Dietary isoflavones alter regulatory behaviors, metabolic hormones and neuroendocrine function in Long-Evans male rats
© Lephart et al; licensee BioMed Central Ltd. 2004
Received: 04 October 2004
Accepted: 23 December 2004
Published: 23 December 2004
Phytoestrogens derived from soy foods (or isoflavones) have received prevalent usage due to their 'health benefits' of decreasing: a) age-related diseases, b) hormone-dependent cancers and c) postmenopausal symptoms. However, little is known about the influence of dietary phytoestrogens on regulatory behaviors, such as food and water intake, metabolic hormones and neuroendocrine parameters. This study examined important hormonal and metabolic health issues by testing the hypotheses that dietary soy-derived isoflavones influence: 1) body weight and adipose deposition, 2) food and water intake, 3) metabolic hormones (i.e., leptin, insulin, T3 and glucose levels), 4) brain neuropeptide Y (NPY) levels, 5) heat production [in brown adipose tissue (BAT) quantifying uncoupling protein (UCP-1) mRNA levels] and 6) core body temperature.
This was accomplished by conducting longitudinal studies where male Long-Evans rats were exposed (from conception to time of testing or tissue collection) to a diet rich in isoflavones (at 600 micrograms/gram of diet or 600 ppm) vs. a diet low in isoflavones (at approximately 10–15 micrograms/gram of diet or 10–15 ppm). Body, white adipose tissue and food intake were measured in grams and water intake in milliliters. The hormones (leptin, insulin, T3, glucose and NPY) were quantified by radioimmunoassays (RIA). BAT UCP-1 mRNA levels were quantified by PCR and polyacrylamide gel electrophoresis while core body temperatures were recorded by radio telemetry. The data were tested by analysis of variance (ANOVA) (or where appropriate by repeated measures).
Body and adipose tissue weights were decreased in Phyto-600 vs. Phyto-free fed rats. Food and water intake was greater in Phyto-600 animals, that displayed higher hypothalamic (NPY) concentrations, but lower plasma leptin and insulin levels, vs. Phyto-free fed males. Higher thyroid levels (and a tendency for higher glucose levels) and increased uncoupling protein (UCP-1) mRNA levels in brown adipose tissue (BAT) were seen in Phyto-600 fed males. However, decreased core body temperature was recorded in these same animals compared to Phyto-free fed animals.
This study demonstrates that consumption of a soy-based (isoflavone-rich) diet, significantly alters several parameters involved in maintaining body homeostatic balance, energy expenditure, feeding behavior, hormonal, metabolic and neuroendocrine function in male rats.
white adipose tissue
brown adipose tissue
phytoestrogen free diet
- (T3, T4):
National Institutes of Health
reverse transcriptase-polymerase chain reaction
Food and Drug Administration
follicle stimulating hormone
Brigham Young University
high performance liquid chromatography
Some phytochemicals are considered to be endocrine disrupters that mimic or modulate the physiological effects of steroid hormones, especially that of estrogens [1, 2]. Of all estrogenic endocrine disrupters examined thus far, phytoestrogens have been extensively studied [1–6].
Phytoestrogens represent hundreds of molecules possessing non-steroidal, diphenolic structures found in many plants (e.g. fruits, vegetables, legumes, whole-grain and especially soy food products) that have similar chemical and structural properties to those of estrogens [1–4]. There are three main classifications of phytoestrogens: 1) isoflavones (derived principally from soybeans), 2) lignans (found in flaxseed in large quantities) and 3) coumestans (derived from sprouting plants like alfalfa) [2–6].
Of these three main classifications, human consumption of isoflavones has the largest impact due to its availability and variety in food products containing soy. Furthermore, the phytoestrogens principally derived from soy foods have received prevalent usage due to their 'health benefits' of decreasing: a) age-related diseases (cardiovascular & osteoporosis), b) hormone-dependent cancers (e.g. breast & prostate) and c) postmenopausal symptoms [2–6]. However, little is known about the influence of dietary (soy-derived) phytoestrogens on neuroendocrine, hormone and metabolic parameters. In spite of this fact, the Food and Drug Administration (FDA) in the United States in October of 1999 authorized the use of-on food labels- the health claim that: soy protein can reduce the risk of coronary heart disease by lowering blood cholesterol levels (when included in a diet low in saturated fat and cholesterol) .
The purpose of this study was to examine, in a comprehensive manner, important hormonal and metabolic health issues by testing the hypotheses that dietary soy-derived phytoestrogens influence: 1) body weight and adipose deposition, 2) food and water intake, 3) metabolic hormones (i.e., leptin, insulin, T3 and glucose levels), 4) brain neuropeptide Y (NPY) levels, 5) heat production [in brown adipose tissue (BAT) quantifying uncoupling protein (UCP-1) mRNA levels] and 6) core body temperature. This was accomplished by conducting longitudinal studies where male Long-Evans rats were exposed (from conception to time of testing or tissue collection) to a diet rich in phytoestrogens vs. a diet low in phytoestrogens.
Long-Evans male and female rats [10 per sex at 50 days old] were purchased from Charles River Laboratories (Wilmington, MA, USA) for breeding. These animals were caged individually and housed in the Brigham Young University Bio-Ag vivarium and maintained on an 11-hour dark 13-hour light schedule (lights on 0600–1900). The animals and methods of this study were approved by the institute of animal care and use committee (IACUC) at Brigham Young University (BYU).
Body weights and food intake were measured on a Mettler 1200 balance [in grams (g) ± 1 g; St. Louis, MO, USA], white and brown adipose tissue and prostate weights were measured on a Sartorious balance [in milligrams (mg) ± 1 mg; Brinkman Inst. Co., Westbury, NY, USA]. Water intake was measured in drinking tubes [in milliliters (ml) ± 1 ml]. White adipose tissue (WAT) was dissected inferior to the kidneys and superior to the testes in the abdominoplevic cavity (representing a majority of intra-abdominal WAT) and then weighed in grams ± 0.01 g. Brown adipose tissue was dissected from between the scapular blades (inter-scapular region) and weighed in milligrams (mg) ± 1 mg.
Serum leptin and insulin levels were determined by kits purchased from Linco Res. Inc. (St. Charles, MO, USA) [from arterial blood samples of 33 and 55 day-old male animals and venous blood samples collected from 75 day-old rats. This was due to exhausting the arterial supplies from the available blood samples for other assays and thus venous blood was assayed at 75 days of age]. Serum thyroid (T3) levels were assayed by a kit purchased from Diagnostic Systems Labs. Inc. (Webster, TX, USA) and glucose levels were detected by a kit (#510) purchased from Sigma Chem. Co. (St. Louis, MO, USA).
Hypothalamic NPY Levels
Subsequent to blood collection (above), after the animals were sacrificed, brains were removed rapidly, frozen on dry ice and then stored at -80°C until microdissection. Coronal slices 300 μm thick were sectioned on a microtome cryostat. The paraventricular nucleus, arcuate nucleus and median eminence regions of the hypothalamus were microdissected by punch technique and homogenized in 100 μl of 0.1 M HCl. Tissue protein was determined by the Lowry method  and NPY was measured using a solid-phase radioimmunoassay in Protein G-coated 96-well plates, as described previously . The NPY antiserum was used at a final concentration of 1:16,000. The sensitivity of the assay is 0.2 pg, with an intra-assay coefficient of variation of 8 %. All samples were run in duplicate in the same assay to avoid inter-assay variation.
Body temperature was monitored by radio telemetry by implanting a very small electronic chip [under the skin above the left thoracic cavity near the heart] that measured and transmitted core body temperature (± 0.1°C) to a notebook-sensor monitor (BioMedic Data Systems Inc., Seaford, DE, USA) within 2 seconds and repeated measurements were made throughout the day and/or the duration of the experiments.
Body Heat Production
Uncoupling protein (UCP-1) mRNA levels were determined in brown adipose tissue (BAT) collected from the interscapular region of each male rat. The BATs were homogenized in Trizol reagent (Invitrogen, Carlsbad, CA, USA) and total RNA was extracted. Two micrograms (2 μg) of total RNA were reverse transcribed (RT) for 60 min at 42°C using Superscript II (Invitrogen, Carlsbad, CA, USA) (200 U). Each 20 μl reaction contained 0.1 M DTT (2 μl), 10 mM dNTP mix (1 μl), 10X PCR buffer (2 μl), random decamers (0.4 μl), and RNaseOUT (Invitrogen) (40 U). A duplex PCR reaction was then performed on each RT product, with 18S rRNA serving as the internal control. Each 50 μl reaction contained the RT product (2 μl), UCP-1 primers (2 μl), 18S primers [2 μl of 3:7 ratio of 18S primer to18S competimer (Ambion, Austin, TX, USA)], 10X PCR buffer (5 μl), 10 mM dNTP mix (0.625 μl), 32P-dCTP (0.1–0.15 μl), and Jumpstart Taq polymerase (Sigma Chem. Co., St. Louis, MO, USA) (1 U). Each tube was then subjected to the following protocol: 95°C for 5 min, 20 cycles of 94°C for 30 sec, 60°C for 30 sec, 72°C for 45 sec, followed by 72°C for a final 10 min. interval. With this profile, the UCP-1 and 18S fragments were amplified within the linear range (20 cycles for UCP-1). The primers for UCP-1 were GTGAAGGTCAGAATGCAAGC (sense) and AGGGCCCCCTTCATGAGGTC (antisense), the resultant fragment was 197 bp. The sequence of the UCP-1 fragment was verified by the DNA Sequencing Center at BYU. The PCR products were then subjected to non-denaturing polyacrylamide gel electrophoresis and the gels were exposed to autoradiographic film. Optical density (O.D.) of each band was determined using the NIH imaging system (Version 1.61). For each sample the O.D. ratio UCP:18S was determined. Each RT-PCR protocol was repeated and O.D. ratio values averaged over at least two runs.
All data are presented as the mean ± SEM with p < 0.05 deemed significant. The data were tested by analysis of variance (ANOVA) (or where appropriate by repeated measures), followed by pairwise comparisons (via Neuman-Keuls analysis) to detect significant differences between the diet treatment groups (p < 0.05).
Body Weight, White Adipose Tissue Weight and Food/Water Intake
Circulating Leptin, Insulin, Glucose and Brain NPY Levels
From the animals collected on 33, 55 and 75 days of age not enough serum was left after other assays were performed to quantify glucose levels on these days. However, circulating glucose levels were assayed in non-fasting 65, 80 or 110 day-old animals, Phyto-600 fed males displayed slightly higher values (that were not significantly different) compared to Phyto-free fed males [age 65 days old- Phyto-600 = 113.5 (± 4.4) vs. Phyto free = 93.5 (± 9.0) mg/dl, n = 8 per group; age 80 days old- Phyto-600 = 137.2 (± 3.8) vs. Phyto-free = 122.5 (± 3.9) mg/dl, n = 10 per group; age 110 days old- Phyto-600 = 123.5 (± 5.0) vs. Phyto-free = 113.8 (± 3.6) mg/dl, n = 5 per group, (mean ± SEM), data not shown graphically].
Circulating Thyroid (T3), UCP-1 mRNA Levels and Core Body Temperature
In non-fasting young adult rats at 65 and 110 days of age, circulating thyroid (T3) levels were determined from venous blood samples. Phyto-600 fed males displayed significantly higher T3 levels compared to Phyto-free fed values [age 65 days old- Phyto-600 = 2.4 ± 0.2 vs. Phyto-free = 1.5 ± 0.4 pg/ml (± SEM), n = 8 per diet treatment; age 110 days old- Phyto-600 = 1.9 ± 0.4 vs. Phyto-free = 0.8 ± 0.3 pg/ml, n = 5 per group, (mean ± SEM) data not shown graphically].
Estrogen is known to play a dual role in regulating body weight, food intake and adipose tissue deposition. On the one hand, estrogens decrease food intake, increase locomotor activity and hence decrease body weight [10, 11]. However, adipose tissue deposition increases with puberty and early pregnancy in women, suggesting that estrogens influence body fat accumulation . Additionally, in aging, estrogens promote adipose deposition and insulin resistance . Conversely, results from aromatase, FSH and ER-knockout studies indicate that estrogens regulate adiposity where the complete lack of estrogens or blocking estrogen hormone action increases adipose tissue deposition [14–18], whereas, estrogen replacement in these models decreases adiposity. Notably, in the present study, male rats fed the Phyto-600 diet displayed significantly decreased adipose tissue and body weights compared to Phyto-free fed animals. While there is not extensive data on phytoestrogens and metabolism, other investigators have reported that genistein, increases lipolysis and decreases lipogenesis in rodent adipocytes  by a tyrosine kinase independent mechanism and these estrogen mimics inhibit glucose uptake by altering membrane-associated glucose transporters [20, 21]. Thus, our data suggests that dietary soy phytoestrogens significantly decrease: 1) body and adipose tissue weights and 2) circulating leptin and insulin levels (that correspond with adipose deposition) compared to Phyto-free fed animals, implying that the hormonal action of phytoestrogens is beneficial to body fat regulation. Recent studies imply that insulin helps to regulate leptin expression in humans  and estrogens appear to enhance the action of insulin [23, 24]. This may account for the decreased incidence of obesity in Asian countries where isoflavone consumption is high compared to Western countries. Decreased adipose tissue deposition by decreasing lipogenesis and increasing lipolysis may help to prevent insulin resistance (by reducing body fat) and the estrogenic actions of dietary phytoestrogens may augment the efficiency of insulin.
It was previously observed in our laboratory that dietary phytoestrogens significantly alter food and water intake [7, 25, 26]. The differential effects of the Phyto-free vs. Phyto-600 diets observed in the present studies on hypothalamic NPY levels, circulating insulin and leptin concentrations and food intake are consistent with the well established interrelationships among these parameters. Thus, relative to animals maintained on the Phyto-free diet, food intake was significantly increased in animals fed the Phyto-600 diet. Phyto-600-fed rats also exhibited higher concentrations of NPY in the arcuate and paraventricular nuclei of the hypothalamus. It is well established that NPY neurons whose perikarya reside in arcuate nucleus and project to PVN comprise an extremely important orexigenic neural pathway . It therefore appears likely that at least one factor contributing to the higher food intake in Phyto-600-fed rats is the increased levels of NPY in this system.
The present studies also suggest a mechanism that may underlie the diet-induced effects on NPY (i.e., plasma insulin and leptin concentrations were significantly reduced in the Phyto-600 fed rats, relative to the Phyto-free animals). A number of previous studies have demonstrated a reciprocal relationship between circulating insulin and leptin titers and NPY concentrations in PVN. Thus, experimentally-induced reductions in either insulin  or leptin  are associated with increased pre-proNPY messenger RNA expression in arcuate nucleus and increased NPY levels in PVN, and moreover, it has been proposed that reductions in insulin and leptin that occur physiologically, e.g., with food deprivation, provide an important signal to the NPY system to initiate feeding [27, 29]. Hence, taken together, the present findings suggest that by reducing secretion of insulin and/or leptin, chronic consumption of the Phyto-600 diet results in up-regulation of the orexigenic NPY circuit in the hypothalamus, which in turn stimulates food intake (and water consumption, since rodents and humans display prandial characteristics).
Uncoupling proteins (UCP-1 through UCP-5) are expressed in various tissues from many different species (mammals, birds, fish, insects and plants) that play important (but controversial) role(s) in the regulation of energy expenditure, or thermogenesis [40, 41]. Uncoupling protein-1 is expressed mainly in BAT. When the influence of dietary phytoestrogens on UCP-1 mRNA levels in BAT was examined, Phyto-600 fed male rats, expressed significantly higher levels of the uncoupling protein (approximately 2-fold) compared to Phyto-free values (but BAT weights were significantly less in the Phyto-600 vs. Phyto-free fed males). To date, we are unaware of any studies that have investigated this aspect of soy consumption on thermogenesis. The decrease in BAT mass in Phyto-600 animals but increased expression of UCP-1 may represent a compensation mechanism for energy expenditure, and there are several neural inputs and hormonal factors that influence UCP-1 in BAT that make it difficult to differentiate the regulatory aspects of UCP-1 expression. For example, sympathetic denervation of inter-scapular BAT markedly reduced UCP-1 mRNA levels and estrogen, T3 and adrenergic agents [norepinephrine (NE)] stimulate UCP-1 expression in BAT [42, 43]. In fact, it has been reported that T3 synergizes with NE to increase UCP-1 in BAT and stabilizes its mRNA transcripts . These factors overlap with the changes seen in Phyto-600 fed vs. Phyto-free fed rats, in the present study, where T3 levels were increased and, presumably, along with the estrogenic influence of circulating isoflavones resulted in stimulating UCP-1 expression in BAT. Previously, we have not observed any significant alterations in circulating estradiol (or LH) levels in Phyto-600 vs. Phyto-free fed intact males . Conversely, it has been reported that increases in hypothalamic NPY decrease UCP-1 [and reduces sympathetic outflow to BAT, but increases adipose tissue lipoprotein lipase activity] . Also, plasma leptin levels are thought to stimulate UCP-1 in BAT [45, 46], results opposite, in general, to that obtained in the present study. Based upon the obtained data sets, it is difficult to identify a common stimulatory or inhibitory pattern for the expression of UCP-1 in BAT of soy fed animals and especially define a functional role for the physiological properties associated with these UCPs in thermoregulation. Therefore, it is reasonable to speculate that multiple factors act collectively to regulate UCPs in BAT that in turn contribute to adaptive changes in body temperature.
This study demonstrates that consumption of a widely used commercially available soy-based rodent diet, (i.e., the Phyto-600 diet rich in isoflavones), alters several hormonal, metabolic and neuroendocrine parameters involved in maintaining body homeostatic balance, energy expenditure and feeding behavior in male rats. Further research is warranted in examining the important aspects of the neuroendocrine and metabolic influences of dietary phytoestrogens via the consumption of soy in humans and laboratory animals. This is especially true when diet is usually not considered as an influencing factor in the experimental design [47–50].
This work was supported, in part, by grants from the USDA (2002-00798; EDL), the BYU Research Office (21-223566 & 19-223566; EDL) The Dean's Graduate Fellowship in Neuroscience (TDL and LB), and the National Institutes of Health (HD-13703; WRC)
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