Centre de Recerca en Nutrició i Ciència dels Aliments, Departament de Bioquímica i Biologia Molecular, Facultat de Biologia, Universitat de Barcelona, Av. Diagonal 645, E-08028 Barcelona, Spain. E-mail: email@example.com
Objective: To establish whether single daily oral doses of oleoyl-estrone result in dose-dependent slimming effects on normal weight rats, and to determine the changes in energy parameters induced by this treatment.
Research Methods and Procedures: The effects of a daily oral gavage of oleoyl-estrone (0, 0.2, 0.5, 1, 2, 5, 10, and 20 μmol/kg per day) in 0.2 ml of sunflower oil given over a 10-day period were studied in groups, each of which contained six adult female Wistar rats initially weighing 190 to 230 g. A group of intact control rats receiving no gavage was included for comparison. Body weight and food intake were measured daily. Rats were killed on day 10 of treatment, and body composition (protein nitrogen, lipids, and water), liver lipids, and plasma parameters (glucose, triacylglycerols, total cholesterol, free fatty acids, 3-hydroxybutyrate, urea, aspartate, alanine transaminases, insulin, leptin, and free and acyl-estrone) were measured.
Results: The administration of oleoyl-estrone resulted in a dose-dependent loss of body fat, because of a partly maintained energy expenditure combined with decreased food intake. The differences in the energy budget were met by internal fat pools. The changes recorded did not affect the levels of the main plasma energy homeostasis indicators: unaltered glucose, triacylglycerols, free fatty acids, 3hydroxybutyrate, and urea. Protein was accrued even under conditions of severe lipid store drainage. There were no changes in transaminases. No lipid accumulation was recorded in the liver. Plasma insulin and leptin levels decreased with increased oleoyl-estrone doses, whereas the levels of free and esterified estrone increased with treatment, although not in proportion to the dose received.
Discussion: Oral treatment with oleoyl-estrone resulted in the specific dose-related loss of fat reserves with little change to other metabolic parameters. These results agree with the postulated role of oleoyl-estrone as a ponderostat signal.
Oleoyl-estrone is a natural hormone derivative found in plasma and tissues (1); its intravenous (IV) administration induces a dose-dependent loss of body fat reserves in lean (1), obese (2), and cafeteria diet-fed rats (3). Loss of body fat is mediated by both a decrease in food intake and the maintenance of energy expenditure (4). Oleoyl-estrone reduces adipocyte size (5) but maintains plasma metabolite levels in the rat (6), sparing protein; in fact, rats receiving oleoyl-estrone treatment maintain their protein growth rates despite being subjected to a massive mobilization of their lipid stores (1) (4). Oleoyl-estrone shows effects on body weight that are markedly different from those of free estrone, suggesting that its function is not directly dependent on supplying the estrone nucleus, but the effect is closely dependent on the chemical structure of the ester (7).
Oleoyl-estrone decreases leptin levels in Zucker Fa/? rats (6) but not in fa/fa rats (8). It also lowers insulin levels, eliciting a glucocorticoid response (6). Leptin enhances the synthesis of oleoyl-estrone in cultured adipocytes (9). Chronic treatment with oleoyl-estrone lowers the reference weight setting in lean rats but not in fa/fa rats (8). This suggests that leptin and oleoyl-estrone share a significant portion of the basic mechanism of body weight control, fitting a ponderostat model in which oleoyl-estrone would be a key signal emanating from adipose tissue (8).
There is a close relationship between body fat mass and circulating acyl-estrone levels in humans (10), a relationship that is lost in the morbidly obese (11). Obese Zucker rats also show circulating levels of oleoyl-estrone that are lower than those expected given their fat mass (8). These data suggest that the synthesis of (or signaling by) oleoyl-estrone is impaired with massive obesity, which supports the postulated role of oleoyl-estrone as a ponderostat signal.
Free estrone is rapidly released in most tissues by the hydrolysis of circulating oleoyl-estrone (12). Estrone induces growth and the accumulation of fat in rats, thus counteracting the effects of oleoyl-estrone (1) (13). Thus, the IV infusion of fat droplets loaded with oleoyl-estrone may result in the unwanted and counteractive effects of the estrone generated during disposal of the injected hormone. The administration of oleoyl-estrone-laced hyperlipidic diets greatly improves the slimming effect of the drug, probably because of increased transfer by the intestine into nascent lipoproteins or carrier proteins (14); the relatively low circulating levels of estrone in the plasma of these animals compared with those of IV-treated rats (6) suggests that oleoyl-estrone compartmentation in the blood is a key element in the effectiveness of the hormone for slimming. In the present study, we determined whether a single daily gavage (a much easier mode of administration than continuous IV infusion) of oleoyl-estrone is effective in eliciting weight loss in rats, forfeiting the eventual problems posed by an overload of estrone.
Research Methods and Procedures
Nine groups of 12-week-old female Wistar rats weighing 190 to 230 g were used. The rats were kept under standard conditions in collective cages and were fed with a standard pellet diet (B&K, Sant Vicent dels Horts, Spain). All animals were weighed daily, and the food consumed by each group was determined by differential weighing. A control group of intact animals (C-I) was maintained with no further manipulation. All remaining groups were given, by means of a stomach tube, a daily gavage of 0.2 ml of sunflower oil containing oleoyl-estrone (Salvat, Esplugues de Llobregat, Spain). Thus, the following dose-groups were established: C-0, controls, received only the oil solvent; T-0.2, treated, received 0.2 μmol/kg per day; T-0.5, treated, received 0.5 μmol/kg per day; T-1, treated, received 1 μmol/kg per day; T-2, treated, received 2 μmol/kg per day; T-5, treated, received 5 μmol/kg per day; T-10, treated, received 10 μmol/kg per day; and T-20, treated, received 20 μmol/kg per day.
At the end of the experiment, the rats were weighed and then killed by decapitation with a guillotine; the blood was collected into heparinized beakers and used for the separation of plasma. The rats were dissected and cleaned of intestinal contents. A liver sample was taken and later used for the estimation of total lipids (15). The rat remains were weighed again and sealed in polyethylene bags that were subsequently autoclaved at 120 °C for 2 hours; next, the whole rat was minced to a smooth paste with a blender.
Plasma was used for the estimation of glucose, total cholesterol, total protein, urea, aspartate transaminase, and alanine transaminase by using a dry-chemistry strip auto analyzer (Spotchem; Menarini, Milan, Italy), and for the estimation of triacylglycerols (kit 11528; BioSystems, Barcelona, Spain), 3-hydroxybutyrate (Roche, Mannheim, Germany), and nonesterified fatty acids (nonesterified-fatty acid C kit; Wako Chemicals, Neuss, Germany). Plasma samples were also used for the measurement of free and esterified estrone (16), insulin (Biotrak RPA547 kit; Amersham, Little Chalfont, UK), and leptin (RL83K kit; Linco Research, St. Charles, MO).
The rat carcass paste was used for the estimation of the proportions of water (differential weighing after 24 hours at 100 °C), lipid (15), energy (bomb calorimeter), and nitrogen, the latter measured as total nitrogen with a NA-1500 elemental analyzer (Carlo Erba, Milan, Italy) and converted into protein using a 5.5 factor (17).
The body composition data obtained in the intact controls (C-I) were used to calculate the initial body composition of all groups at the beginning of the experiment. These values were compared with the actual analyses of the carcasses of all groups after killing. The differences were calculated either as absolute changes in 10 days (in grams) or as a percentage of the initial pool values for protein, lipid, or water, and for total energy content (bomb calorimeter). The changes in crude energy content and the known energy intake were used to determine the proportion of energy accrued and to obtain an estimate of the mean of energy expenditure for the 10-day period (i.e., the difference between energy intake and energy accrued). These data were also expressed, corrected for an allometric factor (18) of body weight (0.75), for direct comparison of energy expenditure data allowing for body weight changes.
Statistically significant differences (p < 0.05) between groups were determined using Student's t test and standard one-way ANOVA programs. Regression plots were calculated with the statistical package of the Prism 2 program (Graphpad Software, San Diego, CA).
Figure 1 shows the body weight changes of rats treated with increasing doses of oleoyl-estrone in a sunflower oil gavage for 10 days. When the absolute change in body weight is expressed versus the daily dose of oleoyl-estrone received on a logarithmic scale, a direct, significant relationship can be appreciated between both parameters. Control and treated rats receiving lower doses of oleoyl-estrone increased their body weights over 10 days, whereas those rats receiving the highest doses lost weight. When the differences in weight accrued between controls (C-0) and treated rats were calculated, a significant dose-related loss of weight was also observed.
Table 1 presents the body composition of control rats and treated rats. The percentage of fat content, absolute fat content, and body energy of the rats decreased in proportion to the dose of oleoyl-estrone given, but the changes in protein and water proportion and content were small or not present. The data in Table 1 show the estimation of change in water, lipid, and protein content (and the percentage of change of the water, lipid, and protein pools) induced by 10 days of oleoyl-estrone treatment.
Table 1. Body composition of Wistar rats receiving a daily gavage of oleoyl-estrone in 0.2 ml of sunflower oil
The percentage values at day 1 that were applied to all groups were those obtained from the intact control group (C-I).
Weight from which the stomach and intestine content have been subtracted.
Values expressed as a percentage of initial values.
Value estimated as 95% of the energy content of the food ingested (3.35 kcal/g); this value also includes 1.67 kcal/d (0.185 g) corresponding to the oil of the gavage.
Calculated (using mean values) from the formula: (BWICI − BWIEXP)/FIEXP; where BWICI is the body weight increase (in grams) of intact control rats, BWIEXP is that of the given experimental group; and FIEXP is the food intake (in grams) of the corresponding experimental group.
Calculated as the difference between energy intake and energy accrued (energy content increase).
Body composition on day 1
(the percentages applied for calculation of absolute values were those obtained from intact rats)
Energy expenditure decreased by <13% in T-20 rats. The changes in energy expenditure were even lower when corrected by body size (11%). These data can be compared with a drop in energy intake of 35%, whereas the data for all the other groups correlated with the dose. The ratio of the difference in weight attained by each group to that achieved by controls in the 10-day period of treatment, divided by the weight of food ingested in the same time, is a rough index of the relationship between tissue mass accrued versus energy ingested. This ratio increases in proportion to the dose of oleoyl-estrone given, which indicates that the decrease in weight was more marked than that of food intake.
Figure 2 depicts the relationship observed between mean daily food intake and accrued energy versus the dose of oleoyl-estrone. The energy accrued in 10 days was also inversely related to the dose of oleoyl-estrone; a dose in the range of 1 μmol/kg per day resulted in no accrual of energy in 10 days, whereas lower doses allowed the accumulation of energy. Higher doses, however, resulted in the net loss of body energy.
Figure 3 shows the correlation of water, lipid, protein, and energy content with the dose of oleoyl-estrone. Water and protein changes (both in absolute values and as percentages) were affected little by the dose of oleoyl-estrone. In both cases there was a net accrual, albeit minimal, of both components even at the highest oleoyl-estrone doses tested. However, the changes in lipid and gross energy content decreased steeply with the dose of oleoyl-estrone, with losses outstripping accruals around the value of 1 μmol/kg per day. The greatest loss of lipid (9 g, 32% of initial lipid pool) was recorded in T-20 rats, which lost no protein at all but consumed ∼17% of their body energy in just 10 days, essentially in the form of lipids.
Table 2 presents some metabolic indicators of the plasma and liver of controls and oleoyl-estrone-treated rats. Overall, there were no significant changes (ANOVA) in glucose, triacylglycerols, 3-hydroxybutyrate, nonesterified fatty acids, and urea. Cholesterol levels, however, decreased with increasing oleoyl-estrone doses. No changes were observed either in alanine transaminase and aspartate transaminase or in liver lipids. Free estrone tended to increase with oleoyl-estrone treatment, but the differences versus controls and the linear changes with the doses given were not significant in any group. Acyl-estrone levels showed a similar pattern, increasing significantly in all treated groups, but the individual variations were large and no direct correlation was found between this parameter and the dose administered. In Figure 4, the changes in insulin and leptin in the plasma of controls and treated rats are shown. Insulin levels tended to increase slightly at the lowest doses tested, but at doses of ≥1 μmol/kg per day, the levels of insulin were lower than in controls and remained unchanged. A similar pattern was observed for circulating leptin, but the leptin levels decreased proportionally to the dose given to the lowest values in the T-20 group (approximately one-third that of the controls).
Table 2. Plasma parameters and liver lipids of Wistar rats receiving a daily gavage of oleoyl-estrone in 0.2 ml of sunflower oil
N = 6 in all groups. Statistical significance of the differences versus intact control rat (C-I) values (Duncan post hoc test).
p < 0.05. The p value column shows the results of one-way ANOVA of all data in a given row.
The oral administration of oleoyl-estrone (compared with IV injection) results in more marked effects on body weight with lower increases in circulating estrone and acyl-estrone (19); this is probably caused by the filtering effect of the liver, which disposes of any excess of estrone arriving through the porta vein, and by the selective loading of acyl-estrone on lipoproteins (our unpublished results). In comparison, IV administration using lipid droplets results in the loading of both oleoyl-estrone and free estrone, because most tissues rapidly hydrolyze oleoyl-estrone (12). The massive presence of free estrone (6) partly counteracts the slimming action of oleoyl-estrone (1). In addition, oral administration does not require surgical procedures. Oleoyl-estrone does not provoke taste-aversion effects in rats but rapidly induces satiety (20). In any case, the use of a stomach cannula with a gavage of a minimal amount of edible oil prevents any taste differentiation of the drug by the animals.
The data presented show that a daily oral gavage of oleoyl-estrone results in a dose-dependent loss of body fat using a dose span of 0.2 to 20 μmol/kg per day. This loss of fat is partially the consequence of maintained energy expenditure combined with decreased food intake. The differences in energy budget were met by internal fat pools. These changes do not affect the levels of the main plasma energy homeostasis indicators: unchanged glucose, triacylglycerols, free fatty acids, 3-hydroxybutyrate, and urea. Protein was accrued even under conditions of severe lipid stores drainage. The oxidation of lipids probably resulted in a higher turnover of lipoproteins, because circulating cholesterol decreased significantly, but both liver and plasma lipids (triacylglycerols and fatty acids) were unchanged. There were also no changes in circulating transaminases, suggesting the absence of a negative effect on the liver, which was also found not to accumulate lipids.
There is a direct relationship between circulating leptin and the size of fat stores (21). However, oleoyl-estrone lowers the expression of the Ob gene, diminishing the circulating levels of leptin (6) (8). Thus, both effects may compound the dose-dependent decrease in leptin levels observed here.
Insulin levels also decreased with oleoyl-estrone treatment, in agreement with previous studies (6) (8), but in this study, the changes were unrelated to the dose. Despite lowered insulin levels, circulating glucose homeostasis was maintained. It is likely that the small amount of food ingested was sufficient to maintain glycemia and prevent ketosis despite the obvious and massive consumption of internal lipids.
Oleoyl-estrone reduces food intake (4), probably by means of the early enhancement of satiety (20). However, it maintains energy expenditure (1) through maintained thermogenesis (4), an effect mediated at least in part by adrenergic signaling (22). It also diminishes insulin resistance (17) by increasing the oxidation of lipids (1) (5) (19) (20). These effects concur in the maintenance of an energy gap (lower energy intake and maintained energy expenditure) (1), which is filled up by the speedy disposal of fat from the adipose tissue (5), under conditions of lowered insulin resistance, increased plasma lipid turnover, and normal glucose and liver glycogen levels (19) (20).
Oleoyl-estrone simply shifts the short-term system of body weight control to enhanced energy consumption within a framework of maintained energy homeostasis. This state of “normality” lends support to the postulate that oleoyl-estrone acts as a ponderostat signal (8), because its administration, even in small amounts (and the consequent rise in circulating levels), induces the shedding of the “excess fat stores” that the raised oleoyl-estrone levels signaled to the body-weight control center.
The loss of fat observed here mimics the effects of the continuous IV injection of oleoyl-estrone observed in lean and obese rats (1) (2) (4) (8). The results are also in agreement with those obtained by lacing a hyperlipidic diet with oleoyl-estrone (20). The data presented here suggest that the slimming effects of oleoyl-estrone were more clear-cut than those of IV injection. The improved slimming effects of oleoyl-estrone may be a consequence of the lower amount of free estrone found under the conditions tested, which minimized its eventual anabolic and other undesirable effects (1) (13).
The clear-cut relationship between dose and effects, however, is not so apparent when we examine the circulating levels of free and fatty-acyl esterified estrone. Oleoylestrone dosage elicits a rise in free and acyl-estrone in plasma, but the increase is not proportional to the dose given. In fact, the lowest concentrations tested resulted in circulating levels of free and acyl-estrone that were similar to the levels observed with doses administered in the middle range. These results point toward the presence of more than one compartment in circulating acyl-estrone; the total acyl-estrone value would be a composite of the active compartment and the other compartment(s), which we assumed were inactive. However, it is safe to assume that the “active” compartment of oleoyl-estrone is directly related to the dose given, because the effects that are elicited seem to indicate this, even though the presence of other carrier or “inactive” compartments may partly obscure the results. Whatever the case, we were not able to demonstrate a direct relationship between circulating levels of acyl-estrone and either the dose given in the gavage or the effects elicited by this dose.
In conclusion, oral treatment of rats with oleoyl-estrone resulted in the dose-related loss of fat reserves with scant modification of other metabolic parameters. The results presented agree with the postulated role of oleoyl-estrone as a ponderostat signal and pave the way for its eventual application as an anti-obesity drug because of its simple and effective means of administration.
This work was financed by Laboratoris Salvat, SA, and by Grants ALI96-1094, BIO98-0316, and 2FD97-0233 from the Government of Spain. We thank Robin Rycroft from the Language Advisory Service at the University of Barcelona for correction of the text.