Several studies in humans and experimental animals have demonstrated that the early life environments may alter overall homeostatic regulatory mechanisms, thereby playing a critical role in influencing the susceptibility of offspring to the later development of certain diseases, as well as fetal programming [1
]. Especially, nutrition during fetal and neonatal periods is one of the major environmental factors that affect the risk of chronic diseases such as obesity, hypertension, cardiovascular diseases, and diabetes in adulthood [2
It has been shown that the consumption of a soy protein diet reduces serum triglyceride and cholesterol in rats [4
]. In particular, soy isoflavone has been shown to alleviate metabolic diseases by reducing serum and hepatic lipid levels [6
]. Similarly, exposure to dietary soy protein isolate (SPI) with isoflavone throughout the in utero, neonatal, and adult periods reduced hepatosteatosis compared with casein [7
]. Although the maternal consumption of genistein or daidzein during pregnancy and lactation was reported to reduce the width to length ratio of myocytes, which may be related to the cardioprotective effects on their offspring during adulthood [8
], most previous studies have investigated only the effect of supplementary genistein in a casein-based diet.
Conversely, another study showed that body weight and fat pad weight between the offspring of dams placed on a soy protein diet (with isoflavone) and the offspring of dams placed on a casein plus genistein diet were different in spite of similar serum genistein levels in dams [9
]. Similarly, dietary soy protein with low isoflavone reduced plasma and hepatic cholesterol and/or triglyceride in rats [10
]. These results suggest that other SPI-derived dietary components may also play important roles in ameliorating metabolic diseases. Indeed, soy protein has a different amino acid composition than casein, with higher amounts of several amino acids, including cysteine, arginine, and glycine, and lower amounts of methionine. It is reported that the effect of dietary soy protein on lipid metabolism may be mediated by its low proportion of methionine among the total sulfur-containing amino acids [12
]. Glycine has also been shown to stimulate insulin secretion [13
], suggesting that the type of dietary protein consumed may play an important role in preventing the development of metabolic syndrome. In addition, previous studies reported the hypocholesterolemic effects of soy bioactive peptides and protein fractions [14
Although several studies have reported that maternal diet affects metabolic disease development in adulthood, few studies have determined the alteration in organs and disease-related metabolism of young offspring. Previously, we have reported that the maternal consumption of low-isoflavone SPI significantly altered the hepatic gene expression profile of offspring. Much less effect was observed in response to genistein supplementation in the maternal diet [15
]. Therefore, the aim of this study was to compare the effects of the early-life consumption of a low-isoflavone SPI diet and a casein diet supplemented with genistein on hepatic glucose and lipid metabolism of male offspring rats at postnatal day (PND) 21.
2. Materials and Methods
2.1. Animals and Diets
Seven-week-old virgin female Sprague-Dawley rats were obtained from the local animal facility (Orient Bio Inc., Seongnam, Korea) and were maintained in a temperature (22 ± 3 °C) and humidity (50 ± 10%)-controlled room with a 12 h dark/light cycle. The experimental procedures used in the present study were approved by Seoul National University Institutional Animal Care and Use Committee (#SNU-081006-4). As previously described [15
], virgin female rats were randomly assigned into three groups after a one week acclimation period. Each group was offered the experimental diets containing casein (CAS diet), low-isoflavone SPI (SPI diet), or casein plus genistein (GEN diet: 250 mg/kg diet), which were prepared on the American Institute of Nutrition-93G formula, except that soybean oil was replaced with corn oil. The composition of diet is shown in Table 1
. After two weeks of feeding, females were allowed to mate with mature males of the same strain (2:1). Female rats were maintained on their corresponding diet throughout pregnancy and lactation. Diets and water were provided ad libitum
. The litter size and pup body weight were recorded at birth, and litters were adjusted to two females and six males to normalize the growth. Dams (CAS-D, SPI-D, and GEN-D groups) and their male offspring at age of PND 21 (CAS, SPI, and GEN groups) were sacrificed after an overnight fast. Blood samples were rapidly obtained by cardiac puncture. Tissues were removed, snap-frozen immediately in liquid nitrogen, and stored at −80 °C until use.
2.2. Serum Biochemical Analyses
Blood was centrifuged at 10,000× g
for 15 min and stored at −80 °C until analyzed. Serum glucose, triglyceride, total cholesterol, and high-density lipoprotein (HDL) cholesterol levels were determined using commercial kits (Asan Pharmaceutical Co., Seoul, Korea). Serum free fatty acid levels were measured using a commercially available kit (Shinyang Diagnostics, Seoul, Korea). Serum hormones, including insulin (Millipore, Temecula, CA, USA), adiponectin (R&D Systems, Minneapolis, MN, USA), and triiodothyronine (T3; GenWay Biotech, San Diego, CA, USA), were measured using an ELISA kit. The insulin resistance index was estimated by the homeostasis model assessment of insulin resistance (HOMA-IR) with the following formula: serum glucose (mmol/L) × serum insulin (mU/L)/22.5. Serum total homocysteine levels were determined by high performance liquid chromatography (HPLC) method according to previously described protocol [16
2.3. Serum Free Amino Acid Analysis
Serum free amino acid levels were determined using the HPLC method, as previously described [17
]. Briefly, serum samples were mixed with 2 mmol/L norvaline as an internal standard and 20% sulphosalicylic acid to precipitate protein. After centrifugation at 12,000× g
for 5 min at 4 °C, the supernatant containing free amino acid was removed and filtered. Samples and free amino acid standard (Agilent Technologies, Santa Clara, CA, USA) were analyzed by the Agilent 1200 HPLC equipped with an Inno C18 column (4.6 mm × 150 mm, 5 μm, Young Jin Biochrom Co., Seongnam, Korea), and the fluorescence detector. The derivatization reagents of amino acids were o-phthalaldehyde (OPA) for the primary amino acids and 9-flurorenylmethyl chloroformate (FMOC) for proline. The analytes were eluted with a gradient of eluent A (20 mm phosphate buffer, pH 7.8) and eluent B (acetonitrile:methanol:water, 45:45:10, v
) at a flow rate of 1.5 mL/min. The fluorescence detector was set at excitation 340 nm and emission 450 nm for OPA, and excitation 266 nm and emission 305 nm for FMOC.
2.4. Oral Glucose Tolerance Test
At 18–19 days of age, randomly selected four males per group with each offspring obtained from a different litter underwent the oral glucose tolerance test. Overnight-fasted offspring were given a single dose of oral glucose (2 g/kg body weight), and blood samples were obtained from the tail vein periodically over a 2-h period prior to (time 0) and 30, 60, 90, and 120 min after the glucose load. Blood glucose levels were determined by using the Accu-Chek Advantage System (Roche Diagnostics, Indianapolis, IN, USA). Total area under the curve (AUC) during the oral glucose tolerance test was calculated using the trapezoidal rule.
2.5. Hepatic Lipid Analyses
Total lipids were extracted according to the method of Folch et al. [18
]. Briefly, hepatic tissue was homogenized in 20 volumes (w
) of ice-cold phosphate buffered saline, and the protein content was measured using the commercial kit (Bio-Rad, Hercules, CA, USA). The homogenates containing 300 μg of protein (1 mg/mL) were incubated in 1.2 mL of methanol-chloroform (1:2, v
) at 4 °C for 3 h. After incubation, 240 μL of 0.88% KCl was added for the aggregation of non-lipid contents, and centrifuged at 1000× g
for 15 min at 4 °C. The bottom layer was transferred to the new tube, and hepatic triglyceride and cholesterol levels were determined by enzymatic colorimetric methods using commercial kits (Asan Pharmaceutical Co., Seoul, Korea).
2.6. Total RNA Extraction, Microarray Analysis and Real-Time PCR
Total RNA of liver tissue was isolated using RNAiso Plus (Takara Bio Inc., Shiga, Japan), and the amount of RNA was measured using Quant-iT™ RNA Assay Kit (Invitrogen, Carlsbad, CA, USA). RNA purity and integrity were analyzed on an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Microarray hybridization was performed with the Illumina RatRef-12 v1.0 Expression BeadChip platform (San Diego, CA, USA). Samples from each group (n
= 4) were analyzed by microarray. A detailed description of microarray hybridization and analysis was given in a previous study [15
]. Further comprehensive analysis was performed on 119 differentially expressed genes with the fold change ≥1.5 between two groups (p
< 0.05). For real-time PCR, cDNA was synthesized using 2 μg of total RNA with the Superscript™ II Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA). Amplification reactions were performed using a StepOne™ Real-time PCR System (Applied Biosystems, Foster City, CA, USA) according to manufacturer’s protocol. The selective gene expressions were determined by TaqMan™- or SYBR®
green-based detection. PCR primers are described in Supplementary Tables S1 and S2
, respectively. Beta-actin was used as an endogenous control. Relative gene expression levels were analyzed using the 2−ΔΔCt
2.7. Tissue Extraction and Immunoblotting
Liver tissues were homogenized in ice-cold protein lysis buffer. Equal amounts of protein were loaded into the lanes of sodium dodecyl sulfate polyacrylamide gel electrophoresis gel, separated, and blotted onto a polyvinylidene difluoride membrane. After being blocked with 5% nonfat milk or bovine serum albumin in Tris-buffered saline, membranes were probed with anti-phosphorylated AMP-activated protein kinase alpha (p-AMPKα: Cell Signaling, Danvers, MA, USA) or beta-actin (Sigma-Aldrich, St. Louis, MO, USA). The membranes were then incubated with a secondary antibody for chemiluminescent detection. The band intensities were quantified with Quantity One software (Bio-Rad, Hercules, CA, USA).
2.8. Statistical Analysis
Statistical analyses were performed using IBM SPSS Statistics version 19.0 software (IBM SPSS Inc., Armonk, NY, USA). For all experiments, one-way analysis of variance (ANOVA) followed by Duncan’s multiple range test or an independent t-test was used to determine statistical significance among the groups. Data were expressed as means ± standard error of the mean (SEM), and differences were considered statistically significant at p < 0.05. Correlation between two variables was analyzed by Pearson correlation coefficient.
Several studies have suggested that the consumption of isoflavone with soy protein has different biological effects than the consumption of isoflavone without soy protein [28
]. Previously, we have reported that maternal intake of low-isoflavone SPI significantly reduced body weights and induced liver growth retardation in offspring, with substantial effects on hepatic gene expression [15
]. Therefore, we investigated whether dietary protein source and isoflavone contents in the maternal diet would regulate the lipid metabolism in their offspring on PND 21. Lipid metabolism-related genes were shown to be significantly upregulated in mouse liver during PND 7–PND 21 [29
In the present study, serum triglyceride, total cholesterol, and HDL cholesterol levels were significantly lower in the SPI group compared with the CAS group. In contrast, only total serum cholesterol levels were significantly lower in the GEN group compared with the CAS group, resulting in a significant increase in the ratio of HDL cholesterol to total cholesterol. No significant differences in hepatic triglyceride and cholesterol levels were observed among the groups. Maternal SPI consumption attenuated serum lipid levels in offspring by reducing hepatic lipogenesis, increasing fatty acid oxidation, and enhancing bile acid catabolism based on the hepatic mRNA levels of related genes.
Although both serum triglyceride and total cholesterol levels were decreased in the SPI group compared to the CAS group, it seems that different mechanisms are involved in maternal effects on lipid metabolism of offspring. Triglycerides in milk are about 100-fold higher than those in the serum of dams and their offspring [30
], suggesting that lower serum triglyceride levels in the SPI group were not due to lower triglyceride levels in the milk of their dams. Meanwhile, dams at 21 days of suckling fed a soy protein diet showed lower cholesterol levels in serum, liver, and milk compared with dams fed a casein diet [31
]. Similarly, maternal flaxseed diet during lactation decreased cholesterol levels in milk and serum of offspring [32
]. Therefore, cholesterol contents in milk could possibly affect serum cholesterol levels.
In consideration of the regulatory role of PPARα on lipid metabolism in adults, we examined whether PPARα activation in offspring is altered by maternal diet. Our results clearly showed increased expressions of target genes of PPARα in the SPI group, which may be responsible for the hypolipidemia observed in the SPI group. PPARα has been shown to regulate the transcription of genes involved in glucose and lipid metabolism, liver inflammation, and hepatocyte proliferation (especially in rodents) [25
]. Besides natural ligands, including fatty acids and their metabolites, various xenobiotics are shown to activate PPARs [33
]. Indeed, we observed increases in the mRNA levels of genes involved in xenobiotics and drug metabolism. The altered levels of xenobiotics were possibly due to the retarded liver development, and may activate PPARα in the SPI group [15
]. Interestingly, the mRNA levels of PPARα-target genes, including Me1
were significantly associated with the relative liver weight of offspring (data not shown). Similarly, the induction of dyslipidemia in offspring by a maternal low-protein diet (9% vs. 18%) during pregnancy involves an altered epigenetic regulation of specific transcription factors, including PPARα, in the liver of offspring [34
]. Indeed, a recent study reported the increased gene expressions of the PPARα pathway (Cyp7a1
) by maternal low-protein diet (8% protein) throughout pregnancy and lactation in the liver of mice offspring [35
]. Furthermore, the maternal consumption of oxidized fat during pregnancy upregulates expression of PPARα-target genes with lowered triglyceride concentrations in both dam and fetal livers [36
Although structure–activity relationship studies showed a direct binding of isoflavones to PPARs [37
], we did not observe the PPARα activation in the livers of GEN diet-fed dams (data not shown) and their offspring. Instead, we observed the significant increase in Ldlr
mRNA levels in the GEN group. Consistently, a previous study showed a higher expression of the Ldlr
gene in male Zucker rats fed a high-isoflavone diet compared with casein-fed rats, but not in rats fed a protein diet with similar amino acid profiles (low methionine/glycine and low lysine/arginine) with SPI for six weeks [38
A previous study proposed that non-isoflavone phytochemicals and their metabolites, or the altered protein/peptide composition during isoflavone-removal processing, may be responsible for the effect of SPI on PPARα activation [39
]. A recent cell culture study reported that the soybean-derived dipeptide Trp-Glu is a key hypolipidemic compound via
PPARα activation [40
]. Here, we did not observe a higher activation of PPARα in dams fed an SPI compared with dams fed a CAS diet (data not shown), which suggested that hormonal or metabolic changes induced in dams may be responsible for PPARα activation in the SPI group.
Maternal consumption of a SPI diet significantly decreased serum BCAA/AAA in dams compared with a CAS diet (1.5 vs. 2.3). In normal animals, the BCAA/AAA ratio in plasma is about 3:1 [41
]. Previous studies reported decreases in maternal BCAA plasma concentrations in intrauterine growth restriction (IUGR) models with dams fed a low-protein diet, and in Ldlr−/−
dams fed a Western diet [42
]. Similarly, IUGR induced by calorie restriction decreased plasma concentrations of BCAAs [44
]. In the same study, BCAA supplementation effectively increased plasma BCAA levels and improved the physiological functions of uterus and placenta, resulting in increased litter size and embryo weight [44
]. These results suggest that the altered amino acid profile, especially lowered BCAA concentrations, may be responsible for some of the phenotypes observed in the offspring of IUGR models. We consistently observed a significant negative correlation between the serum AAA levels in dams and the body weight of offspring at PND 21. Moreover, maternal AAA concentration was significantly correlated with serum triglyceride levels and PPARα-target gene expressions in offspring. Further research is warranted to investigate the underlying mechanism for the association between maternal AAA levels and PPARα activation in offspring.