Noble Rats Display Decreased Weight Gain and Visceral Adiposity via Lifelong Exposure to an Isoflavone-Rich Diet

generalized body fat appears to play a key role between obesity being link to cancer [6,8]. Many biomarkers, hormones and chemical signals are involved in body weight gain, adipose tissue deposition and cancer outcomes [9,10]. For example, obesity in postmenopausal women has been associated with a higher risk of breast cancer that is linked to estrogen formation within the breast [11,12]. Also, obesity is associated with decreased testosterone levels in males [13]. This suggests a complex interaction between adipokines, inflammatory factors, hormones and other chemical signaling that is just beginning to be revealed.


Introduction
Noble Rats Display Decreased Weight Gain and Visceral Adiposity via Lifelong Exposure to an Isoflavone-Rich Diet.The World Health Organization defines obesity as an abnormal or excessive fat accumulation in adipose tissue, to the extent that health is impaired (Obesity-Report WHO consultation, 2000) [1].Obesity frequency has been steadily increasing over the past decades and in 2007-2008, the prevalence of obesity among US adults was 33.8% and for overweight individuals 68% [2].Furthermore, obesity is associated with the development and progression of various cancers, including tumors of the colon, esophagus, pancreas, liver, breast, prostate, kidney, endometrium and cardiovascular disease [3][4][5][6][7].In fact, visceral rather than All animals were housed in clear plastic cages with wire lids (20 cm x 24 cm x 40 cm standard shoebox with woodchip bedding).The young adult male and female rats were housed individually except during mating.Following delivery, offspring pups were housed with their mothers until age 21 days, at which point they were weaned and placed in separate cages (three to four animals per cage by sex by diet treatment).At 40 days the animals were separated to two animals per cage and finally, at 60 days, placed in individual cages by diet treatment.

Mating
Animals were diet-matched and mated to ensure offspring would be exposed solely to only one of the two diet treatments.During the mating procedure, males were housed within hanging wire cages over cardboard mats.One female was introduced into each male cage, and the cardboard was checked for the appearance of vaginal plugs twice a day (at about 8 a.m. and 5 p.m.) indicating successful mating/insemination.Inseminated females were removed from the male's cage, weighed and replaced with another female.This continued until vaginal plugs were found for all females.All males were fertile and inseminated 4 litters by diet treatment.Pregnancy was confirmed by a 30-40 g weight gain over the first 5-7 days of gestation.

Diet treatments
After arrival at our animal facilities, male and female rats to be mated were immediately placed on either a high soy-isoflavonoid diet (High-Iso) containing isoflavones at a concentration of approximately 600 ppm as glycones and aglycones (Harlan Teklad 8604, Madison, WI, USA) or a low-isoflavonoid diet (Low-Iso) with approximately 15 ppm of soy isoflavones as glycones (Zeigler Bros. Inc., Gardners, PA, USA, Phytoestrogen Reduced Rodent Diet) [26].The Low-Iso diet contains casein and corn and wheat provided the protein content for this formulation [26].Offspring were kept on the maternal diet, thus ensuring strict exposure to only one diet treatment from conception to adulthood.The specific nutritional content for these diets is described elsewhere [26].The diets were balanced and matched for protein, fat, carbohydrate, amino acids, vitamins and minerals (Table 1).

High-Iso
Low-Iso

Body weight gain, visceral adipose tissue weight and blood sample collection
Tissue collection occurred at 145 days of age.Animals (n=8to-10) by diet treatment by sex (representing 4 litters) were weighed (±0.1 grams).To determine final body weight gained each animal's birth weight was subtracted from their body weight at 145 days of age.Then the animals were anesthetized (ketamine/acepromazine at 75/2.5mg/kg IP) prior to collecting blood samples and VAT.An incision was made from the pelvis to the bottom of the rib cage after which arterial blood was collected from the heart.Subsequently, white adipose tissue (or VAT) was dissected from the caudal surface of the diaphragm to the base of the pelvic cavity and recorded in grams (±0.01 grams) for each animal.

Determination of isoflavone blood levels
After an arterial blood sample (5-7ml) was collected from each animal, the sample was allowed to clot and then centrifuged to obtain serum, which was then labeled and frozen at -20 O C until analysis was performed.Quantification of total isoflavonoid (daidzein, genistein, and equol) serum levels were performed at ESA Biosciences, Inc. Chelmford, MA, USA, using HPLCelectrochemical detection (ECD) with a lower limit of detection of 5ng/ml for each aglycone.Samples (by diet treatment and sex) were analyzed in duplicate in a single assay along with internal control samples; the intra-assay coefficient of variation was approximately 4%.

Statistical analysis
All data sets were analyzed with Minitab (State College, PA, USA) software [by ANOVA (unstacked data)], followed by Fisher's pairwise comparisons to determine significance and all data were expressed as the mean ± standard error of the mean (SEM); p<0.05 was considered significant.

Serum isoflavone levels
Serum isoflavone levels in males and females fed the High-Iso diet were significantly higher compared to animals fed the Low-Iso diet (Figure 1).For example, total isoflavone levels in female Noble rats fed the High-Iso diet were approximately 16.8-fold higher compared to the Low-Iso values (p<0.001).Additionally, the total isoflavone levels in male Noble rats fed the High-Iso diet were approximately 13.9-fold higher compared to the Low-Iso values (p<0.001, Figure 1).Equol represented the majority of the total isoflavone values: a) In females, equol represented 77% of the total isoflavone values followed by daidzein (15%), then genistein (8%), and, b) In males, equol represented 72% of the total isoflavone values followed by daidzein (21%) then genistein (7%).
The present data confirm that rodents, in general, produce high circulating levels of the polyphenolic molecule, equol when fed a soy-containing diet [27,28].
The present total isoflavone results by diet treatments were similar to those reported elsewhere for male Long-Evans rats (@ 75 or 100 days old) fed the same diets [20,29].However, male Noble rat total isoflavone values in the present study fed the

Noble Rats Display Decreased Weight Gain and Visceral Adiposity via Lifelong Exposure to an Isoflavone-Rich Diet
Copyright: © 2014 Lephart High-Iso diet were approximately 25 to 30% lower compared to male Long-Evans rats on the same High-Iso diet [20,29].This may suggest a difference in isoflavone metabolism and/or clearance between the two rat strains or potential differences in agerelated parameters.Also, the equol metabolite represented 73% of the total isoflavones in the present study from male Noble rats fed the High-Iso diet which were similar to those reported for adult male Long-Evans rats on the same diet at 77% for 75 day old or 82% for 100 day old animals [20,29].

Body weight gain and VAT weight
Male and female Nobel rats fed the High-Iso diet displayed significantly lower body weight gain compared to animals fed the Low-Iso diet (p<0.042 and p<0.038, respectively; Figures 2 &  3).The reduction in body weight gain was greater in the female Noble rats at approximately 13% while in male Noble rats the decrease was approximately 8% in the High-Iso groups compared to animals fed the Low-Iso diet.The present results were similar to previous studies examining young adult and aging (300 day old) Long-Evans rats for decreased body weight gain using the same two diet treatments [20,29].
When VAT weight was examined, VAT deposition was significantly reduced by 61% in females (from approximately 5.4 to 2.1 grams; p<0.011) and 21% in males (from approximately 3.9 to 3.1 grams; (p<0.027)fed the High-Iso diet versus the Low-Iso diet (Figures 4 & 5).The present results were similar to previous findings where feeding Long-Evans rats significantly reduced VAT weights when fed a High-Iso vs. a Low-Iso diet [20,29].While genistein has been shown to have weight reducing and diabetic protecting properties [30], it is interesting to note that equol represents the major circulating isoflavonoid in the present study that has been shown to have beneficial weight and VAT reducing actions [19,31].
In the present study, the greater VAT deposition in females compared to males, in general, suggests that estrogens play an important role in promoting adipose tissue deposition.For

Noble Rats Display Decreased Weight Gain and Visceral Adiposity via Lifelong Exposure to an Isoflavone-Rich Diet
Copyright: © 2014 Lephart example, estrogens promote the accumulation of subcutaneous fat [32] while the loss of estrogens is associated with increased visceral fat [33]through its receptors, estrogen receptor alpha (ER α) and estrogen receptor beta (ER β) [34,35].While ER α plays a major role in body weight regulation [36], ER β functions more as a SERM in the regulation of adiposity [37].It is known that genistein, daidzein and especially equol are selective ER β agonists [27,28,38] that may play a pivotal role in the significant VAT reduction seen in the present results.
In this manner, the link between visceral fat and chronic disease is well established, especially the connection of VAT deposition in relationship to increased cancer risk [6,8].By examining the body weight and VAT reducing properties of a diet high in isoflavones may suggest that the Noble rat model might be influence by consumption of this soy-derived parameter.It is intriguing to consider whether investigators realize how important a laboratory diet may be in altering study outcomes [39,40].For example, long-term exposure to an isoflavone-rich diet has been shown to enhance testicular and prostate health in Long-Evans rats [41].Male Noble rats exposed to such an isoflavone-rich diet may alter the outcome of spontaneous and hormonal induced tumors of the reproductive tract, thus influencing cancer parameter outcomes.This hypothesis remains to be tested, but recent evidence suggests that this may be the case since equol has the potential to improved prostate health in animal and human studies [42,43].Thus, further research is warranted in this important field of study.
In summary, visceral rather than generalized body fat appears to play a key role between obesity being link to cancer.The present study used the Noble rodent model that examined the influence of lifelong dietary exposure to soy-derived High-Iso compounds on body weight gain and VAT weight in adult female and male animals.Body weight gain and VAT weight were significantly lower in the High-Iso vs. the Low-Iso diet treatment groups, presumably by the isoflavonoid molecules acting as SERMs especially for reductions in VAT deposition.While further research is warranted, the present findings suggest potential impact of isoflavone diets on further Noble rat studies involving spontaneous and hormone-induced tumorigenesis.Finally, the concept that consumption of plant foods a way to reduce the risk of chronic disease and cancer is supported by the findings of the present study, particularly by isoflavonoid molecules and specifically by the polyphenolic compound, equol.

Figure 2 :Figure 3 :Figure 4 :Figure 5 :
Figure 2: High Isoflavone (High-Iso) versus Low Isoflavone (Low-Iso) lifelong dietary consumption in female noble Rats: influence on body weight gain.Symbol (*): significantly lower body weight gain from birth to adulthood in animals fed the High-Iso diet versus animals fed the Low-Iso diet (p<0.038).

Table 1 :
Composition of High Soy-Isoflavonoid (High-Iso) diet or a Low-Isoflavonoid (Low-Iso) diet fed to noble rats.