In the present study, restricting protein in the diets of rats during fetal life led to a low birth weight. Overfeeding during lactation, induced by small litter size resulted in a higher body weight at weaning and in adulthood for the offspring, regardless of the protein content of the mother’s diets during pregnancy. However, based on Lee’s index, the protein restriction during intrauterine life resulted in obesity at weaning, independent of postnatal nutrition. It is well established that early overfeeding induces a dramatic increase in body weight during the suckling period[7, 24, 25].
Postnatal overfeeding and normal nutrition after weaning corrected the features typical of protein malnutrition (hypoalbuminemia and low serum total protein), and resulted in a higher final body weight, Lee’s index, and visceral fat mass. An increased fat mass and adipocyte number has been observed in a similar animal model[26–28]. These animals were shown to exhibit enhanced 11-β-hydroxysteroid dehydrogenase type 1 (11β- HSD1) mRNA in adipose cells, an enzyme that converts cortisone to its active form of cortisol. It has been suggested that exacerbation of adipose tissue in obese rodents and humans occur due to elevated glucocorticoid production as a result of increased 11β- HSD1[29, 30]. We verified that prenatal protein restriction did not alter body weight per se but increased visceral fat deposits. However, prenatal protein restriction combined with postnatal overfeeding did not modify the body weight, Lee’s index, and visceral pad weight and content. Therefore, our results are in disagreement with human studies that showed that growth-restriction in utero followed by postnatal catch-up growth contributes to increased central fat mass.
Obesity is the result of an imbalance between energy intake and energy expenditure; excess energy is stored as fat whenever energy intake exceeds energy expenditure. Hyperphagia, among other factors, seems to have contributed to the excessive body weight gain that occurred in our overfed groups. Interestingly, an increased appetite was also observed in the prenatal protein restricted rats, when the food consumption data were normalized for body weight. This finding contradicts the observation that in rodents, unlike in humans, the development of the hypothalamic appetite regulatory system occurs predominantly after birth. Early postnatal overnutrition, induced by small litter size[6, 7, 24, 25, 27, 33], causes persistent hyperphagia and alters the hypothalamic energy homeostasis mechanism in adulthood. The hypothalamus is the primary center in the brain that regulates food intake and body weight homeostasis. It is subject to the influence of several peripheral factors, including circulating levels of leptin, that which are proportional to the total fat mass. We verified that the circulating leptin levels were not modified by early protein restriction or by overfeeding during lactation. Moreover, the serum leptin concentrations were not equivalent to the absolute and relative weight and lipid content of the RWAT and EWAT. These results were not surprising, at least for postnatal overfed rats, because, hyperleptinemia occurs only at weaning, when subcutaneous adipose tissue is increased and leptin is produced mainly by subcutaneous adipose tissue[37, 38]. It is noteworthy that the hyperphagia in rats exhibiting unaltered serum leptin levels, as observed in the animals that were submitted to intrauterine protein restriction or early postnatal overfeeding, suggests of leptin resistance.
Spontaneous activity levels, O2 consumption and CO2 production were reduced in the animals submitted to prenatal protein restriction that exhibited a lean profile. In contrast, in the overfed rats that showed a phenotype of obesity, the activity levels did not change, but the O2 consumption and CO2 production increased. The higher O2 consumption, CO2 production and energy expenditure in the overfed animals could be explained by higher body weight. One possible explanation for the different phenotypes exhibited by our equally hyperphagic animals is the different levels of facultative thermogenesis. This hypothesis is reinforced by the diverse profile of IBAT weight and lipid content in the overfed and protein restricted rats. It has been demonstrated that IBAT thermogenic activity is accompanied by a simultaneous elevation in fatty acid synthesis and IBAT weight[39, 40]. Thus, we suggest that IBAT thermogenic activity was enhanced by the prenatal protein restriction and suppressed (decreased) by overfeeding during lactation. Reduced responsiveness to sympathetic stimulation and down-regulated adrenoreceptors in WAT, commonly observed in obese rodents[41–43], could have contributed to the increase in lipid deposits in our overfed animals. This effect is manifested by a decrease in fatty acid mobilization, and our overfed animals exhibited reduced FFA levels. FFA release from adipose tissue is also down-regulated by fasting hyperinsulinemia and could, therefore, be another factor contributing to low FFA levels and reduced fatty acid mobilization, at least in the CO group.
Independent of prenatal nutrition, postnatal overfeeding resulted in impaired glucose tolerance. Curiously, the overfed groups (CO and LO) exhibited opposite basal and stimulated serum insulin profiles and, varying hepatic insulin sensitivity, but similar peripheral insulin sensitivity. These observations are based on the HOMA-IR and Kitt values, respectively. Insulin sensitivity estimated by HOMA-IR predominantly expresses the ability of basal insulin to suppress hepatic glucose production in a fasting state, whereas Kitt is more influenced by the disposal of glucose after insulin application, reflecting peripheral insulin resistance. Increase in the body weight and Lee’s index were not accompanied by peripheral insulin resistance, and the increase in visceral fat depots was not a determinant of liver insulin resistance. It appears that hepatic insulin resistance was determined by the serum insulin concentration, corroborating the observation that insulin sensitivity and insulin secretion are reciprocal and inversely related. Another adequate explanation for the unrelated visceral fat deposits and liver insulin resistance is the mild increase in visceral adiposity observed in our overfed animals. The LC group had relative RWAT and EWAT weights and lipid contents similar to the LO group, however only the LC group exhibited hepatic insulin resistance. Remarkably, whereas prenatal protein restriction and overfeeding during lactation resulted in hyperinsulinemia in the basal state and after glucose challenge, fetal protein restriction combined with overfeeding during lactation, produced a state of basal insulin deficiency. Although the insulin deficiency was counteracted by the increase in peripheral and hepatic insulin sensitivity in the LO group, it was not enough to warrant glucose homeostasis. The insulin AUC after glucose challenge was similar in the LO and CC groups. However, these results are not indicative of normal secretion, because the insulin AUC after GTT provides an idea of the amount of insulin that acts on the tissues but cannot provide information on the dynamic of the hormone in terms of secretion, extraction and clearance. In addition to the alterations in glucose metabolism, this study found an increased cardiovascular risk in CO rats, because the Castelli II index was elevated in that group. Prenatal protein restriction also conferred an increased cardiovascular risk, considering the high triglyceride levels.
In conclusion, our results suggest that overfeeding during lactation increased visceral fat and body mass, altered lipid metabolism and contributed to glucose intolerance by distinct mechanisms. When postnatal overfeeding was combined with normal intrauterine nutrition, impaired glucose tolerance resulted from insulin resistance. When postnatal overfeeding was combined with intrauterine protein restriction, it resulted in deficient pancreatic function.