obesity-resistant inbred Fisher F344 dams. Male offspring were crossed twice more against female F344 dams. The resultant N3 (F.DIO) rats were then inbred 3 more times. On low fat chow, 1wk old male and female DIO rats weighed 86% and 59% more than respective F344 rats. By the N3 (F.DIO) generation, they were only 12% and 1% heavier, respectively. After 3 additional inbreeding cycles, chow-fed F.DIO males had an exaggerated insulin response to oral glucose compared to F344 rats. Following 3wk on a 31% fat (HE) diet, male N3 F.DIO rats gained 16-2% more carcass and adipose weight with 98% higher plasma leptin levels, while F.DIO females gained 36-54% more carcass and adipose weight with 13% higher leptin levels than comparable F344 rats. After 3 inbreeding cycles, F.DIO males still gained more weight on HE diet and developed a 3- fold greater insulin response to oral glucose than F344 males. Preservation of the DIO and glucose intolerance traits through successive backcrosses and inbreeding cycles to produce the F.DIO strain lends further support to he idea that they inherited in a polygenic fashion. 2

3 INTRODUCTION Diet-induced obesity (DIO) in the rat has proven to be a useful experimental surrogate for human obesity. When outbred Sprague-Dawley rats are placed on a diet with increased fat and caloric density (high energy [HE] diet), half of them overeat initially and develop DIO. The rest are diet-resistant (DR), gaining no more weight than rats fed a low fat, low caloric density chow diet (11). The development of DIO is also associated with hyperinsulinemia and insulin resistance (5;11), as well as hyperleptinemia (7). However, if outbred Sprague-Dawley rats are kept on chow throughout their lives, the DR and DIO weight gain phenotypes do not develop (11). This suggests that there is an interaction of diet composition with an underlying genetic predisposition to be obesity-prone or obesity-resistant. A polygenic mode of inheritance of the DIO and DR traits is suggested by the ability to selectively breed for these traits. After only 3-5 generations of breeding the highest (DIO) and lowest (DR) weight gainers on HE diet with opposite sex rats of the same weight gain phenotype, there is complete penetrance of the DIO and DR traits (8). Thus, these selectively bred DIO rats do appear to inherit their weight gain phenotype as a polygenic trait, similar to inheritance of most human obesity (3). Since that original study, we have subsequently bred more than 3 generations of DIO and DR rats with absolute maintenance of the DIO and DR phenotypes, respectively. 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 4-6wk of life, even when fed chow from weaning (9;13). Thus, by 1-12wk of age, selectively bred chow-fed DIO rats become considerably heavier than comparable DR 3

4 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 HE diet (8;13). The current studies were undertaken with the hope of using these selectively bred DIO rats to confirm that the DIO phenotype is, indeed, a heritable trait. Towards this end, we crossed selectively bred DIO rats with the relatively obesity-resistant inbred Fisher 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 which gains weight on low fat chow almost comparably to F344 rats but maintains 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 (Fisher F344 x DIO). METHODS Animals, diets and breeding schemes. Breeding was carried out originally using male rats from our in-house colony of rats bred selectively for the DIO trait (8). Animal usage was in compliance with the animal care committee of the E. Orange VA Medical Center and the guidelines of the American Physiological Society (1). Unless otherwise specified, rats were fed Purina rat chow (#51) and water ad libitum from weaning and were housed on a 12:12 light-dark schedule with lights out at 18. Purina rat chow contains 3.3 kcal/g with 23.4% as protein, 4.5% as fat and 72.1% as carbohydrate which is primarily in the form of complex polysaccharide (1). HE diet was used for metabolic and weight gain phenotyping. This diet is composed of 8% corn oil, 44% sweetened condensed milk and 48% Purina rat chow (Research Diets #C1124F, New Brunswick, 4

5 NJ). It contains 4.47 kcal/g with 21% of the metabolizable energy content as protein, 31% as fat and 48% as carbohydrate, 5% of which is sucrose (1). For the majority of experiments, rats were weighed weekly. Food intake was measured biweekly for 3wk on either chow or HE diet. At the end of each study, rats were decapitated in the non-fasted state between 8 and 11h with collection of trunk blood for plasma glucose, insulin and leptin levels. Epididymal (males) or ovarian (females), retroperitoneal, perirenal and mesenteric fat pads and livers were removed and weighed. For production of the first (N1) generation, each of 4 selectively bred DIO males was mated with 3 Fisher F344 females (Charles River Labs, Kingston, NY). From the 12 potential breeding pairs, there were 9 successful pregnancies producing 2 male and 19 female N1 offspring. These offspring were placed with 4 of the original dams in litters of 9-1 pups each at a male:female pup ratio of ~1:1. Offspring of all subsequent breeding cycles were similarly handled with regard to litter size and sex ratio. At 1wk of age, half of all N1 males and females were placed on HE diet. The other half were kept on chow for 3wk. 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 3wk at 1wk of age for weight gain phenotyping. Of these N2 rats, data from the 1 highest male and 1 highest female weight gainers after 3wk of HE diet were used for comparison to other groups (Figure 1). The 1 highest male N2 weight gainers were then crossed with 1 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 placing 1wk old, randomly selected F.DIO male (n=8-9) or 5

6 female (n=1-11) and F344 male (n=8) and female (n=8) rats on either HE diet or continuing them on chow for 3wk. From this first generation of F.DIO rats, 3 successive additional generations of F.DIO rats were produced from pairings of 4-5 male and 8-1 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). Spontaneous wheel running. At 1wk of age, 7 chow-fed F.DIO generation 2 offspring and 7 chow-fed F344 male rats were given continuous access to a running wheel in their home cage for 14d. Animals were allowed ad libitum access to chow and water during this time. Data were collected remotely by a computerized system (Mini Mitter Co.) and the data were expressed as the number of revolutions/ 12h for the dark (181-6h) and light (61-18) cycles, respectively. Oral glucose tolerance test. Groups of 8 male F.DIO (generation 3) and 8 male F344 rats were fed chow from weaning until 1wk 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 (.5g/kg body weight) and repeated tail blood samples of.25ml were taken at 15, 3, 6, 9 and 12min. They were then placed on HE diet for 3wk 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 measure using radioimmunoassay kits (Linco). 6

7 Statistics. Parameters were compared among various groups (DIO, F344, N1, N2, N3 [F.DIO]) by one way analysis of variance (ANOVA) with post hoc Scheffe 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 using the trapezoidal rule (GraphPad Prism 3. software). Glucose and insulin curves were also compared using repeated measures ANOVA. Feed efficiency was calculated by dividing the body weight gain by the calories of diet ingested during 3wk on either chow or HE diet. RESULTS Energy homeostasis and metabolic parameters. In male rats, there was a progressive reduction in chow-fed body weight of the offspring of each successive backcross with the F344 rats. At 1wk of age, chow-fed male DIO rats weighed 89% more than F344 males (Figure 1). The chow-fed N1 males weighed 67%, N2 males weighed 24% and the N3 (F.DIO) males weighed 12% more than comparable 1wk old, chow-fed F344 males (Table 1; F[1,31]=18.94; P=.1). When fed either chow or HE diet for 3wk, there was a significant genotype (F[1,31]=9.4; P=.5) and diet x genotype (F[1,31]=4.23; P=.48) effect comparing the first generation of F.DIO rats to F344 males (Table 1; Figure1). F.DIO males gained 95% more body weight, while F344 males on HE diet gained only 41% more body weight than their respective chow-fed counterparts. The distribution of body weight gains on chow for 3wk ranged from 24-55g in F344 and from 7-5g in F.DIO rats (Figure 2). There was a highly variable weight gain in F344 males 7

8 over 3wk on HE diet (42-7g; Figure 2) and a narrower spread in F.DIO males (58-77g; Figure 2). After 3 generations of inbreeding, 1wk old chow-fed male F.DIO x F.DIO male offspring weighed 338+6g; 7% more than comparable chow-fed F344 rats (316+5g; p=.5). After 3wk on HE diet, these F.DIO rats gained 35% more weight (7+3g) than F344 rats (52+4g; P=.5). Overall, caloric intake of 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=.1). However, post hoc analysis showed that it was only the F.DIO rats that ate more on 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=.44). But again, post hoc analysis showed that only the F.DIO males on 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[1,2]=9.21; P=.7). However, while there was no statistically significant difference in fat pad weights between chow-fed F.DIO and F344 rats, 3wk on HE diet increased F.DIO fat pad weights by 85% and F344 fat pad weights by only 71% compared to 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 percent of total body weight, total fat pad weights did not differ between chow-fed F.DIO and F344 males. But F.DIO rats on HE diet had heavier fat pads as a percent of body weight than comparable F344 rats (Table 1; F[1,2]=8.91; P=.8). The increase in fat pad weights associated with HE diet intake was reflected in higher plasma leptin levels in 8

9 F.DIO (132%) and F344 (7%) rats as compared to their respective chow-fed controls (F[1,2]=71.16; P=.1). However, F.DIO males had higher leptin levels than F344 males, regardless of diet (F[1,2]=.3). On HE diet, F.DIO leptin levels were 98% higher than comparable F344 rats by post hoc analysis (Table 1). In addition to heavier adipose pads, F.DIO males on HE diet had 2% heavier livers than their chow-fed counterparts, while there was no diet effect on liver weights in F344 males (Table 1). Fasting (Figure 3) and non-fasting (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 (Figure 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 following an oral glucose load. Intake of HE diet for 3wk altered neither fasting (Figure 3) nor non-fasting (Table 1) basal glucose levels in F.DIO or F344 rats. However, F.DIO fasting insulin levels rose by 168% (Figure 3) and non-fasting levels by 176% (Table 1). Furthermore, their glucose response to oral glucose was increased by 6% and their insulin response by 37% compared to their own chow-fed responses (Table 1, Figure 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=.11), although the increase in F344 rats (96%) over their chow-fed responses was less than that in F.DIO rats (Table 1, Figure 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 3wk on HE diet. 9

10 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 1wk of age, chow-fed female DIO rats weighed 59% more than F344 females (Figure 4). The chowfed 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 1% more than comparable chow-fed F344 females (Table 2, Figure 4; F[1,31]=18.94; P=.1). Intake of HE diet for 3wk resulted in a significant genotype (F[1,32]=13.21; P=.1) and diet (F[1,32]=9.71; P=.4) effect (Table 2; Figure 4). F.DIO females gained 152%, while 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 3wk (4-29g) than in F344 females (7-2g; Figure 5). However, unlike the males, intake of HE diet for 3wk produced a wider spread of body weight gain in F.DIO (26-58g) than F344 females (16-35g; Figure 5). Overall, F.DIO females consumed more calories than F344 females, regardless of diet (Table 2; F[1,32]=3.42; P=.1) and this was accentuated by intake of HE diet (F[1,32]=17.5; P=.1). F.DIO females ate 16% more calories as chow and 22% more as HE diet than F344 females, respectively. 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 to 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=.14). This difference was significant by post hoc comparison only on HE diet where only F.DIO females had higher feed efficiency (115%) than their respective chow-fed controls. 1

11 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[1,2]=15.14; P=.1). There was no significant difference in total fat pad weights between chow-fed F.DIO and F344 females (Table 2). While fat pad weights were increased in both strains by intake of 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 percent of body weight comparably on HE diet (Table 2). In keeping with the development of diet-induced obesity 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=.1). 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 34% higher leptin levels than their comparable chow-fed controls. Non-fasting basal insulin levels were also higher in F.DIO than F344 females, regardless of diet (F[1,32]=16.25; P=.1). This effect was due primarily to the fact that HE diet intake increased nonfasting insulin levels only in F.DIO rats. Non-fasting 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[1,2]=8.64; P=.1). HE diet exposure increased liver weight in F.DIO females by 13% but had no effect on F344 females compared to chow-fed controls. Running wheel activity. Chow-fed male F.DIO and F344 rats were evaluated for their dark vs. light cycle activity across 14d of continuous access to running wheels in their home cages (Figure 6; Table 1). There was enormous inter-individual variability in both 11

12 groups across the 14d. F.DIO males ranged from ,199 revolutions and F344 males from ,99 revolutions per 24h across the 14d 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=.32). Since the light cycle running made up only 4% of total running in F.DIO s and 1% in F344 s, the two strains did not differ from each other in total 24h running across the entire 14d period. DISCUSSION We have crossed selectively bred DIO with obesity-resistant F344 rats to produce rats which are more obesity-prone and glucose intolerant than the parent F344 strain. In comparison to the parent DIO strain (8,13), this new F.DIO strain does not readily gain weight on low fat chow but still maintains a robust tendency to develop diet-induced obesity when fed a diet of moderate fat and caloric density. At least in males, these phenotypic traits were maintained through 3 additional cycles of inbreeding F.DIO rats with each other. Given this breeding scheme and the persistence of the DIO trait, we believe that the F.DIO rats represent a new strain of obesity-prone, glucose intolerant rats. Unlike the parent DIO strain (8), both F.DIO male and female rats exhibit only a slight increase in body weight and carcass adiposity when fed low fat chow. However, even in the absence of obesity, chow-fed F.DIO rats have an abnormal hyperinsulinemic response to an oral glucose load, a feature not present in either of the DIO (8) or F344 parent strains. Despite their lack of elevated fasting or non-fasting glucose and insulin levels, their hyperinsulinemic response suggests that non-obese F.DIO rats might have 12

13 incipient insulin resistance. This apparent insulin resistance becomes markedly exaggerated when they develop diet-induced obesity on HE diet. Obese F.DIO rats have elevated fasting and non-fasting insulin levels and increased areas under both their plasma glucose and insulin curves following a glucose load. Surprisingly, the moderate increase in adiposity of F344 males on HE diet was associated with a small but clearly abnormal insulin response to glucose. This diet-induced abnormality of glucose tolerance in F344 rats is different from the selectively bred DR rats which do not develop abnormal glucose tolerance on HE diet (8). The fact that both DIO and F344 parent strains are prone to glucose intolerance might explain why the resultant F.DIO strain develops glucose intolerance even when fed only a low fat diet from weaning. 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 to respective F344 rats during a 3wk period on chow. Since 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 to the F344 parent strain. But feed efficiency on chow does not differ between the genotypes. Thus, while 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). 13

14 During 3wk on HE diet, both male and female F.DIO rats become more obese than do 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 HE diet strongly suggests that they truly develop diet-induced obesity. While both F344 males and females on HE diet also have heavier fat pads and/or leptin levels than comparable chow-fed controls, these changes are small in comparison to comparable F.DIO rats. This is in keeping with our prior report in male F344 rats showing that they are relatively obesity-resistant compared to outbred DIO rats (11). While F.DIO male rats have a narrow, and probably unimodal range of weight gains on 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 HE diet. Female F344 rats also have a wide range of weight gains on HE diet. Although the numbers of animals tested was too small to be certain, the tight clustering of male weight gains on 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 non-genetic 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 HE diet and the preservation of the DIO phenotype after 3 successive backcrosses against obesity-resistant F344 rats followed by 3 successive generations of inbreeding F.DIO with F.DIO rats. 14

15 As with weight gains on chow or HE diet, there is also a large inter-individual 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 24h 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 since they also run less than selectively bred DR rats at 4-5wk of age, before DIO rats become obese (unpublished observation). But, 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 which exhibits dietinduced obesity 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 3wk on 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 3 successive backcrosses against the obesity-resistant inbred F344 strain followed by 3 successive cycles of inbreeding, suggest that both the DIO and glucose intolerant phenotypes are inherited as a polygenic trait in this model. 15

16 ACKNOWLEDGMENTS The authors thank Antoinette Moralishvilli, Ghoufeng Zhou, and Matthew Klein for their expert technical assistance. This work was funded by NIDDK grant (DK 366) and the Research Service of the Veterans Administration (BEL, AAD-M) and NIDDK grants DK26687, DK57621 (SC and JM) and an NIDDK NRSA grant DK61229 (JM). 16

17 REFERENCES 1. American Physiological Society. Guiding principals for research involving animals and human beings. Am.J.Physiol. 283: R281-R283, Boozer, C. N. Decreased voluntary wheel running by Sprague-Dawley rats selectively bred for increased susceptibility to diet-induced obesity. FASEB J. 12: A54, Bouchard, C. and L. Perusse. Genetics of obesity. Ann.Rev.Nutr. 13: , Burcelin, R., V. Crivelli, A. Dacosta, A. Roy-Tirelli, and B. Thorens. Heterogeneous metabolic adaptation of C57BL/6J mice to high-fat diet. Am.J.Physiol. 282: E834-E842, Chang, S., B. Graham, F. Yakubu, D. Lin, J. C. Peters, and J. O. Hill. Metabolic differences between obesity-prone and obesity- resistant rats. Am.J.Physiol. 259: R113-R111, Ghibaudi, L., J. Cook, C. Farley, M. Van Heek, and J. J. Hwa. Fat intake affects adiposity, comorbidity factors, and energy metabolism of Sprague-Dawley rats. Obes.Res. 1: , Levin, B. E. and A. A. Dunn-Meynell. Reduced central leptin sensitivity in rats with diet-induced obesity. Am.J.Physiol. 283: R941-R948,

18 8. Levin, B. E., A. A. Dunn-Meynell, B. Balkan, and R. E. Keesey. Selective breeding for diet-induced obesity and resistance in Sprague-Dawley rats. Am.J.Physiol. 273: R725-R73, Levin, B. E. and E. Govek. Gestational obesity accentuates obesity in obesity-prone progeny. Am.J.Physiol. 275: R1375-R1379, Levin, B. E., S. Hogan, and A. C. Sullivan. Initiation and perpetuation of obesity and obesity resistance in rats. Am.J.Physiol. 256: R766-R771, Levin, B. E., J. Triscari, and A. C. Sullivan. Relationship between sympathetic activity and diet-induced obesity in two rat strains. Am.J.Physiol. 245: R367-R371, Meaney, M. J. Maternal care, gene expression, and the transmission of individual differences in stress reactivity across generations. Ann.Rev.Neurosci. 24: , Ricci, M. R. and B. E. Levin. Ontogeny of diet-induced obesity in selectively-bred Sprague-Dawley rats. Am.J.Physiol. In Press:

19 FIGURES Figure 1. Generation of the male F.DIO strain. Male DIO and female Fisher F344 rats were bred to produce the first generation (N1). The highest weight gaining male N1 rats were backcrossed against female F344 rats and the male offspring of these matings were successively bred with F344 females to produce N2 and N3 (F.DIO) offspring. A: body weights at 1wk of age when fed chow from weaning (n=8-1/ group). B: Body weight gain when fed either chow or HE diet for 3wk beginning at 1wk of age (n=8-1/ group). NB: the N2 generation data are from the 1 highest weight gainers on HE diet which were subsequently used to breed the N3 generation. Data are means + SEM. Bars with differing superscripts differ from each other at P=.5 or less by post hoc Scheffe test after ANOVA showed significant intergroup differences. Figure 2. Body weight gain histograms of male F.DIO and F344 rats on chow or HE diet for 3wk. Histogram of body weight gains of the F344 (n=8 each for chow and HE diet) and N3 (F.DIO; n=9 chow, n=8 HE diet) rats described in the legend of Figure 1. N= number of rats in each 5g weight gain bin. Figure 3. Oral glucose tolerance in male F.DIO and F344 rats on chow or HE diet. Generation 3 F.DIO rats and inbred F344 rats were fed chow from weaning to 1wk of age and then continued on chow or fed HE diet for 3wk. Data are mean + SEM plasma glucose (A. chow-fed; B. HE diet) and insulin (C. chow-fed; D. HE diet) levels following.5g/kg oral glucose gavage in overnight-fasted rats (n=8/group). P=.5 or less when 19

20 plasma levels in F.DIO rats were compared to those in F344 rats at a given time point by post hoc Scheffe test. Figure 4. Body weights of female parent strains (DIO and F344) and successive generations of matings on chow and HE diet. Details are the same as Figure 1 except that they apply to female rats of the same genotypes; n=8-11/ group. Figure 5. Body weight gain histograms of female F.DIO and F344 rats on chow or HE diet for 3wk. Histogram of body weight gains of the F344 (n=8 each for chow and HE diet) and N3 (F.DIO; n=11 chow, 1 HE diet) rats described in the legend of Figure 4. N= number of rats in each 5g weight gain bin. Figure 6. Spontaneous running activity of male F.DIO and F344 rats. Rats from F.DIO generation 2 were compared to inbred F344 rats. Home cage running wheel activity was monitored cumulatively over 12h periods during the dark (18-6) vs. light (6-18) phases across 14d. Data are mean + SEM for n=7/group. P=.5 or less when running in F.DIO rats was compared to F344 running over the specified 1h period. 2

21 Males F344 Chow F344 HE F.DIO Chow F.DIO HE Initial body weight (g) a a b b Final body weight (g) 32+8 a 34+9 b b c Body weight gain (g) a b a b % Body weight gain Food intake (kcal) a a a b Feed efficiency (g/kcal1) a a a b Fat Pads Epi (g) a b a b RP (g) a b a c PR (g) a b a b Mes (g) a b a c Total (g) a b a c % Body weight a b a c Liver (g) a a a b % Body weight Leptin (ng/ml) a b ab c Insulin (ng/ml) a a a b Glucose (mg/dl) 12+4 a 19+4 a 11+3 a 11+5 a Insulin AUC a b b c Glucose AUC a a a b Dark Running a (rev/12h) a Light running a (rev/12h) b Table 1. Characteristics of male Fischer F344 and F.DIO rats on chow and HE diet. Male rats were fed chow from weaning to 1wk of age. Then half were fed HE diet or chow for an additional 3wk (n=8-1/ group) and sacrificed. Initial body weight is weight at 1wk of age on chow; Final body weight is after 3wk on chow or HE diet; % Body 21

22 weight is the percent gain of body weight during the 3wk on chow or HE diet; Food intake is the caloric intake during that 3wk period; Feed efficiency is the amount of body weight gain (g)/ caloric intake (kcal) x1 during that 3wk period; Fat pad weights: EPI = epididymal; RP= retroperitoneal; PR= perirenal; Mes= mesenteric; Total is the total weight of 4 depots; % Body weight is the total weight of 4 depots as a percent of body weight; Liver: % Body weight is the liver weight as a percent of total body weight; Plasma leptin, insulin and glucose levels were obtained from trunk blood of non-fasted rats between 8 (lights on) and 11. Glucose and Insulin AUC- areas under the curve for oral glucose tolerance for glucose (mg/dl/min) and insulin (ng/ml/min) from tail blood following an oral glucose load of.5g/kg in overnight fasted rats; Running wheel dark (18-6) vs. light (6-18) running are the cumulative 14d spontaneous running activities (revolutions per 12h period x 14d). All data are from the first F.DIO (N3) generation except for running wheel (generation 2) and glucose and insulin area under the curve data (generation 3). Data are mean + SEM. Data with differing superscripts differed from each other at P<.5 or less by Scheffe post hoc test after ANOVA showed significant intergroup differences. 22

23 Females F344 Chow F344 HE F.DIO Chow F.DIO HE Initial body weight(g) a a 19+4 b b Final body weight (g) a a 24+3 a a Body weight gain (g) a b a c % Body weight a gain b a c Food intake (kcal) a b b c Feed efficiency (g/kcal1) a a a b Fat Pads Ovarian (g) a b a b RP (g) a b a c PR (g).8+.1 a b.9+.2 a b Mes (g) a a a b Total (g) a b a c % Body weight a b a b Liver (g) a a a b % Body weight Leptin (ng/ml) a b a c Insulin (ng/ml) a a,b a b Glucose (mg/dl) 18+4 a 11+3 a 11+3 a a Table 2. Characteristics of female Fischer F344 vs. F.DIO rats on chow and HE diet. All methods, abbreviations and data comparisons are the same as those in Table 1. 23

24 1wk Body Weight (g) A Males a b c d b DIO F344 N1 N2 N3 3wk Body Weight Gain (g) B a b Chow HE Diet c a a d c d DIO F344 N1 N3 FIGURE 1 24

25 F344 Male Chow F344 Male HE Diet N 2 N Body Weight Gain (g) Body Weight Gain (g) F.DIO Male Chow F.DIO Male HE Diet N 2 N Body Weight Gain (g) Body Weight Gain (g) FIGURE 2 25

26 A Chow B 3wk HE Diet Plasma Glucose (mg/dl) F344 F.DIO Plasma Glucose (mg/dl) Plasma Insulin (ng/ml) C Minutes Chow Minutes Plasma Insulin (ng/ml) D Minutes 3wk HE Diet Minutes FIGURE 3 26

27 A Females 3 a b c b b 1wk Body Weight (g) 2 1 DIO F344 N1 N2 N3 3wk Body Weight Gain (g) B a b Chow HE Diet c d a,d e c a DIO F344 N1 N3 FIGURE 4 27

28 F344 Female Chow F344 Female HE N N Body Weigh Gain (g) Body Weigh Gain (g) F.DIO Female Chow F.DIO Female HE 3 3 N 2 2 N Body Weigh Gain (g) Body Weigh Gain (g) FIGURE 5 28

29 A B 4 Dark Cycle 5 Light Cycle Revolutions per 12h F344 F.DIO Revolutions per 12h F344 F.DIO FIGURE Days Days 29