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Regional Fat Pad Growth and Cellularity in Obese Zucker Rats: Modulation by Caloric Restriction Dorothy B. Hausman,* Jacqueline B. Fine,† Krishna Tagra,† Shea S. Fleming,† Roy J. Martin,* and Mario DiGirolamo†

Abstract HAUSMAN, DOROTHY B., JACQUELINE B. FINE, KRISHNA TAGRA, SHEA S. FLEMING, ROY J. MARTIN, AND MARIO DIGIROLAMO. Regional fat pad growth and cellularity in obese Zucker rats: modulation by caloric restriction. Obes Res. 2003;11:674-682. Objective: To investigate, in young obese male Zucker rats, the effects of chronic food restriction and subsequent refeeding on: 1) parameters of nonadipose and adipose growth, 2) regional adipose depot cellularity [fat cell volume (FCV) and number], and 3) circulating leptin levels. Research Methods and Procedures: Obese (fa/fa) and lean (Fa/?) male Zucker rats were studied from age 5 to 19 weeks. After baseline food intake monitoring, 10 obese rats were subjected to 58 days of marked caloric restriction from ad libitum levels [obese-restricted (OR)], followed by a return to ad libitum feeding for 22 days. Ten lean control rats and 10 obese control rats were fed ad libitum for the entire experiment. All rats were fed using a computer-driven automated feeding system designed to mimic natural eating patterns. Results: After food restriction, OR rats weighed significantly less than did lean and obese rats and showed a significant diminution in body and adipose growth as compared with obese rats. Relative adiposity was not different between obese and OR rats and was significantly higher than that of lean rats. The limitation in growth of the adipose tissue mass in OR rats was due mostly to suppression of fat cell proliferation because the mean FCV in each of the four

Received for review November 18, 2002. Accepted in final form March 7, 2003. *Department of Foods and Nutrition, University of Georgia, Athens, Georgia and †Department of Medicine, Emory University School of Medicine, Atlanta, Georgia. Address correspondence to Dr. Dorothy B. Hausman, Department of Foods and Nutrition, 263 Dawson Hall, University of Georgia, Athens, GA 30602. E-mail: [email protected] Copyright © 2003 NAASO

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depots was not affected. Serum leptin levels of OR and obese rats were not different from each other but were significantly higher than those of lean rats. Discussion: Marked caloric restriction affects obese male Zucker rats in a manner different from that of nongenetic rodent models (i.e., Wistar rats). In comparison with the response to caloric deprivation of Wistar rats, these calorically restricted obese male Zucker rats appeared to defend their relative adiposity and mean FCV at the expense of fat cell number. These findings indicate that genetic and/or tissue-specific controls override the general consequences of food restriction in this genetic model of obesity. Key words: adipose cellularity, restriction-refeeding, genetic obesity, leptin

Introduction In both genetic and nongenetic rodent models of obesity, ad libitum feeding leads to age-related increases in body weight, body fat, fat cell volume (FCV)1 and number, and circulating leptin levels (1– 8). The response to food restriction, however, distinguishes genetic from nongenetic rodent models. For nongenetic rodent models (e.g., Wistar and Sprague Dawley rats), underfeeding early in life leads to reduced rates of body weight and body fat enlargement and limits age-related increases in FCV and number (1,9 –11). Circulating leptin levels reflect the limitation in fat mass accretion (2,3). In contrast, when genetically obese fatty Zucker rats (fa/fa) are subjected to caloric restriction, they still develop the complete obese syndrome, with a fat mass that is persistently elevated relative to lean body weight (4,12,13). Some reports have indicated that restricting food intake in these rats produces a decrease in FCV, whereas

1

Nonstandard abbreviations: FCV, fat cell volume; OR, obese-restricted; ORR, obeserestricted-refed.

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other studies indicate that FCV remains unchanged, even when the food restriction is coupled with exercise (13–15). The development of adipose mass and its cellularity is influenced by diet, energy expenditure, hormonal milieu, innervation, and local, region-specific regulators, such as leptin, TNF-␣, IGF-I, IL-6, and others (5,16 –21). Because obese male Zucker rats respond to food restriction with persistence of relative adiposity, we have chosen to study the changes in circulating levels of the adipose regulator, leptin, and to relate these changes to the tissue cellular parameters (FCV and fat cell number). Toward this end, we have used the male Zucker model in conjunction with an automated feeder to further analyze: 1) effects of early chronic food restriction on adipose tissue growth and cellularity, 2) region-specific effects of the food restriction on adipose depot growth and cellularity, 3) relation of altered nutritional plane to absolute and relative circulating leptin levels, and 4) the region-specific adipose response to resumption of ad libitum feeding by previously food-restricted obese rats.

Research Methods and Procedures Animals and Diet Thirty-one male Zucker rats [11 lean (Fa/?) and 20 obese ( fa/fa)] were obtained from the University of Georgia breeding colony at the age of 5 weeks. The animals were housed individually in stainless steel hanging rack cages, equipped with automated feeders and automatic water spouts, in the animal facility at Emory University. The animal room was maintained on a 12-hour-light/12-hourdark schedule at an ambient temperature of 21 °C to 23 °C. All of the rats had ad libitum access to granular Laboratory Rodent Diet (no. 5001; PMI威 Nutrition International, Richmond, IN). The chow was available in metal feeding cups secured inside their home cages. After an initial acclimation period, food intake was recorded to the nearest 0.01g daily to determine average daily consumption (grams per day) for each rat and the average for the lean and the obese groups. At the end of the food intake monitoring period (5 days), the 20 obese rats were subdivided into two groups [obese and obese-restricted (OR)] that were matched for mean body weight and variance. Individual body weights were monitored three times per week for the duration of the experiment. Restriction Refeeding Protocol For 58 days, the lean and obese groups were fed rodent chow ad libitum with automated individual feeders, attached outside the cage and connected to a computer-driven relay box as described previously (9). The feeders were programmed to deliver an excess of food per 24 hours based on the average daily food consumption values for these groups. The feeders for the OR group were programmed to

deliver 65% of the calories consumed by the obese group during the baseline food intake monitoring period. This level of restriction was chosen based on our experience with studies of food restriction in lean and obese rats. Specifically, results of pilot experiments had determined that our body weight goal in the OR rats (weight equal or slightly lower than that of the control lean rats) could be achieved with an initial food restriction of 35% to 40%. To preserve the predominantly nocturnal eating pattern of rodents, the computer was programmed to deliver ground chow to all cages in discrete meals in the evening and at night and one feeding in the afternoon at 2 PM. Body weight and food intake (corrected for spillage) of individual rats were measured every other day. At the end of the 58 days of food restriction, subsets of animals (n ⫽ 5) from each group, matched for mean body weight and variance of the remaining rats, were killed and terminal measures were taken, as described below. For the remainder of the study (22 days), the rats in the lean and obese groups continued to receive rodent chow ad libitum, and those in the OR group were released from food restriction and allowed to eat ad libitum. At the end of the study, the remaining animals (n ⫽ 5 to 6) in each group were killed. This protocol resulted in an overall 44% reduction in food intake from ad libitum-fed obese intake levels and a 29% reduction from ad libitum-fed lean levels in the OR group. Considering that the levels of most nutrients in the constant nutrient formulation diet are in excess of nutrient requirements (22,23) and considering the inclusion of a margin of safety in nutrient recommendations, this level of restriction was not believed to be severe enough to compromise overall nutrient status of the restricted animals. These studies were approved by the Institutional Animal Care and Use Committee of Emory University. Terminal Measures of Body Weight, Adiposity, Lean Body Growth, and Adipose Tissue Cellularity At each specified time-point (end of food restriction and end of refeeding phases), rats were euthanized in the fed state between 9 and 11 AM. After administration of xylazine (13 mg/kg) and ketamine (87 mg/kg), mixed just before intraperitoneal administration, the terminal body weight and body length (nose to anus) of each rat was measured. Blood was then collected using cardiac puncture, and the serum was separated by centrifugation and stored at ⫺70 °C for subsequent analysis of serum leptin levels (nanograms per milliliter) by radioimmunoassay (rat leptin kit; Linco Co., St. Louis, MO). After euthanasia by exsanguination and heart removal under deep anesthesia, the liver, kidneys, and testes were removed and weighed. Four white adipose depots were carefully dissected as follows: 1) the paired epididymal, by a longitudinal cut above the epididymus; 2) the paired retroperitoneal, by first separating the perirenal fat and then OBESITY RESEARCH Vol. 11 No. 5 2003 2003

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collecting the retroperitoneal depot in toto; 3) the mesenteric, by cutting the intestine below the duodenal-jejunum junction and stripping the fat by gently pulling the intestinal loops apart; and 4) the paired subcutaneous inguinal, by carefully dissecting all the fat in the inguinal region up to a horizontal line parallel to the xyphoid cartilage. The tissues were dissected of visible vessels, collected in warm saline, blotted, and weighed to the nearest milligram. Determination of Adipose Tissue Composition and Cellularity A modification of the method of Hirsch and Gallian (24) was used for the quantification of adipose tissue cellularity measures (fat cell size and number). In brief, 40- to 50-mg tissue fragments from each adipose depot of each rat were obtained as individual samples and fixed with osmium tetroxide (2% solution) and incubated for 7 d at 37 °C. The tissue fragments were separated from the cells using filtration through a 250-␮m mesh sieve onto a 25-␮m sieve. The cells were then counted with a Coulter electronic particle counter (model ZM; Coulter Electronics, Hialeah, FL), using a 25-␮m lower threshold for both the lean and obese animals. This lower cutoff point, although eliminating debris and cellular fragments, may also eliminate the “very small fat cells” described by DeMartinis and Francendese (25); however, that fraction is small and elusive by any present method (26). Analogous tissue samples (80 to 100 mg) were also processed in duplicate for lipid content and the defatted dry residue content using the method of Folch et al. (27). From this, tissue lipid, defatted dry residue, and water were estimated in absolute and relative terms for each depot. Statistical Analyses Means, SD, and SE of the mean were calculated in the usual way. Unless indicated otherwise, the significance of differences between group means was determined by ANOVA (SigmaStat; SPSS, Chicago, IL). Group differences or linear correlations were considered statistically significant when p ⱕ 0.05. Post hoc comparisons of significant differences among groups were made using the Student-Newman-Keuls multiple comparisons test.

Results Effects of Early-Onset Caloric Restriction on Body Weight of Obese Zucker Rats Figure 1A shows the body weight curves for lean, obese, and OR rats over the duration of the experiment. Separation of mean body weights for the three groups was seen as early as 10 days after the start of the restriction paradigm. Continued restriction of the OR group produced rats of the obese phenotype that weighed significantly less than both the obese and lean groups (p ⬍ 0.001) before refeeding. 676

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Figure 1: Body weights and mean daily food intake of lean, obese, and obese food-restricted male Zucker rats over the duration of the experiment. After ⬃1 week of monitored food intake with ad libitum feeding, the automated feeder protocol was initiated. Lean and obese rats were fed ad libitum, and OR rats were fed 65% of the calories consumed by the obese control group for 58 days. At the end of the restriction phase, OR rats were allowed to eat ad libitum (arrow) for 22 days. (A) Group mean body weight ⫾ SEM for 5 to 6 animals in each group. (B) Food intake expressed as kilocalories per day ⫾ SEM. ad lib, ad libidum.

Calories Consumed by Lean, Obese, and OR Rats during the Restriction Phase The average food intake values, expressed as kilocalories per day, are shown in Figure 1B. Before the start of the restriction phase, the average kilocalories per day consumed by the obese rats were, as expected, significantly greater than those consumed by lean rats (119 ⫾ 8 vs. 94 ⫾ 6, p ⬍ 0.05). It should be noted that, even though initially the OR

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Table 1. Measures of lean body growth of male Zucker rats fed either ad libitum, food restricted, or food restricted and refed

Time 1: end of restriction Lean Obese OR Time 2: end of refeeding Lean Obese ORR

Body length (cm)

Tail length (cm)

Testes weight (paired; g)

Kidney weight (paired; g)

Liver weight (g)

23.64 ⫾ 0.44a 23.56 ⫾ 0.56a 19.82 ⫾ 0.12b

19.16 ⫾ 0.25a 16.30 ⫾ 0.48b 13.80 ⫾ 0.46c

2.90 ⫾ 0.09a 2.76 ⫾ 0.15a 2.68 ⫾ 0.12a

1.07 ⫾ 0.02a 3.35 ⫾ 0.20a 1.17 ⫾ .07a 4.14 ⫾ 0.31a 0.82 ⫾ 0.02b 2.27 ⫾ 0.08b

13.72 ⫾ 0.77a 23.69 ⫾ 2.04b 12.51 ⫾ 0.48a

23.80 ⫾ 0.22a 23.34 ⫾ 0.21a 21.60 ⫾ 0.62b

20.32 ⫾ 0.45a 17.16 ⫾ 0.63b 13.42 ⫾ 0.55c

3.03 ⫾ 0.03a 2.93 ⫾ 0.11a 2.73 ⫾ 0.20a

1.15 ⫾ 0.04a 3.59 ⫾ 0.12a,c 1.35 ⫾ 0.08a 4.52 ⫾ 0.30c 1.03 ⫾ 0.04a 3.22 ⫾ 0.14a

13.87 ⫾ 0.34a 26.75 ⫾ 0.66b 17.93 ⫾ 0.95c

Heart weight (g)

Values represent means ⫾ SEM for five to six animals in each group. Values without a common superscript are significantly different, as determined by post hoc analysis ( p ⬍ 0.05), for each parameter measured.

rats were fed 65% of the amount of food consumed by ad libitum-fed obese rats, the relative restriction in food intake became greater as the obese rats increased food intake with time, whereas OR rats were given fixed amounts of chow. Calculation of food restriction in OR rats over the entire period of observation showed that OR rats ate, on average, 56% of the amounts ingested by the obese rats. The average daily caloric consumption by OR rats was also significantly lower than that of lean rats (67 ⫾ 5 vs. 94 ⫾ 6 kcal, p ⬍ 0.05). Although the average amount of calories consumed was different for the three groups of rats during the restriction phase, there were no significant differences between the groups when food intake was normalized to body weight (data not shown). Effects of Early Caloric Restriction on Measures of Lean Body Growth Measures of body length and weights of selected nonadipose organs are shown in Table 1. There were no differences between the body lengths of lean and obese rats. Restricting young OR rats to 56% of the calories consumed by their ad libitum-fed obese counterparts produced a significant decrease in body and tail length (p ⬍ 0.05). When absolute weights of selected organs (testes, heart, liver, and kidneys) were compared across groups, the OR rats had significantly reduced liver weights compared with obese rats (p ⬍ 0.05) and significantly lower kidney weights compared with both the lean and obese rats (p ⬍ 0.05 for both). The significant differences between the OR and obese rats disappeared when organ weights were standardized to body weight, but the difference between weights of lean and obese rats remained (data not shown; p ⬍ 0.05).

Effects of Early Caloric Restriction on Selected Adipose Depot Weights Although the calories consumed, body weights, and body lengths of the OR rats were significantly less than those of both the lean and obese rats, the weights of the four white adipose depots of the OR rats were intermediate to those of the lean and obese groups (Figure 2A; p ⬍ 0.05 for both). When the weights of the individual adipose depots were expressed relative to total body weight, the significant differences between the obese and OR rats disappeared (Figure 2B). Thus, even with a body weight lower than the lean rats, the obese food-restricted rats defended their relative degree of adiposity. Additionally, there were no significant differences in the compositions of these depots (i.e., relative content of lipid, water, and defatted dry residue; data not shown) for the obese and OR groups of rats. Effects of Early Caloric Restriction on Measures of Adipose Depot Cellularity We examined aspects of adipose depot cellularity that could be potentially responsible for the significantly smaller depot weights of the OR group relative to the obese group. Figure 3A shows the total fat cell number per depot for the three groups. As expected for Zucker rats at 14 weeks of age, the obese rats had significantly more fat cells per depot for the inguinal, epididymal, and retroperitoneal depots compared with lean rats (p ⬍ 0.05). In addition, early caloric restriction in the OR rats significantly decreased fat cell number in these three depots as compared with those of the obese rats (p ⬍ 0.05). In contrast, the number of fat cells in this depot of obese OBESITY RESEARCH Vol. 11 No. 5 2003 2003

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Figure 2: Adipose depot weights of the three groups of rats at the end of the 58-day restriction phase. (A) Adipose depot weights are shown as absolute group means ⫹ SEM. (B) Adipose depot weights are shown as group mean values ⫹ SEM as percent of total body weight (relative adiposity). Ing, subcutaneous inguinal depot; Epi, epididymal depot; Retro, retroperitoneal depot; Mes, mesenteric depot. Mean values, within a depot, without a superscript in common indicate a significant difference (p ⬍ 0.05). ad lib, ad libidum.

rats was not different from that of the mesenteric depot of lean rats. Furthermore, caloric restriction of OR rats did not produce a significant suppression of fat cell number in the mesenteric depot, as compared with obese rats (p ⫽ 0.12). As shown in Figure 3B, the mean FCV was significantly larger in all depots of obese rats compared with lean rats. Interestingly, food restriction did not produce a significant reduction in mean FCV for any depot of the OR rats compared with the obese group. Thus, it is apparent that the differences in inguinal, epididymal, and retroperitoneal adipose depot weights for the OR vs. obese rats were attributable solely to differences in fat cell number. 678

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Figure 3: Depot-specific adipose tissue cellularity of obese male Zucker rats subjected to early caloric restriction compared with that of lean and obese control rats. (A) Fat cell number data are shown as group mean fat cell number (⫻106) ⫹ SEM for each adipose depot studied. (B) FCV data are shown as group mean FCV (in picoliters) ⫹ SEM for each adipose depot studied. Ing, subcutaneous inguinal depot; Epi, epididymal depot; Retro, retroperitoneal depot; Mes, mesenteric depot. Mean values, within a depot, without a superscript in common indicate a significant difference (p ⬍ 0.05). ad lib, ad libidum.

Body Weight Change of Calorically Restricted Rats in Response to Refeeding As described above, the restriction phase lasted for 58 days, after which the remaining OR rats were allowed to eat ad libitum for 22 days. The body weight curves for the rats during this phase are shown in Figure 1 after the identifying arrow. After refeeding, the body weights of the OR rats slightly rose to, but did not surpass, those of the lean group and remained significantly lower than those of the obese group (p ⬍ 0.05). Calories Consumed by Previously Restricted Rats on Refeeding The caloric intakes of lean, obese, and OR rats during the refeeding phase are shown in Figure 1B. Mean food intake

Food Restriction and Regional Adiposity in Obesity, Hausman et al.

levels of the OR group approached, but did not exceed, those of the lean rats and remained significantly lower than those of the obese rats (p ⬍ 0.05). This may be because of the fact that the OR rats were already eating the same amount of calories per body weight as the lean and obese rats during the restriction phase (data not shown). Relative Changes in Adipose Depot Weights and Cellularity after Release from Caloric Restriction Relative changes in fat depot weight during the refeeding phase were determined by calculating the relative difference between mean values for a given depot at the end of the refeeding phase and the analogous depot at the end of the restriction phase. For the obese rats, the only significant change in fat depot weight during this phase was observed for the inguinal depot, where a 16% increase was observed (p ⬍ 0.05). In contrast, the OR group showed significant increases of 38% and 39%, respectively (p ⬍ 0.05), in the weights of both the inguinal and epididymal pads during the refeeding phase. The relative change in fat cell number for each depot for the OR and obese rats during the refeeding phase was also determined. As for adipose depot weights, the only depot from the obese rats that showed a significant change in fat cell number during this phase was the inguinal depot, which had a 20% increase in cell number (p ⬍ 0.05). The OR group showed significant increases of 29% and 53%, respectively (p ⬍ 0.05), in fat cell number for both the inguinal and retroperitoneal depots. As for the relative change in mean FCV for each depot for the two groups, both the obese and OR rats showed a significant increase in fat cell size for the mesenteric depot (23% and 34%, respectively; p ⬍ 0.05). The OR group also showed a significant increase in mean epididymal fat cell size (25%; p ⬍ 0.05). No change in mean FCV was seen for the inguinal and retroperitoneal depots. Thus, the OR-refed (ORR) rats exhibited adipose tissue growth that was depotspecific and was, in some cases, due to both cellular hyperplasia and cellular hypertrophy. Circulating Leptin Levels Figure 4 shows circulating leptin levels for lean, obese, and OR rats at the end of the restriction and refeeding phases. As expected, the obese rats had 4 to 5 times higher mean leptin levels than did the lean rats. The mean leptin level of OR rats, however, was not different from that of obese rats (p ⬍ 0.05). Furthermore, mean leptin levels of OR rats were unchanged after 22 days of refeeding (Figure 4A). When the serum leptin was expressed relative to the sum of adipose depots (Figure 4B), lean and obese rats had similar mean levels, whereas the normalized mean leptin level of ORR rats was nearly double that of the other two groups (p ⬍ 0.05).

Figure 4: Circulating leptin values as a function of genotype and nutritional status. Serum leptin levels (nanograms per milliliter) are shown for lean and obese ad libitum-fed rats and OR rats at the end of the restriction and refeeding phases. (A) Absolute group means ⫹ SEM are shown. (B) Group means ⫹ SEM normalized to the sum of the weights of the four adipose depots harvested. Values without a superscript in common indicate a significant difference within a given time period (p ⬍ 0.05). ad lib, ad libidum.

Discussion The results of this study confirm and expand the previously held notion that genetically obese animals, such as ob/ob or db/db mice (28) or obese Zucker rats (4,12,13), maintain their degree of relative adiposity, even when subjected to marked food restriction at a level similar to or lower than the food consumed by their lean counterparts. In addition, several novel findings were obtained: 1) although absolute fat mass was lower in the OR rats than in the obese OBESITY RESEARCH Vol. 11 No. 5 2003 2003

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control rats fed ad libitum, circulating leptin levels were unaffected by food restriction; and 2) the reduced growth of fat mass, in each depot studied, was due only to a suppressed fat cell number, with FCV remaining virtually unchanged. As expected, the circulating levels of leptin were markedly higher in the obese rats than in the lean rats because of the high differential in fat mass (Figure 4). The circulating leptin levels of OR rats, however, were similar to those of obese rats, despite the fact that the adipose depot mass of OR rats was smaller (Figure 2A) than that of obese rats. This discrepancy is not believed to be due to short-term feeding effects on circulating leptin concentrations. In this study, all rats were killed in the morning within a 2-hour time period and were in the fed state at time of sample collection. Although the time from last meal may have varied slightly, it was regulated somewhat in the restricted fed animals through the use of the automated feeding system, which mimicked the natural eating patterns of the animals. Furthermore, although an influence of meal timing on leptin diurnal rhythms has been observed in some studies (29,30), others have found no effect of food consumption on plasma leptin levels (31–35). Rather, the discrepancy between similar circulating leptin concentrations and dissimilar total adipose tissue weights in the two obese groups in the present study is perhaps explained by the observation (Figure 2B) that adipose mass relative to body weight is similar for obese and OR rats. Indeed, when expressed relative to the sum of adipose depots (Figure 4B), the circulating levels of leptin in lean and obese rats were similar, but the leptin levels in OR rats were nearly doubled. It is generally acknowledged that circulating leptin levels are proportional to the fat mass in humans and in rodents (1,2). In the OR rats in this study, this relationship appears to be disrupted and may reflect an altered physiological modulation of leptin production during food restriction in this genetic model of obesity. The most striking result of our study is that, despite major reductions in growth of fat mass in the four depots of the OR rats, there were no changes in mean fat cell size, which was similar in each depot to that of the unrestricted obese rats (Figure 3B). Other investigators have reported that exercise coupled with calorie restriction leads to a lower fat mass without affecting mean FCV in male Zucker rats (14), but variable results were obtained in female Zucker rats (15,36). Likewise, a previous study by Cleary et al. (13) using a food restriction of 30% in male Zucker rats indicated that the weight of the epididymal pads was reduced in the obese food-restricted rats compared with obese controls, whereas the weight of the retroperitoneal pads was unchanged. The fat cell size was not affected by food restriction in the obese rats, and the cell number was reduced only in the epididymal pads of the obese rats. Our study, employing a slightly higher degree of food restriction (35% 680

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Table 2. Modification of body growth parameters and adipose mass and cellularity by marked food restriction in normal male Wistar rats and in obese male Zucker rats

Body weight Parameters of lean body mass Body fat (absolute) Body fat/body weight Fat cell number Fat cell volume Leptin

Wistar rats

Zucker rats

2 2 2 2 2 2 2

2 2 2 3 2 3 3

The directional changes indicated by the arrows reflect the changes described for Wistar rats (10; JB Fine, K Tagra, S Fleming, M DiGirolamo, unpublished data) and for Zucker rats (39,40).

initially), led to clearer separation of the effects of food restriction on adipose tissue cellularity in four depots. Adipocyte size remained unchanged in the obese food-restricted rats in all four depots that had significantly reduced depot weight, and the fat cell number was significantly reduced in all depots (inguinal, epididymal, and retroperitoneal) except the mesenteric depot. The finding that caloric deprivation alters adipocyte cell number, but not cell volume, is contrary to the finding in nongenetic animal models that food restriction reduces the lipid content (and cell size) of white adipose tissue, in parallel with a reduction in cell number (11). Our results may reflect a preferential nutrient partitioning for adipose tissue of obese Zucker rats, but they could also represent genetically altered regulation of adipocyte metabolism, in which lipid accumulation is preserved, in the obese and OR Zucker rats, even at the expense of cellular proliferation. An earlier hypothesis of a higher lipoprotein lipase activity in adipocytes of obese Zucker rats would be consistent with the present findings (37,38). Table 2 summarizes the effects of marked caloric restriction on a nongenetic model of obesity (male Wistar rat) compared with the effects on obese male Zucker rats seen in the present study. Although Wistar rats respond to caloric restriction with a general reduction in body parameters, adipose mass and cellularity, circulating leptin levels, and adipose tissue proliferative activity, the obese Zucker rats present a different pattern of response (39,40). In particular, relative adiposity remains unchanged with an unmodified FCV, and circulating leptin concentrations do not follow the reduced absolute fat mass and suppressed fat cell number. It is of interest that leptin levels were unchanged in fa/fa rats during short-term fasting (41).

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We also studied the effects of release from caloric restriction. When animals that had been markedly restricted (receiving 56% of ad libitum intake) for 2 months were given ad libitum access to food for 1 month, the OR animals showed minimal catch-up growth, only minor increases in food consumption (not exceeding that of lean control rats), and a food intake per body weight that matched the other two groups. This is in contrast to other studies of food restriction in nongenetic animal models, where resumption of ad libitum feeding produces significant increases in food consumption and catch-up growth (42,43). This may reflect the fact that OR rats had already achieved a significant degree of obesity and that relative adiposity was preserved. The increment in fat mass after resumption of ad libitum feeding was small and showed a region-specific pattern. In two of the depots studied (retroperitoneal and inguinal depots), fat mass increase took place mostly by cellular hyperplasia; in two others (mesenteric and epididymal), the fat mass increased mostly by cellular hypertrophy. This is reminiscent of the region-specific growth patterns of adipose depots from male Wistar rats, a nongenetic model of obesity (5). Even after resumption of ad libitum feeding, the mean fat cell size in the four adipose depots did not change significantly in comparison with the obese unrestricted Zucker rats. The increase in cell number for the ORR rats varied from depot to depot and was higher in the inguinal and retroperitoneal depots. Thus, the male Zucker rat appears to defend fat cell size in all of the depots studied. Fat cell number is the component of adipose tissue cellularity that is most readily modulated by nutritional intervention in this model. Specifically, adipose depots of young male obese Zucker rats (with the exception of the mesenteric) show increases in fat cell number under conditions of ad libitum feeding and a significant reduction in fat cell number when subjected to early and marked nutritional deprivation. Mesenteric adipose tissue appears to be unique in that the fat cell number of obese rats does not differ from that of lean rats. Furthermore, food restriction in OR rats does not significantly influence the fat cell number for this depot as it does for the other three depots (Figure 3A). Previous studies with Wistar rats in our laboratory (5) have shown that hypertrophy, rather than hyperplasia, is the preferred modality of growth for the mesenteric tissue. This may be the case even for this genetic model of obesity. In summary, the results of this study indicate that genetic obesity, in this animal model, develops by a combination of marked adipocyte enlargement and additional cell proliferation to produce large increments in fat depot mass in association with increased circulating levels of leptin. The findings that marked caloric restriction affects only fat cell number without reduction of FCV indicates that genetic or tissue-specific controls override the general consequences of food restriction. Finally, the observation that, on release of food restriction, specific patterns of adipose depot growth

and restoration resemble those of nongenetic animal models fed ad libitum, suggests that certain common and universal regulatory processes exist in both genetic and nongenetic models of obesity.

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