Division of Energy Balance and Obesity, Rowett Research Institute, Aberdeen Centre for Energy Regulation and Obesity (ACERO), Bucksburn, Aberdeen AB21 9SB, United Kingdom. E-mail: email@example.com
Objective: To investigate the effect of a high-energy (HE) diet on caloric intake, body weight, and related parameters in outbred male Sprague-Dawley (SD) rats.
Research Methods and Procedures: Twenty-eight SD rats were fed either chow (C) for 19 weeks or HE diet for 14 weeks and then C for 5 weeks. Blood hormones and metabolites were assayed, and expression of uncoupling protein-1 and hypothalamic energy-balance-related genes were determined by Northern blotting and in situ hybridization, respectively.
Results: HE rats gained body weight more rapidly than C animals with a range of weight gains, but there was no evidence that weight gain was bimodally distributed. Caloric intake was transiently elevated after introduction of the HE diet. Transfer of HE rats back to C resulted in a drop in caloric intake, but a stable body weight. In terminal analysis, two of four dissected adipose tissue depots were heavier in rats that had previously been fed HE diet. Blood leptin, insulin, glucose, and nonesterified fatty acids were not different between the groups. Uncoupling protein-1 mRNA was elevated in interscapular brown adipose tissue from HE rats. There was a trend for agouti-related peptide mRNA in the hypothalamic arcuate nucleus to be higher in HE rats.
Discussion: Contrary to other studies of the SD rat on HE diet, body weight and other measured parameters were normally distributed. There was no segregation into two distinct populations on the basis of susceptibility to diet-induced obesity. This characteristic may be dependent on the breeding colony from which animals were sourced.
Significant advances have been made in understanding the mechanistic underpinnings of energy homeostasis and obesity in laboratory animal models. Many studies have investigated laboratory rodents carrying spontaneous mutations or target genes that have been subject to transgenic manipulation (1). Specific genes that are essential for body weight regulation have been identified, and mutations to some of these genes constitute the causative lesion in obese human subjects. However, the number of individuals in the human population bearing such identified mutations is generally very small, although it is now established that up to 3% to 5% of very obese human subjects may carry mutations in the melanocortin-4 receptor gene (2).
Despite the advances that have been made in studying monogenic models of obesity, it is clear that most human obesity is polygenic and represents the interaction between multiple genes and environment, of which diet is a major component. Accordingly, there is a pressing need to investigate polygenic rodent models of obesity, with diet-induced obesity (DIO)1 models coming under increasing scrutiny. The Sprague-Dawley (SD) rat model of DIO provides an experimental system for investigating the basis of differential susceptibility or resistance to DIO (3, 4) and the different effects of body weight gain on the level of body weight that will subsequently be defended (5, 6, 7).
Work with the outbred SD rat has focused on animals purchased from Charles River Laboratories in the United States (Wilmington, MA) (4, 5, 6, 8, 9). The same company also supplies SD rats from breeding establishments outside of the United States. Research characterizing the SD rat model of DIO has frequently employed a high-energy (HE) diet that combines a relatively high-fat and -energy content with a high content of sugar and simple carbohydrates. Studies using this animal/diet combination have reported that populations of rats rapidly diverge into two distinct groups on the basis of body weight gain (8); this parameter has been reported as being bimodally distributed (9), with one-half of the rats becoming obese and the rest remaining lean. From such observations, a strategy has emerged of defining the top 40% of weight gainers as DIO susceptible and the bottom 40% as DIO resistant (5, 6, 8).
This study used SD rats from Charles River Laboratories in the United Kingdom (Kent, United Kingdom) and examined body weight trajectories on basal chow (C) and HE diets and the defense of body weight on return from the HE diet to the C diet. Experiments were designed to test the hypothesis that outbred SD rats have particular body weight responses to the HE diet and that they partition themselves into two weight-gain-defined populations, irrespective of their source colony. Residual differences between rats that had been fed the HE diet for 14 weeks followed by C for 5 weeks and those that remained on the C diet throughout the 19-week experiment were assessed through measurement of body tissues, blood metabolites and hormones, hypothalamic neuropeptide and receptor gene expression, and uncoupling protein-1 (UCP-1) mRNA in interscapular brown adipose tissue (IBAT). Many characteristics of the SD DIO model observed in this study were consistent with those reported in the literature. Significantly, however, there was no evidence of effective separation of subpopulations of susceptible and resistant individuals after exposure to the HE diet.
Research Methods and Procedures
Animals and Experimental Protocol
All procedures were licensed under the Animals (Scientific Procedures) Act of 1986 and were approved by the Rowett Research Institute's Ethical Review Committee.
In Experiment 1, 28 male outbred SD rats (Charles River Laboratories, Kent, United Kingdom) weighing 280.7 ± 2.19 g were housed individually at 22 to 23 °C on a 12 h:12 h light:dark cycle and fed Purina Chow 5001 (PMI Nutrition International, Nottingham, United Kingdom; 3.34 kcal/g, with 23% energy as protein, 12% as fat, and 65% as carbohydrate) ad libitum for 4 days. Rats were divided into two weight-matched groups: 20 rats were fed an HE diet (Research Diets C11024: 8% corn oil, 44% sweetened condensed milk, and 48% Purina Chow 5001; 4.5 kcal/g, with 15% energy as protein, 33% as fat, and 52% as carbohydrate; Research Diets, New Brunswick, NJ) for 14 weeks and then the C diet for 5 weeks, whereas the remaining 8 rats were fed the C diet for 19 weeks. Body weights were measured daily throughout most of the study. Food intake was measured daily for three periods during the experiment. Animals were killed, blood plasma and serum were collected, and brains were frozen on dry ice for storage at −80 °C. The following tissues were dissected and weighed: subcutaneous, epididymal, retroperitoneal, and mesenteric white adipose tissue (WAT), IBAT, liver, testes, and soleus and gastrocnemius muscles.
In Experiment 2, 80 male SD rats, initially weighing 192 ± 0.89 g, were fed the C diet for 7 days. All animals were then transferred to the HE diet (C11024; Research Diets) for 14 days. Two animals were withdrawn from the study for reasons unrelated to the experimental procedure. The rats were subdivided into two groups balanced for body weight, one of which (38 animals) continued to be fed the HE diet for a total of 50 days.
Circulating Hormones and Metabolites
Serum leptin concentrations were measured using a commercially available rat-specific radioimmunoassay kit (Linco RL-83K; Biogenesis, Poole, United Kingdom). The sensitivity of the assay was 0.5 ng/mL, and the intra-assay coefficient of variation (CV) was 1.7%. Plasma insulin was measured by radioimmunoassay (10) using antiserum to porcine insulin raised in guinea-pigs (65–104; ICN, Thame, United Kingdom), [125I]-labeled ovine insulin (Sigma, Poole, United Kingdom), and porcine insulin (Sigma) as standard. The bound hormone was separated using a mixture of ovine anti-guinea-pig IgG serum (SAPU, Carluke, United Kingdom) and normal guinea-pig serum (SAPU). Assay diluent was 0.05 M sodium phosphate buffer (pH 7.4) with 0.5% bovine serum albumin. The sensitivity of the assay was 0.2 μIU/mL, and the intra-assay CV was 0.5%. Plasma glucose and nonesterified fatty acids were determined using the fully automated KONE analyser (11, 12). The sensitivities of the assays were 0.34 and 0.04 mM, respectively, with intra-assay CVs of 0.35% and 2.0%, respectively.
UCP-1 Gene Expression
Total RNA was isolated from 100- to 150-mg samples of IBAT with TRIzol (Invitrogen, Karlsruhe, Germany) following the manufacturer's instructions (13, 14). RNA pellets were redissolved in Sol D [6.3 M guanidinium thiocyanate, 40 mM sodium citrate (pH 7), 0.8% sarcosyl, and 8 mM 2-mercaptoethanol], precipitated with 1 volume isopropanol, washed in 75% ethanol, and dissolved in diethyl pyrocarbonate-treated water (15). Ten micrograms of RNA from each sample were electrophoresed in a 1% agarose gel [5% formaldehyde, 0.02 M 3-(N-morpholino)-propanesulfonic acid, 5 mM sodium acetate, and 1 mM Na2EDTA (pH 7)]. Using a Northern downblot procedure (16), the RNA was transferred to a nylon membrane (Hybond N; Amersham Pharmacia Biotech, Little Chalfont, United Kingdom) overnight in 10× saline sodium citrate (SSC) (1.5 M NaCl and 0.15 M sodium citrate, pH 7), and was cross-linked to the membrane (Cross-Link UV-Stratalinker; Stratagene, Amsterdam, The Netherlands). A rat full-length UCP-1 cDNA (GenBank accession no. M11814) was random prime-labeled with [α-32P]deoxycytidine 5′-triphosphate (rediprime DNA labeling system; Amersham Pharmacia Biotech). Blots were prehybridized for 1 h and hybridized overnight at 64 °C in the same solution [0.5 M sodium phosphate buffer (pH 7.0), 1 mM EDTA, 7% sodium dodecyl sulfate (SDS), and 1% bovine serum albumin]. After hybridization, membranes were washed three times at room temperature: for 20 minutes in 2× SSC/0.1% SDS, for 10 minutes in 1× SSC/0.1% SDS, and for 10 minutes in 0.5× SSC/0.1% SDS. Finally, membranes were washed for 5 minutes at 58 °C in 0.1× SSC/0.1% SDS and were exposed to an X-ray film at −80 °C (X-OMAT AR-Film; Eastman Kodak, Rochester, NY). Signals were measured on a Phosphor Imager (Storm 860; Amersham Pharmacia Biotech) and quantified using Image Quant Version 5.0 and Image Quant Tools Vers. 2.2 (Amersham Pharmacia Biotech). The signals of HE rats were expressed as relative units of the mean signal intensity of C animals. Ethidium bromide staining of total RNA served to confirm uniform gel loading.
Hypothalamic Gene Expression
Hypothalamic gene expression for a panel of energy-balance-related neuropeptides and receptors was quantified using in situ hybridization as described in detail elsewhere (17, 18, 19). Riboprobes complementary to partial fragments of neuropeptide Y, corticotrophin-releasing factor, agouti-related peptide (AgRP), pro-opiomelanocortin, cocaine- and amphetamine-regulated transcript, and melanocortin receptors 3 and 4 were generated from cloned cDNAs as described previously (18, 20, 21, 22). Leptin receptor long-form riboprobes were generated using a partial rat cDNA sequence (23). A 306-bp fragment of growth hormone releasing hormone was cloned from rat hypothalamic cDNA using the primers 5′-TGCTGCTCTGGGTGCTCTTT-3′ and 5′-CGTCCGCTGAAGGCTTCA-3′ (GenBank NM010285), with 35 cycles of 94 °C for 30 seconds, 55 °C for 30 seconds, and 72 °C for 1 minute and then one cycle at 72 °C for 5 minutes. The DNA fragments were ligated into a pGEM-T-easy vector, transformed into JM 109 cells, and sequenced. Forebrain sections were collected from the very caudal extent of the arcuate nucleus (ARC) through to the rostral extent of the paraventricular nucleus (PVN) onto three sets of 10 slides. The first two sets of slides spanned the full extent of the ARC, approximating −4.52 to −2.30 mm, relative to Bregma, according to the atlas of the rat brain (24). The third set of slides continued through to −1.40 mm relative to Bregma and included both rostral and caudal extents of the PVN. Sections were fixed, acetylated, and hybridized overnight at 58 °C using 35S-labeled antisense riboprobes (1 to 1.5 × 107 cpm/mL). Slides were treated with RNase A to remove unhybridized probe and desalted with a final high stringency wash in 0.1× SSC at 60 °C for 30 minutes. The slides were air dried and apposed to Hyperfilm β-max (Amersham Pharmacia Biotech). Autoradiographic images were quantified using the Image-Pro Plus system (Media Cybernetics, Silver Spring, MD). This determined the intensity and area of the hybridization signal on the basis of set parameters; the integrated intensity was then computed using standard curves generated from 14C autoradiographic micro-scales (Amersham Pharmacia Biotech). Depending on the probe used, image analysis was performed on five to six sections spanning the ARC or two to three sections from the PVN.
Means ± SE are reported, and the probability value used for statistical significance was p < 0.05. Data were analyzed by Student's t test using Microsoft Excel (Microsoft Corp., Redmond, WA) except for UCP-1 data, which were analyzed using STATISTICA for Windows (StatSoft, Inc., Tulsa, OK). The data distribution for Experiment 1 due to the smaller population size was assessed by the Kolmogorov-Smirnov Normality test, using SigmaStat statistical software (Jandel, Erkrath, Germany). Data distribution for Experiment 2 was assessed by both the Kolmogorov-Smirnov Normality test (SigmaStat) and χ2 analysis, using Microsoft Excel (Microsoft Corp.). Correlation was used to determine whether a significant relationship existed between AgRP gene expression and final body weight gain.
In Experiment 1, rats were transferred to the HE diet at approximately 300 g (day 0; Figure 1). The body weight of HE- and C-fed rats began to diverge immediately, reaching significance at day 7 (p < 0.05). A body weight differential of 50 to 61 g between HE and C groups was maintained throughout days 33 to 99. Once the HE rats were returned to the C diet (day 99), the weight differential began to close, reflecting continuing weight gain in the C group and relative weight stability in the HE group after transfer back to C diet. By the end of the study (HE animals back on C diet for 35 days), the body weight differential averaged 34 g. Rats exhibited a range of weight gains on either diet (14 days HE: 80 to 144 g; 14 days C: 13 to 102 g; 50 days HE: 172 to 284 g; 50 days C: 98 to 233 g). Variation in weight gain on the HE diet can be interpreted as relative susceptibility or resistance to DIO (5, 6, 8). These previous studies have selected the upper 40% and lower 40% of weight gainers, defining these as susceptible and resistant phenotypes, respectively. Here, analysis of weight gain on the HE diet using this template gives rise to diverging body weight trajectories (Figure 2). Differences between the weight gain groups averaged 39 g at 14 days and 63 g at 50 days. However, despite this difference, there was no evidence of a bimodal distribution of body weight gain within the population of rats fed the HE diet for 14 (K-S distance = 0.145, p = 0.32) or 50 days (K-S distance = 0.177, p = 0.10; Figure 3). Both weight gain groups were weight stable when returned to the C diet.
Transfer to the HE diet resulted in an immediate increase in caloric intake (Figure 4). Mean daily caloric intake over days 0 to 10 was higher in rats fed the HE diet (HE, 105.6 ± 2.96 vs. C, 90.4 ± 1.6 kcal/d; p < 0.05). However, by days 86 to 93, the effect of diet on caloric intake had dissipated. Transfer of HE rats back to the C diet resulted in a decrease in caloric intake; intake over days 99 to 106 was lower than that of rats fed the C diet throughout the experiment (HE, 71.8 ± 0.77 vs. C, 87.0 ± 1.04 kcal/d; p < 0.05).
Epididymal and retroperitoneal WATs were significantly heavier in rats fed the HE diet for the first 14 weeks of the study compared with rats fed the C diet throughout (Table 1). Similar trends were observed for subcutaneous and mesenteric fat. The pooled weight of these four WAT depots accounted for ∼25% of the body weight differential between HE and C rats. Paired testes weights were 10% greater in rats fed C throughout. Within the HE group, statistical analysis with the Kolmogorov-Smirnov Normality method revealed that the weights of epididymal, retroperitoneal, mesenteric, and subcutaneous WAT, liver, testes, and soleus and gastrocnemius muscles were all normally distributed (data not shown). There were no differences in measured circulating hormones or metabolites between HE and C rats (Table 2), although insulin and leptin concentrations tended to be higher in HE animals. Hormone concentrations in the latter group were normally distributed (data not shown).
Table 1. Terminal tissue weights (grams) for outbred male Sprague-Dawley rats (Experiment 1)
Rats were fed either chow ad libitum for 19 weeks (C; n = 8) or high-energy diet ad libitum for 14 weeks and then chow ad libitum for 5 weeks (HE; n = 20).
11.0 ± 1.81
12.7 ± 0.89
9.2 ± 1.21
12.1 ± 0.54
4.7 ± 0.97
5.2 ± 0.41
9.2 ± 1.53
13.5 ± 0.83
Total dissected WAT
33.8 ± 5.25
43.5 ± 2.37
0.4 ± 0.04
0.4 ± 0.03
18.4 ± 1.42
19.2 ± 0.59
4.1 ± 0.12
3.7 ± 0.06
0.4 ± 0.02
0.5 ± 0.01
7.0 ± 0.31
7.1 ± 0.13
Table 2. Terminal blood metabolites and hormones for outbred male Sprague-Dawley rats (Experiment 1)
Rats were fed either chow ad libitum for 19 weeks (C; n = 8) or high-energy diet ad libitum for 14 weeks and then chow ad libitum for 5 weeks (HE; n = 20).
8.1 ± 0.10
8.1 ± 0.16
1.5 ± 0.19
1.7 ± 0.10
8.7 ± 1.36
9.9 ± 1.01
13.2 ± 2.50
17.3 ± 1.45
Total RNA was isolated from 100 to 150 mg IBAT and used in the quantification of UCP-1 gene expression. UCP-1 mRNA was higher in IBAT from HE animals than from C animals (F(1, 16) = 4.92, p < 0.05; Figure 5). Hypothalamic sections were subjected to in situ hybridization using 35S-labeled riboprobes to assess expression of a panel of neuropeptide and receptor genes (Figure 6). There was a trend for AgRP gene expression in the ARC to be higher in HE animals than in controls fed the C diet throughout, although this difference did not attain statistical significance (t = 1.525; p = 0.14). AgRP mRNA levels were positively correlated with body weight gain in HE rats (p < 0.05, y = 0.6512x − 12.415; R2 = 0.25).
In Experiment 2, rats were transferred to the HE diet at a body weight of 269 ± 1.11 g (Figure 7). Selection of the upper 40% and lower 40% of weight gain groups after 14 days on the HE diet gave rise to diverging body weight trajectories, with weight differences of approximately 44 and 110 g at 14 and 50 days, respectively. Statistical analysis revealed a normal distribution of weight gain at both 14 days (K-S distance = 0.09, p = 0.08; χ2 unimodal distribution χ2 = 10.31, df = 6, p = 0.11 and bimodal distribution χ2 = 24.60, df = 6, p = 0.0004; n = 78) and 50 days (K-S distance = 0.11, p = 0.23; χ2 unimodal distribution χ2 = 5.05, df = 5, p = 0.41 and bimodal distribution χ2 = 20.26, df = 5, p = 0.001; n = 38) on the HE diet (Figure 8). Growth rate of the animals in Experiment 2 at day 50 was slightly higher than in Experiment 1, but there was again no evidence of a bimodal distribution. Energy intake on diet transfer (C to HE) increased on average by 24%, but by day 13 on HE, intake had returned to a level similar to that recorded on C before introduction of HE (Figure 9).
Outbred male SD rats supplied by Charles River Laboratories in the United Kingdom displayed a range of body weight trajectories on an HE diet that is relatively high in fat and sugar. The variation in rate of weight gain in these animals was very similar to that reported elsewhere; e.g., body weight differential between the top and bottom 40% of weight gainers was very similar to published data. (For example, Figure 1 of ref. (5) depicts differentials of ∼33 and 72 g after 14 and 50 days on HE, respectively.) However, weight gain on the HE diet was normally distributed in both Experiments 1 and 2, as were a number of terminal measures. There was no evidence of a bimodal distribution in relative susceptibility or resistance to DIO, i.e., of effective separation into groups of susceptible and resistant animals. This was a surprising result in the light of earlier reports, and under these circumstances, analysis of these rats (sourced in the United Kingdom) as arbitrary subgroups is inappropriate.
The response to a novel diet is likely to be important to susceptibility to DIO, yet hitherto, energy intake of SD rats has not been studied in detail. Evidence from studies that report weekly energy intake (5) demonstrates that the HE diet evokes hyperphagia. However, the precise duration and amplitude of this response has not been described. The present studies demonstrated that daily caloric intake was increased immediately on presentation of the HE diet, but that this hypercaloric intake was transient. Energy intake of the C-fed animals was maintained around 80 to 102 kcal/rat per day, similar to values calculated elsewhere (5). Experiment 1 confirmed that SD rats defend the body weight that they attain on the HE diet when returned to the base C diet, as shown previously (3, 5, 6). Defense of body weight occurs even though caloric intake drops in animals formerly fed the HE diet, suggesting that components of energy expenditure or assimilation efficiency must also be altered when returned to C.
The body composition, blood, and gene expression data collated in Experiment 1 relate to the context of converging body weight trajectories, with all animals consuming the same diet at the time of sampling. Feeding the HE diet and subsequently returning animals to C gave rise to two groups of animals with different body adiposity, particularly in the size of abdominal adipose tissue depots. However, although insulin and leptin were somewhat higher in the HE group, differences were not statistically significant. HE rats had higher UCP-1 mRNA in IBAT than animals fed the C diet throughout. Mouse strains that are susceptible or resistant to DIO both have increased IBAT UCP-1 mRNA in response to a high-fat diet (25), suggesting that elevated UCP-1 expression in Experiment 1 may be indicative of residual thermogenic capacity as a consequence of earlier exposure to the HE diet. Although not statistically significant, gene expression of the anabolic peptide, AgRP, was ∼31% higher in rats that had previously been fed the HE diet. Such an elevation would normally be a characteristic of animals in negative energy balance, rather than those in a weight stable, possibly hypocaloric, state. However, as many energy balance peptides have complementary effects on both energy intake and energy expenditure, it may be that the trend toward an increase in AgRP gene expression is related to predicted decreased energy expenditure.
Published studies with the Charles River SD rat strain imply that exposure to the HE diet rapidly results in effective separation into two subpopulations on the basis of weight gain (5, 6, 8, 9). No such segregation is evident in our study, despite using the same strain of rat, the same diets, and, as much as possible, the same experimental manipulations. The normally distributed weight gain ranges exhibited in our experiments agree with the expected distribution of a polygenic trait in an outbred population. As far as can be determined, the source of origin of these animals (Charles River breeding establishments in either the United Kingdom or the United States) seems to be the major difference between the studies in question. However, if the present United Kingdom origin rat data are artificially manipulated into “resistant” and “susceptible” subgroups (as discussed earlier), the differences in mean weight gain and parameters such as food intake between the subgroups are very similar to published data from rats sourced in the United States. This implies that the sensitivity of the UK SD rat to the HE diet is very similar to that of SD rats from the United States. It will probably not be possible to resolve the discrepancies between the apparent weight gain distributions of the various studies (i.e., bimodal vs. normal) without a direct comparison at a single site of equivalent animals sourced from the different breeding establishments.
The distribution of weight gain on the HE diet shown by the animals in the current study seems to mimic susceptibility to obesity on a Western diet in the human population more accurately than a bimodal segregation. As such, these animals will be valuable models of DIO in the human population, allowing mechanistic studies that complement the phenotyping and genotyping of individuals with differing susceptibility to DIO. Such an integrated approach will be critical to further progress in understanding the rapid development of the obesity epidemic. It is clear that, in humans, the development of obesity on a high-fat, HE diet is not a biological inevitability (26), and we need to understand how the body responds to a Western-style diet that is high in fat, sugar, and calories.
This work was funded by the European Commission, Quality of Life and Management of Living Resources, Key action 1 “Food, nutrition and health” program (QLK1-2000-00515). We thank Nicole Steinberg and Sigrid Stöhr for technical assistance and Dr. Graham Horgan (Biomathematics and Statistics Scotland) for help with statistical analysis.
Nonstandard abbreviations: DIO, diet-induced obesity; SD, Sprague-Dawley; HE, high energy; C, chow; UCP-1, uncoupling protein-1; IBAT, interscapular brown adipose tissue; WAT, white adipose tissue; CV, coefficient of variation; SSC, saline sodium citrate; SDS, sodium dodecyl sulfate; AgRP, agouti-related peptide; ARC, arcuate nucleus; PVN, paraventricular nucleus.