The administration of oleoyl-estrone (OE) (a naturally occurring ester of estrone and fatty acids stored in adipose tissue) to rats at pharmacological doses, induces a transitory decrease in food intake, resulting in a noticeable decrease in body weight that is not paralleled by any concurrent losses in body protein 1. Since rats follow this same pattern under different nutritional and physiological conditions 23 hints at OE as a ponderostat (set-point of body weight) regulator 4. Although receptors other than the estrogen receptor mediate OE effects 5 its mechanism of action remains unknown, and involves pathways different from those activated by food restriction alone 6.

It is well known that the availability of food during pregnancy affects the postnatal development of their litter 7. The consequences of starvation or food deprivation are well documented, in particular the high correlation between maternal energy restriction and adult obesity 8. Obesity is related to insulin resistance, hypertension and dislipemia, factors that alone or combined decrease the life span 9. These effects are mediated, in part, by oxidative processes linked to inflammation 10. On the other hand, there are evidences that energy restriction is associated with longer life span in rodents 11. Thus, it can be argued that the control of factors eliciting the onset of obesity can also help decrease morbidity and mortality. We have intended to determine whether OE-treatment may affect pregnant rat descendants' growth pattern, since OE reduces voluntary energy intake.

In Fig. 1A are depicted the variations in weight of pregnant rats along time. Specifically, control rats increased their weight 118 ± 4 g from day 0 to day 21, whereas OE-treated rats increased 65 ± 4 g. After parturition, both groups' dams had the same weight. Fig. 1B shows that the pups from OE-treated rats had a growth pattern similar to that of controls from birth up to day 20. After weaning, the treated group slowed its growth rate, significantly diverging from controls from day 27 onwards.

The changes in daily food intake caused by treatment can be seen in Fig. 2. Control animals ingested 1328 ± 62 g of standard chow pellets during the entire study period, whereas treated dams ingested 1193 ± 113 g (i.e. 89.8% of controls). There were significant differences between groups on days 1, 16, 17 and 19 of pregnancy.

Changes in serum metabolites during pregnancy and lactation period, both in dams and their pups are shown in Table 1. Transition from pregnancy to lactation induced significant changes in almost all serum metabolites in the dams, with increases in urea and cholesterol and decreases in protein, non-esterified fatty acids and triacylglycerols. OE treatment induced significant decreases in glucose and HDL-cholesterol in late pregnancy, and in ketone bodies and non-esterified fatty acids at weaning, whereas protein levels were increased on day 20 after delivery. Transition from fetal life to suckling pups brought significant changes (essentially increases) in all parameters tested, except for urea and 3-hydroxybutyrate. Pups from OE-treated mothers showed significant increases in protein and HDL-cholesterol and decreases in glucose on day 30 (PP30); the descendants from OE-treated dams showed lowered glucose values in fetal life and in ketone bodies on day 20, and increased protein values at weaning.

In Table 2, the levels of serum hormones are depicted. A significant decrease in insulin and adiponectin progressed from late pregnancy to lactation. OE treatment decreased leptin and adiponectin during late pregnancy. Developing pups showed increased adiponectin both in control and OE-treated groups. OE treatment caused a significant increase in insulin at weaning and decreased leptin and adiponectin on day 30 (PP30).

Body composition of pregnant rats on days 11 and 21 of pregnancy, as well as the changes caused by OE treatment are depicted in Table 3. These changes were determined by comparing an ideal composition of each rat on day 11 (using the mean P11 body composition but their actual day 11 weight) with their measured composition and weight on the final day of treatment (P21). Our data show that OE treatment induced a limited increase in body weight, essentially a consequence of low lipid accrual. Since protein content did not change, and lipid content was reduced in OE-treated rats, the decrease in total energy content in OE-treated rats was a consequence of the decreased lipid content.

Changes induced in the weight, cell number, and cell mass of different adipose tissue depots, both in the dams and their pups can be seen in Table 4. Although adipose tissue weights increased throughout pregnancy, during the transition to lactation they markedly decreased except for IBAT. The loss of WAT was parallel to decreases in cell number counts in all the locations, but also by increases in brown adipose tissue cell number. OE treatment reduced cell number in all WAT locations in late pregnancy, but not during lactation. In the pups, we observed the expected increase in different adipose tissue locations together with higher cell numbers in retroperitoneal and perigonadal sites, but only increased mean cell mass in the perigonadal site. The pups from OE-treated mothers showed decreases in the weight (and a tendency towards increased cell number) of mesenteric and perigonadal pads (day 30), and decreased cell numbers in retroperitoneal location.

The aim of this study was to determine the effects of OE treatment on pregnant rat metabolism; as expected, the treatment with OE decreased food intake and dampened increases in body weight during pregnancy. In fact, there is discordance between the magnitude of total food restriction and the weight loss this generates: i.e. a 10% food limitation seems too small to cause a nearly 50 % difference in weight increase (OE vs. controls) at the peak of pregnancy. This can be viewed as a confirmation that the slimming effects of OE are potent per se, i.e. not only because it decreases dietary energy availability; in fact, they are additive to those caused by food restriction, as has been shown using pair-fed 6 and in food-deprived lactation models 16. Thus, the main effect of OE treatment was the observed decrease in different adipose tissue depots, since in late pregnancy the weight of individual adipose depots measured only 50% of those of controls. These data are validated by our balance energy analysis, in which the difference in total weight was a consequence of both a slower rate of protein accretion and a net loss of lipid. This maybe critical in the dams' catabolic context of the last third of pregnancy 17, when OE treatment selectively increased the catabolism of lipid and exerted only limited effects on protein metabolism and on the maintenance of glucose homeostasis, in a way similar to that previously described 2.

The mild effects of OE treatment on intermediate metabolism during pregnancy give support to the assumption of OE acting selectively on lipid stores. In addition, they establish a pattern different from that of the more aggressive food restriction models, in which normal development of the feto-placental unit depends on the extent of restriction 18. This may help explain the important changes that pups of food restricted mothers undergo following delivery 19. These changes, which may deeply determine their otherwise normal development 20 and body balance control in adulthood 21 are minor in our model. As a consequence, we have to assume that OE treatment causes a selective mobilization of maternal lipid stores in order to protect the near-normal development of the conceptus at the expense of mother's reserves. Since the differences in food intake of pregnant rats are maximal in late pregnancy, it is unlikely that the normal weight shown by pups at delivery will be a consequence of altered maternal food intake pattern; thus, the stable weight must be a consequence of OE treatment. Furthermore, this protective maternal pattern allows the normal growth of fetuses, resulting in smaller differences in weight at delivery, as well as in similar normal growth rates with controls up to weaning, a buffering model that differs completely from that described for the growth of pups from dams treated with OE during lactation 16.

Which mechanism induces this selective pattern? Although we cannot answer this question yet, the activation of apoptotic mechanisms by OE treatment in adipose tissue 22 may contribute to lipid mobilization. Moreover, OE treatment produces minor changes in the metabolic parameters of pregnant rats, as shown by the low levels of HDL-cholesterol and glucose, which were the only significant changes, observed in agreement with previous reports 2. In addition, the maintenance of insulin levels under OE treatment reinforces the maintenance of insulin-glucose homeostasis in spite of decreased leptin and adiponectin levels. This fall in leptin during late pregnancy is consistent with previous results 23, and probably is a direct consequence of adipose tissue depletion. The decrease in leptin levels of lactating dams, however, may result from its suppressed release in response to food intake 24. In addition, the decreased adipose depots suggest that OE may be ineffective in reducing this response.

It is possible that the differences in various adipose tissue sites reflect their metabolic specialization, since the metabolic activity of adipose tissue is not uniform nor is their cell mass and the presence of non-adipocyte cells 25. Thus, while the retroperitoneal location has long been thought to serve as a storage site for readily usable lipids during the lactation period 26, our results show that during late pregnancy it remains undersized in the same proportion as the perigonadal or mesenteric sites; thus, contributing in a general way to the total loss of fat.

The consequences of OE treatment on pregnant rats were minimal, since after delivery they recovered their food intake levels up to weaning. However, OE-treated dams also exhibited the expected decrease in total and specific site lipid stores 1623 resulting from the use of these lipids to sustain milk production.

The minimal differences in plasma parameters of pups from OE-treated rats vs. controls, and the minor changes observed in the weight gained up to day 20 after birth, are indicative of the maintenance of normal growth and unaffected metabolism. Thus, the low leptin levels observed in pups 23 must be a consequence of the initially limited adipose tissue masses that progressively grow thereafter.

Food intake restriction has been found to improve metabolic and functional parameters in rats 27. Consequently, if this restriction is regarded as an ambiental condition suffered by pregnant rats, we can assume that the normal development of their pups would be affected. Thus, maternal OE treatment could induce selective signals that limit the normal growth of adipose tissue depots in their pups, thereby improving their chances of survival, and limiting the appearance of complications associated with the onset of obesity. These effects are specifically related to OE, since they are not paralleled by estrogen signaling: OE neither binds the estrogen receptor 5 nor increases body weight 28. Furthermore, as OE treatment preserves body protein, any possible reduction in life span due to protein restriction in utero 29 is very probably minimized. These findings enhance the possible utilization of OE as experimental drug to prevent the development of obesity in later life.

The pregnant rats can buffer the effects of OE treatment, mobilizing their own energy stores that can be used by their offspring, and generating simultaneously a metabolic imprinting in their pups that limits the accretion of adipose tissue, thereby reducing their pups' risk for developing obesity in adulthood.

Dr. García-Peláez and Dr. Remesar are shareholders of a University-based spin-off: Oleoyl-Estrone Developments SL, created to develop structural analogous of the OE for their eventual application in the treatment of obesity. Oleoyl-Estrone Developments SL financed in part the study presented here in spite of not having potential interest as patentable material.

BGP took part in animal handling and biochemical determinations, RV contributed to design the experience, analyze the data and to biochemical analysis, XR contributed to experimental design, animal managing and drafted the manuscript. All authors made contributions to the final version of manuscript. All authors read and approved the final manuscript.