Effect of nutritional recovery with soybean flour diet on body composition, energy balance and serum leptin concentration in adult rats

Currently, around 600 million people worldwide suffer major nutritional problems including energy deficits and starvation, whilst approximately 300 million people have chronic problems such as energy surpluses and obesity and, in some cases, these problems co-exist [1, 2]. The increase in obesity and the reasons why some individuals appear to gain weight so easily is a much-debated topic [3].

Epidemiological studies show a higher incidence of obesity among men whose mothers experienced food deprivation in pregnancy during the Dutch famine in the Second World War [4]. In addition, central adiposity and other features related to a metabolic syndrome in adults born underweight were also documented [5, 6]. These findings suggest that malnutrition in early life could promote obesity in adulthood.

Body weight, food intake and metabolism are regulated by the hypothalamus, that processes central and peripheral signals. Within the hypothalamus, neurons residing in the ARC (arcuate nucleus) – PVN (paraventricular) – PF/LH (perifornical/lateral hypothalamus) axis communicate with each other and are subjected to the influence of several peripheral factors, including leptin [7]. Leptin is a hormone mainly produced by fat cells in which plasma levels are a function of fat mass [8, 9].

It has been suggested that programming of leptin concentrations by early diet may be one mechanism that links early nutrition with later obesity [10]. Protein restriction is associated to increase in body fat, serum leptin concentrations and food intake, which suggests a state of leptin resistance [11]. When rats are undernourished during the first two weeks of pregnancy and re-fed during the third week, the male offspring develop hyperphagia and obesity in adulthood if maintained either on a high fat diet or on a standard low fat and high carbohydrate diet [12–15]. Protein and energy restriction during lactation increase expression of leptin receptors in the pituitary and maintain normal serum leptin concentrations in adult life [16]. Thus, poor nutrition in early life and the type of diet utilized during the recovery phase appear to be critical in the determination of obesity in adulthood.

Considerable energy has been invested in finding new therapeutic approaches to overcome obesity. Studies have shown that the consumption of soybean reduces the body weight and adiposity [17, 18]. These effects were attributed either to soy protein, isoflavones and/or others components (saponins, fiber, etc.) that act together or separately. Soybean did not reduce food intake, but increased the activity of uncoupling protein 1 (UCP-1) in brown adipose tissue, enhanced lipolysis and inhibited the lipogenesis in the white adipose tissue [19–23]. In addition, isoflavones reduce serum leptin concentrations in male obese rats, because it diminishes the adiposity [21].

This study was undertaken to evaluate body composition, serum leptin concentration and energy balance in adult rats which had been subjected to protein restriction during intrauterine life and lactation and had recovered on whole soybean flour diet.

The total energy intake during the recovery phase was lower in LC and LS rats, compared to CC and CS rats (F_1,26 = 188.99, p < 0.0001) and in CS and LS groups than in the CC and LC groups (F_1,26 = 11.14, p < 0.01). During the recovery phase, the total energy intake was similar in the LS and LC groups and significantly greater than in the LL group (Figure 1A). When expressed per gram of body weight, the energy intake was higher in the LC and LS groups, in relation to the CC and CS groups (F_1,26 = 37.58, p < 0.0001) (CC = 3609 ± 120 Kj/100 g body weight, CS = 3593 ± 251 Kj/100 g body weight, LC = 3987 ± 154 Kj/100 g body weight, LS = 4264 ± 350 Kj/100 g body weight). Rats from LS and LC groups had energy intake/100 g body weight similar among them, but significantly lower than the LL group (6432 ± 1230 Kj/100 g body weight, p < 0.001) (Figure 1B).

Also, rats from LC and LS groups had smaller carcass energy (F_1,26 = 178.68, p < 0.0001), energy gain (F_1,26 = 74.89, p < 0.0001), gain as lipid (F_1,26 = 20.12, p < 0.0001), gain as protein (F_1,26 = 27.31, p < 0.0001), calculated energy expenditure (F_1,26 = 149.34, p < 0.0001) and energy efficiency (F_1,26 = 8.36, p < 0.01) than rats from CC and CS groups. These variables were lower in the CS and LS groups than in the CC and CS groups (F_1,26 = 19.77, p < 0.0001, F_1,26 = 19.77, p < 0.001, F_1,26 = 5.87, p < 0.03, F_1,26 = 6.42, p < 0.02, F_1,26 = 4.35, p < 0.05 and F_1,26 = 11.77, p < 0.01, respectively) (Figures 1C–1H). Carcass energy (Figure 1C), energy gain (Figure 1D), gain as protein (Figure 1F) and energy efficiency (Figure 1H) were lower in the LS group than in LC group (p < 0.05), but higher than in the LL group (p < 0.05). The gain as lipid (Figure 1E) was similar in the LS and LL groups but significantly lower than in the LC group (p < 0.05). In the LS and LC groups, the calculated energy expenditure (Figure 1G) did not differ and was significantly higher than LL group (p < 0.05).

In this study, protein restriction during fetal and neonatal life led to a low weight at birth and at weaning, in agreement with our previous observations [29]. At weaning, the poor nutrition status was confirmed by hipoalbuminemia and hipoinsulinemia, features commonly found in malnourished infants and in animals [30, 31]. In contrast to what has been observed in those subjected to protein restriction during growth phase after weaning which showed increases in the proportion of body fat, in this study we verified decreases in the total fat mass and percentage of body fat, but no alteration in the proportion of body protein [11, 32]. Thus, it seems that protein restriction in early life affects body fat to a greater degree than it does body protein.

Undernourished rats and those subjected to protein-restricted during intrauterine life show similar and higher body weights, respectively, after nutritional recovery, compared to control rats [13, 15, 33]. In this and in previous studies, nutritional rehabilitation after weaning did not correct the body weight deficit, regardless of dietary protein quality [34, 35]. Despite low body weight, the proportion of protein carcass remained similar in recovered and control rats, in contrast to study showing that rats submitted to prenatal and early postnatal protein malnutrition present reduced fat-free dry mass protein after nutritional rehabilitation [36]. Based on these observations, prenatal period and caloric restriction are more critical for obesity genesis than the postnatal period and protein restriction.

Obesity is thought to be result of an energy intake – energy expenditure imbalance. Although both recovered groups have showed higher energetic intake relative to body weight and lower energy expenditure, they exhibited lower proportion of carcass fat and fatty deposits than control rats, an indication that our model did not induce obesity. However, this study ended when the rats were 90 days old and it is possible that body composition changes as rats grow old.

Soybean diet has been associated to reduction in the fat deposits due to the action of isoflavone that increases energy expenditure by altering the brown-fat activity and the thyroid function [21]. In this study, soybean diet was more effective than the casein diet in reducing the proportion of fat deposits, although the animals maintained on soybean diet had eaten proportionally the same amount of diet and had showed lower energy expenditure than those fed the casein diet. It has been reported that genistein, an isoflavone of soy, decreases lipogenesis, enhances lipolysis and counteracts the antilipolytic action of insulin in isolated rat adipocytes genistein [22, 37]. Recently, we reported that our rats fed with the soybean diet show considerable amount of genistein in the sera [38] and express less mRNA of acetyl-CoA carboxylase in the liver [39]. Thus, the reduced adiposity seen in our animals maintained on soybean diet could be an effect of this isoflavone on lipid metabolism.

However, we observed a lower energy efficiency of soybean diet than casein diet. Especially in the LS group, the low energy efficiency was reflected in less energy gain as protein and proportion of carcass protein, and lower energy gain as lipid. We have recently verified that our soybean diet has lower digestibility than the casein diet (RV Veloso, MQ Latorraca, VC Arantes, MAB Reis, F Ferreira, AC Boschero and EM Carneiro unpublished results), and perhaps this effect was critical to accretion of carcass protein in animals that suffered previous nutritional deficit. Nevertheless, since the rats maintained with the soybean or casein diet showed serum albumin and total protein concentrations similar to control rats and higher as compared to malnourished rats, it became clear that the soybean diet was efficient for the recovery of the nutritional status of the rats. The lower accretion of lipid in depots and in carcass in animals recovered with soybean diet could be beneficial if in our animal model age determine changes in the body composition, favoring the development of obesity.

In agreement to what has been observed by others [40, 41], we verified that basal concentration of serum leptin did not differ among weaned protein-deprived rats, in the adults rats recovered from early protein restriction and in the control rats. Although isoflavones have been associated with reduction in leptin levels [18, 21], our rats fed with the soybean diet had similar leptin levels to those fed the casein diet. It is known that insulin and glucose together regulate leptin production and secretion [42]. Moreover, genistein increases insulin secretion by isolated islets in the presence of basal, physiologic and supraphysiologic glucose concentrations [43, 44]. In this study, rats kept on a soybean diet showed high serum insulin concentrations, as compared with those fed with a casein diet. In contrast, recovered and malnourished rats showed reduction in the serum insulin concentrations, as previously described [34]. Equivalent serum leptin levels in rats exhibiting reduced proportion of fat depots and low insulin/glucose ratio, such as it was seen in our LC and LL groups in relation to CC and CS, are consistent with the concept that early protein restriction enhances the leptin response, by increasing insulin sensitivity of adipocyte glucose uptake [41]. On the other hand, in rats maintained on soybean diet, the same effect was perhaps favored by high insulin levels, which appeared to stimulate leptin production [45].

Circulating levels of leptin are proportional to the total fat mass, and food intake and energy expenditure are regulated by leptin [46, 47]. In this study, recovered and malnourished rats, as well as those fed with soybean diet that exhibited lower fat mass, had similar serum leptin levels when compared to control rats and animals fed with casein diet. This suggests that although these animals possess fewer adipocytes, they obtain a higher amount of leptin than their controls. Also, we observed that energy expenditure appear to be related to body weight. Interestingly, serum leptin concentrations were not associated to energy expenditure or to energy intake. Even so, maintenance of normal serum leptin concentrations can be beneficial to prevent leptin resistance and obesity.

Thus, our results indicate that protein restriction during intrauterine life and lactation periods did not provoke obesity in adulthood, despite the increase in food intake in relation to body weight and reduction of energy expenditure. Nutritional recovery with soybean diet did not interfere in the serum leptin concentration, food behavior and energy expenditure, but decreased the body weight at the expense of lower energy efficiency with repercussion on lean mass.