Dose-dependent effects of a genistein-enriched diet in the heart of ovariectomized mice
© The Author(s) 2012
Received: 28 July 2012
Accepted: 16 October 2012
Published: 30 October 2012
The isoflavone genistein is used as a pharmacological compound and as a food supplement. The duration and the level of exposure of humans to genistein are considerable. However, the magnitude of genistein-supplemented dietary interventions necessary to induce any changes in the heart has not been studied so far. The aim of this study was to investigate the dose-dependent effects of dietary genistein in the disease- and stress-free mouse heart. Female C57BL/6J mice at the age of 2 months were ovariectomized and randomly assigned to feed on diets with seven different genistein doses (0.01, 0.03, 0.1, 0.3, 1, 3 and 10 g genistein/kg food) for 3 months. Mice with intact ovaries or ovariectomized fed on soy-free diets were used as controls. Ovariectomy led to an increase in body weight, while the two highest genistein doses prevented this increase. Absolute uterus weight was decreased in the ovariectomized group and all genistein groups except for the 10 g/kg food group compared with the intact ovaries/soy-free group. Considering cardiac mass, although the 3 and 10 g/kg food groups had significantly lower absolute heart weight than all other groups, heart-to-body-weight ratios did not differ between these two groups and the intact ovaries/soy-free group, while all remaining groups had smaller ratios. Next, we observed dose-dependent effects of genistein on cardiac gene expression. The present findings indicate that exposure of female mice to the soy isoflavone genistein influences body weight and cardiac mass and gene expression in a dose-dependent manner. Human exposure to dietary genistein supplements may influence cardiac function.
KeywordsCardiac mass Diet Genistein Soy
Cardiovascular disease (CVD) represents a major cause of morbidity and mortality in the developed world (Lakatta 2002; Yusuf et al. 2001). In women, the incidence of CVD rises substantially with menopause. Due to this, it has been generally believed that the loss of oestrogen at menopause might be a major contributing factor to the increased risk for CVD. Consequently, several combinations of hormone therapies with naturally occurring and/or synthetic products have been used widely. However, unexpected negative findings from large randomized clinical trials (Anderson et al. 2004; Rossouw et al. 2002) and conflicting results from animal studies have led to controversy about the use of hormone therapies.
Along this line, soy-rich foods have been studied extensively for their ability to reduce cholesterol levels (Zhan and Ho 2005). Epidemiological studies suggest that the coronary benefits of soy extend beyond lipid lowering (Zhang et al. 2003). Soy is a rich source of isoflavones and phytoestrogens (Murphy et al. 2002). Genistein (GEN) is an isoflavone derivative found in plants, which has been shown to inhibit tyrosine kinases (Akiyama et al. 1987) and could, therefore, lead to detrimental effects in the heart (Sereno et al. 2008; Force et al. 2007) and to interact with oestrogen receptors (Davis et al. 1999). The latter activity of GEN has led to its use as a substitute of oestrogen in hormone therapy regimes. However, GEN is not only used as a pharmacological compound but also as a food supplement. Therefore, the duration and the level of exposure of humans to GEN are considerable.
The effects of GEN have been generally studied in the classical target organs of oestrogen, such as mammary gland, uterus and bone. However, little is known about the potential role of dietary GEN directly in the disease- and stress-free heart. In the present study, we investigated the dose-dependent effects of dietary GEN in the hearts of female C57BL/6J mice. We tested the hypothesis that GEN will affect body weight, cardiac mass and gene expression in a dose-dependent manner.
Materials and methods
Animals and diets
Quantitative real-time RT-PCR
Total RNA was isolated from left ventricles using the RNeasy Total RNA Kit (Qiagen, Hilden). cDNA was synthesized using the M-MLV Reverse Transcriptase (Promega, Madison) and random primers (Invitrogen, Karlsruhe). Reactions were performed as described previously (Kararigas et al. 2011; Nguyen et al. 2012) using SYBR Green (Applied Biosystems, Foster City). For Myocd analysis, we used the Mm_Myocd_1_SG primers, for Esr1 the Mm_Esr1_2_SG primers and for Esr2 the Mm_Esr2_1_SG primers; all from the QuantiTect Primer Assay (Qiagen, Hilden). For Igf1 analysis, we used the following: forward primer 5′-CTTCAACAAGCCCACAGGCTA-3′, reverse primer 5′-GCTCCGGAAGCAACACTCAT-3′ and probe 5′-CTCCAGCATTCGGAGGGCACCTC-3′.
All data were analysed statistically using the R version 2.14.2 software. Data are shown as the mean ± SEM. Comparisons between multiple groups were performed using analysis of variance with Tukey’s post hoc test adjusting for multiple comparisons, considering P ≤ 0.05 significant.
In the present study, we investigated the dose-dependent effects of GEN in the hearts of OVX mice under physiological conditions. We tested the hypothesis that GEN will affect body weight, cardiac mass and gene expression in a dose-dependent manner. For this purpose, we selected seven concentrations of GEN to include a wide range of GEN levels with relevance to human consumption and doses of GEN that have been previously shown to exert oestrogenic-like effects in rodents (Nguyen et al. 2012). Mice that received GEN were ovariectomized and a further OVX group on soy-free diet was used as one of two controls. The second control group was soy-free-fed mice with intact ovaries (Fig. 1).
Food consumption and actual GEN intake
Food consumption and actual GEN intake
Food consumption (g/mouse/day)
Actual GEN intake (mg/mouse/day)
Dose-dependent effects of dietary GEN on body weight
Dose-dependent effects of oral GEN treatment on uterus and heart weight
Dose-dependent effects of dietary GEN on gene expression in the heart
In the present study, we investigated the dose-dependent effects of GEN in the heart of disease- and stress-free mice. We employed seven different concentrations of GEN in OVX mice and found that significant effects were exerted on body and organ weight and cardiac gene expression by the three highest GEN concentrations.
In particular, we discovered that although the average daily food intake per mouse was comparable in most groups, mice fed on a diet with the two highest GEN doses exhibited a significant decrease in food intake. While this was a surprising finding and there is no obvious explanation for this difference, we anticipate that GEN might exert direct effects on the brain, which in turn has a major role in the control of food intake (Del Parigi et al. 2002). Subsequently, we found that OVX mice had an increase in their body weight compared with mice with intact ovaries. However, this increase was hindered in the GEN 3 and 10 g/kg food groups, which may be the result of the combination of lower food intake in these groups and unrecognized molecular pathways involved in the control of body weight and adiposity signals that may be regulated by high GEN levels. In fact, it has been suggested that controlling meal size may be relevant for the development of efficacious therapeutic tools to reduce eating (Lutz 2006). Along this line, the leptin receptor long form and the SH2-tyrosine phosphatase Shp2 might be potential targets of GEN in the brain, whose modulation by leptin and oestrogen signalling regulates food intake and energy balance (He et al. 2012; Ring and Zeltser 2010). Based on our findings, we propose that GEN supplementation of a well-balanced healthy diet might be instrumental in whole body weight loss approaches. However, deciphering the underlying molecular pathways holds the promise for the development of novel therapeutic means.
Assessing absolute heart weight, we found that mice fed on the two highest GEN doses had significantly smaller hearts than any other GEN group. This finding associated with the effects on body weight fits the obvious model that a bigger body requires a bigger heart and vice versa. However, heart-to-body-weight ratios revealed that there were no significant differences between the groups with the two highest GEN concentrations and the intact ovaries/soy-free-fed control group. This suggests that in the absence of endogenous hormones and particularly oestrogen as a result of ovariectomy, GEN might be crucial for the maintenance of cardiac stability. Preserving the size and structure of an organ would be of utmost importance for the organ’s function. Therefore, we believe that GEN supplementation of a normal diet may be beneficial for cardiac function, especially in postmenopausal women. However, this hypothesis needs to be tested in a study where cardiac function is also assessed.
On the other hand, it should be taken into account that hormonal actions in the heart may be sex specific and not always beneficial (Kararigas et al. 2010, 2012). In fact, in a genetic model of hypertrophic cardiomyopathy, it was shown that a soy-based diet was beneficial in females but harmful in males (Luczak et al. 2011). The observed beneficial effects in females were postulated to be attributed to increased Igf1 pathway expression (Luczak et al. 2011). Downstream activating signalling of Akt by Igf1 in cardiomyocytes is protective against injury (Fujio et al. 2000). To this extent, premenopausal women have been shown to have significantly higher levels of nuclear-localized phosphorylated Akt in cardiomyocytes compared with age-matched men or postmenopausal women (Camper-Kirby et al. 2001). In the present study, we found that the three highest doses of GEN, that is, 1, 3 and 10 g/kg food, led to increased Igf1 expression compared with both control groups. Based on these findings, we put forward that the GEN-induced Igf1 expression is beneficial for the heart. However, considering sexual dimorphism in hormonal effects, it would be very interesting to verify the role of GEN in the disease- and stress-free heart of male mice.
The potential use of GEN as a natural selective oestrogen receptor modulator (SERM) seems to be promising. In long-term hormone therapy regimens, the use of oestrogen may exert negative effects in the breast and the endometrium. However, a clear advantage of GEN is that it may behave as an oestrogen receptor antagonist in both of these tissues. Along this line, in a previous study, we did not observe any oestrogenic-like effects of GEN in the uterus, as assessed by the expression of the insulin-like growth factor 1 (Igf1) gene (Nguyen et al. 2012). This indeed supports the notion that genistein is not an oestrogen receptor agonist in the uterus, whose inner cell layer is the endometrium. In addition, the incidence of uterine dysplasia was low in ovariectomized rats fed GEN, suggesting a weak oestrogen receptor agonist role of GEN also in the rat uterus (Aidoo et al. 2005).
In conclusion, we have identified dose-dependent effects of dietary GEN on body weight and directly on cardiac mass and gene expression. Further research is required to study GEN effects on cardiac function and to elucidate the molecular factors mediating these effects.
This study was supported by the European Union-funded research project EUGeneHeart (LSHM-CT-2005-018833).
Conflict of interest
The authors declare that they have no conflict of interests.
This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited.
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