Centre de Recerca en Nutrició i Ciència dels Aliments, Universitat de Barcelona, Barcelona, Spain

Correspondence to: M Alemany, Departament de Bioquimica i Biologia Molecular, Facultat de Biologia, Universitat de Barcelona, Av. Diagonal 645, 08028 Barcelona, Spain.alemany@bio.ub.es

OBJECTIVE: To test whether oleoyl-estrone affects body weight when given orally, which may help curtail the secondary growth-boosting effects of derived estrone.

DESIGN: The rats were fed for 15 days with a powdered hyperlipidic diet (16.97 MJ/kg metabolizable energy) in which 46.6% was lipid-derived and 16.1% protein-derived energy (HL group), containing 1.23±0.39 mol/kg of fatty-acyl esters of estrone. This diet was supplemented with additional oleoyl-estrone to produce diets with 2.5 mol/kg (diet OE2.5), 4.4 mol/kg (diet OE4.4), and 33.3 mol/kg content in fatty-acyl estrone (diet OE33).

SUBJECTS: Twelve-week old female Zucker lean (Fa/?) rats initially weighing 200-235 g.

MEASUREMENTS: Food intake and body weight changes; urine and droppings production and nitrogen content. Body composition (water, lipid, protein) and total energy. Energy and nitrogen balances. Plasma chemistry including free amino acids.

RESULTS: Oral administration of oleoyl-estrone in a hyperlipidic diet resulted in significant losses of fat, energy and, ultimately, weight, which were dependent on the dose of oleoyl-estrone ingested. Treatment induced the maintenance of energy expenditure combined with lower food intake, creating an energy gap that was filled with internal fat stores whilst preserving body protein. The decrease in food intake was not a consequence of food aversion but of diminished appetite. Energy expenditure was practically constant for all groups except for the OE33, which showed values about 25% lower than the controls. In most of the groups studied, there was a net protein deposition in spite of severe lipid and energy drainage. Amino acid levels agreed with this N-sparing shift. In spite of lowered energy intake, the N balance was positive or near zero in all groups, with a sizeable N-gap in controls and in lower-dose groups that disappeared in the OE33 group.

CONCLUSION: Treatment of rats with a hyperlipidic diet containing added oleoyl-estrone resulted in the dose-related loss of fat reserves with scant modification of other metabolic parameters and preservation of body protein. The results agree with the postulated role of oleoyl-estrone as a ponderostat signal and open the way for its development as anti-obesity drug.

International Journal of Obesity (2000) 24, 1405-1412

obesity; body weight; slimming; oleoyl-estrone; energy balance; nitrogen balance; energy expenditure

Introduction

Oleoyl-estrone, found in plasma and adipose tissue, induces a dose-dependent loss of body fat reserves when chronically injected.^1,2 The ability to induce the loss of weight is closely dependent on its structure, since modification of the fatty acid or the steroid nucleus deeply alters its effectivity.³ Treatment with oleoyl-estrone of lean,¹ obese,⁴ and cafeteria diet-fed rats⁵ reduces their fat content, sparing protein^1,2 and decreasing fat cell size,⁶ but maintaining unchanged plasma metabolite levels in the rat.⁷ Circulating oleoyl-estrone, present mainly in the lipoproteins, is related to body mass in humans,⁸ but not in the morbidly obese (unpublished results). The loss of body fat due to starvation also decreases the mass of circulating acyl-estrone.⁹

Treatment with oleoyl-estrone induces both a decrease in food intake and the maintenance of energy expenditure.² The loss of appetite is not directly mediated by NPY¹⁰ nor CRH.¹¹ Oleoyl-estrone decreases insulin and leptin levels, eliciting a marked glucocorticoid response,⁷ also decreasing the circulating levels of corticosterone-binding globulin.¹² Adrenalectomy combined with oleoyl-estrone treatment results in accelerated loss of body mass and the failure to maintain energy homeostasis,¹³ stressing the counteractive role of glucocorticoids with respect to oleoyl-estrone slimming effects.

Chronic treatment with oleoyl-estrone decreases the expression of the Ob gene⁷ in lean but not in fa/fa rats,¹⁴ and lowers the ponderostat reference setting in lean but again not in fa/fa rats.¹⁵ Since fa/fa rats lose fat under oleoyl-estrone treatment it may be assumed that oleoyl-estrone does not act through leptin, but that both hormones share a close functional interrelationship. Leptin enhances the synthesis of oleoyl-estrone in cultured adipocytes,¹⁶ but its synthesis and circulating levels are decreased by oleoyl-estrone.⁷

The main problem that oleoyl-estrone treatment poses is how it is administered. Constant i.v. infusion in liposomes induces dramatic effects on body weight and preserves most other homeostatic constants. However, oleoyl-estrone is rapidly taken up by tissues and hydrolysed to estrone,^17,18 which induces body weight increases¹ when given alone. It may be assumed, then, that an increase in circulating estrone levels¹⁹ counteracts the down-setting of the ponderostat elicited by oleoyl-estrone. The presence of oleoyl-estrone in lipoproteins leaves open the possibility of naturally loading the chylomicra derived from the digestion of dietary fats, by giving the compound with a hyperlipidic diet to mimic, to some extent, the high-fat diets consumed by humans. This approach, however, is paradoxical because hyperlipidic diets induce fat deposition.²⁰ In the present study we test whether oleoyl-estrone affects body weight when given orally, which that may help curtail the secondary growth-boosting effects of derived estrone.

Materials and methods

Four groups of 12-week-old female Zucker lean (Fa/?) rats initially weighing 200-235 g were used. The rats were kept under standard conditions in metabolic cages (Techniplast Gazzada, Guguggiatte, Italy) and were fed with a powdered hyperlipidic diet (control diet, HL; B&K, Sant Vicent dels Horts, Spain), which contained 22.18% fat, 17.23% protein, 4.93% fiber, 1.86% minerals, 36.31% starch and 3.43% sugars, with a gross energy content (bomb calorimeter, C-7000, IKA, Heitersheim, Germany)of 19.79 MJ/kg, and metabolizable energy of 16.97 MJ/kg; 46.6% of the energy was derived from lipids, 37.2% from carbohydrate and 16.1% from protein. Samples of HL diet were used for the evaluation of naturally occurring acyl-estrone esters by means of extraction with anhydrous methanol in a Soxhlet followed by saponification and RIA analysis of the estrone released.²¹ The diet contained a mean 1.23±0.39 (n=5) µmol/kg of fatty-acyl esters of estrone. A part of this diet was supplemented in origin with oleoyl-estrone (Salvat, Esplugues de Llobregat, Spain); analysis of the supplemented diet showed a 33.3±3.0 µmol/kg (n=5) content in acyl-estrone (diet OE33). By mixing both plain and supplemented diets in various proportions (1:10 and 1:25), two additional diets containing a mean 4.4 µmol/kg (diet OE4.4), and 2.5 µmol/kg (diet OE2.5), respectively, were obtained. Each diet was fed for 15 days to a group of five rats. The weights and food consumption of the rats were measured daily; urine and droppings were also recovered, weighed and stored daily. The dose of oleoyl-estrone ingested by the rats was calculated from the food intake and acyl-estrone content of the diet.

At the end of the experiment, the rats were anesthetized with ethyl ether, and blood was taken through heart puncture and used to obtain plasma. The rats were then killed and dissected, cleaned of intestinal contents, weighed again and sealed in polyethylene bags that were subsequently autoclaved at 120°C for 2 h; the whole rat was then minced to a smooth paste with a blender.

Plasma was used for the estimation of glucose, total cholesterol, total protein, triacylglycerols and urea using a dry-chemistry strip auto analyser (Spotchem, Menarini, Firenze, Italy), as well as for the estimation of esterified estrone²¹ and insulin (rat insulin kit, Amersham, UK). Plasma amino acids were determined with an ALPHA-PLUS (Pharmacia, Uppsala, Sweden) amino acid analyzer and a ninhydrin method.²²

The rat carcass paste was used for the estimation of the proportions of water (differential weighing after 24 h at 100°C), lipid,²³ energy (bomb calorimeter) and nitrogen, the latter measured as total N with a Carlo Erba NA-1500 elemental analyzer, and then converted into protein using a factor of 5.5.²⁴ The total estrone ester content of the carcass paste was also measured using the same procedure as used for the diet.

The nitrogen content of the diet, droppings and urine were also measured and used for the estimation of nitrogen balance.²⁵

Two groups of two rats each were exposed to either the HL diet of to the OE33 diet after an overnight fast, determining for 2 h the overall food intake per group at 10 min intervals. The experiment was repeated after 48 h of return to normal pellet diet. The rates of food ingestion by the two groups of animals were used to determine whether the changes in food intake were due to food aversion or to a change in appetite.

Statistically significant differences (P<0.05) between groups were determined using one-way ANOVA programs followed by the Tukey post-hoc test.

Results

Figure 1 shows the effect of oleoyl-estrone presence in the HL diet on the immediacy of its ingestion by rats subjected to 12 h of starvation. Both HL and OE233 diets were consumed at the same initial rate, thus establishing that the presence of oleoyl-estrone in the diet did not provoke taste aversion. However, after about 40 min, consumption of oleoyl-estrone-laced HL diet tapered off whilst that of plain HL diet stabilized to just nibbling after about 70 min.

Figure 2 shows the body weight changes induced by treatment with oleoyl-estrone in a hyperlipidic diet. Rats in the OE2.5 group practically did not change their weight during the 2 week study, in contrast to the increase shown by the HL controls. The OE4.4 group showed a limited weight loss, but the rats in the OE33 group lost about 30% of their weight in 12 days, although they recovered about 5% at the end of the study.

Table 1 shows the mean values for food and water intake, as well as excreta production. Food intake decreased with increasing doses of oleoyl-estrone. These differences resulted in the ingestion of about twice the amount of oleoyl-estrone than HL controls for group OE2.5, three times for the OE4.4 rats and only 10-fold higher for the OE33, in spite of this diet having 27 times more oleoyl-estrone than the HL.

The plasma parameters (Table 2) showed little change; only the highest dose of oleoyl-estrone (group OE33) induced significant changes¾lowered glucose and increased triacylglycerols and urea. The OE2.5 and OE4.4 groups showed lower triacylglycerol levels than controls. Circulating acyl-estrone levels were increased to about twice those of controls in the OE4.4 rats and about 10-fold in the OE33 group. Insulin levels tended to be lower witholeoyl-estrone treatment, but there were no significant differences between the groups.

In Table 3, the body composition analyses ofthe experimental animals are presented; for direct calculations, the percentages of body components were corrected for a standard initial weight of 220 g. There was a marked decrease in the percentage of lipids and energy content of the rats in groups OE2.5, OE4.4 and especially OE33, with little change in the proportion of protein, and increasing water concentration with higher doses of oleoyl-estrone. In 15 days of treatment body weight decreased in proportion to the dose of oleoyl-estrone. The losses of protein were minimal, and only recorded in the OE33 group; all other groups accrued protein (Figure 3). The main differences in the treated groups were found in lipid and energy, with differences of up to 87% in lipid content and 56% in total energy with respect to the HL group.

The estimated values for energy balance are shown in Figure 4. Energy intake decreased in direct proportion to the dose, as did energy accrual, but energy expenditure remained fairly uniform in the HL, OE2.5 and OE4.4 groups, to decrease in the OE33 rats, but to a lower extent than energy intake, thus explaining the drainage of energy to fill up the wide energy gap.

The nitrogen balances of the rats in the five experimental groups are presented in Table 4. In spite of lower intake (OE33 group ingested only 26% of the energy eaten by the HL rats) at the higher doses, the proportion of dietary N absorbed was fairly constant. The range of urinary N losses was smaller than that of N intake (OE33 excreted 65% of the HL). Accrual was minimal: 7% of intake in HL vs - 24% in OE33. These differences generated an 'N gap'^25,26 that ranged from 43% of N ingested in HL to 0 in the OE33 group.

Table 5 shows the plasma amino acid levels in the rats of the HL group, and Figure 5 the changes observed in the aminograms with respect to those of HL controls for the four doses of OE tested. The aminograms corresponding to the OE2.5 and OE4.4 groups were fairly similar to those of controls, with little quantitative or qualitative change. However, at the largest dose, OE33, the changes were more marked, with a mean amino nitrogen concentration similar to that of controls, but showing lower branched chain and glutamate+glutamine levels and high aspartate+asparagine concentrations, a relative predominance of non-essential glucogenic amino acids and decreased arginine vs increased citrulline and ornithine.

The analysis of estrone balance is depicted in Table 6. The total intake of dietary estrone (essentially estrone fatty esters) was about two-fold that of HL controls for groups OE2.5 and OE4.4, and seven-fold for the OE33 group. However, the total amount of estrone esters remaining in the rat carcasses was fairly similar for all groups, about 1 nmol/g. In spite of increased intake, the rats receiving higher doses of oleoyl-estrone accumulated less estrone, progressively increasing the excretion of this compound. When the estrone content is related to the mass of lipid, however, the expected concentration of estrone esters in the dwindling fat stores of treated rats rose.

Discussion

The lower intake of food observed in the rats fed the HL diet laced with oleoyl-estrone is not a consequence of taste aversion, since the rates of consumption were initially identical for HL and OE33 diet; however, the effects of the latter on appetite were observed sooner after ingestion than in HL diet fed rats. This early effect on appetite can help explain the lower food intake of the rats orally treated with oleoyl-estrone.

Oral administration of oleoyl-estrone in a hyperlipidic diet to Zucker lean rats resulted in significant losses of fat, energy and, ultimately, weight. This behavior mimics the effects of the continuous i.v. injection of oleoyl-estrone observed in lean and obese rats.^1,2,4,15 However, as expected, the oral administration of oleoyl-estrone, at least at the highest doses used, resulted in higher effectiveness of the hormone, since the effects on weight and energy loss were observed with doses that were only a fraction of those used in the i.v. procedures.^2,7 Here, the rats receiving the highest dose (OE33), ie 0.626 µmol/kg day of oleoyl-estrone, lost 24% of their weight, along with practically all their lipid reserves, in 2 weeks, whilst a standard i.v. dose of 3.5 µmol/kg·day induced more limited losses.^7,15 This discrepancy may be a consequence of the 'natural' incorporation of oleoyl-estrone into an 'active' lipoprotein pool in the blood via intestinal chylomicra, which did not occur following artificial administration in chylomicra-sized lipid emulsions. Another important factor explaining the higher success of the oral administration lies in the limitation of the counteracting effect of estrone, since estrone induces growth and deposition of fat, thus hampering the slimming effects of oleoyl-estrone.¹ In the i.v. procedure, injected oleoyl-estrone was rapidly converted into estrone,^17,18 the levels of which rose considerably; in the present study, however, most of the dietary estrone was excreted, thus minimizing its anabolic effects. Oleoyl-estrone compartmentation may also play a role in the enhanced effect of the oral administration, since the circulating levels of oleoyl-estrone increased because of treatment less than one order of magnitude, compared with the three orders of magnitude described for the i.v. procedure.^7,15

The higher effectivity of oleoyl-estrone given orally in a hyperlipidic diet, however, poses other problems, which we tentatively attribute to the 'estrone effect' or anabolic counteracting of oleoyl-estrone energy wasting effects. The administration of fairly diluted doses of oleoyl-estrone in hyperlipidic diets results in enhanced increases in body weight,¹⁹ an effect that has been attributed to the anabolic effects of free estrone.¹

The effects of oleoyl-estrone on the body energy budget are summarized in Figure 3, as described elsewhere:^1,2 maintained energy expenditure combined with lower food intake creates an energy gap that is filled with internal fat stores and preserves body protein. When we correct the energy expenditure data for body mass (using the mean of initial and final weights) and apply an allometric correction such as BW^0.75 for comparative purposes,²⁷ we obtained the following values: HL, 40.8 mW/g^0.75; OE2.5, 41.1 mW/g^0.75; OE4.4, 42.4 mW/g^0.75; OE33, 30.7 mW/g^0.75. These results show that energy expenditure was practically constant for all groups except for the OE33, about 25% lower than the controls, which contrasts with the practical disappearance of lipid reserves (the 4 g of lipid remaining in OE33 rats includes all the structural, eg membrane, lipids in the body) and the loss of more than half of the body energy compared with the controls. The decrease in energy expenditure is thus less marked than could be expected for the observed decrease in energy intake. Starvation induces more marked losses of energy expenditure in the short term,²⁸ and severe undernutrition (comparable to the reduction of more than three-quarters of the energy ingested observed in the OE33 group) also markedly reduces energy expenditure. This is not the case with oleoyl-estrone treatment, since in spite of important depletion of the fat reserves, protein synthesis, amino acid availability and glucose levels were all maintained within physiological limits. The slight rise of triacylglycerols in the OE33 group contrasts with the normal-to-low values for all the other groups, which are indicative of active lipid mobilization by most tissues without compromising glucose homeostasis. 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' gives support to the postulate of oleoyl-estrone as a ponderostat signal,¹⁵ since 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's weight control center.

The differences between energy budget administration during oleoyl-estrone treatment and that of food deprivation or starvation are not limited to energy expenditure, but also affect the handling of body protein. In most of the groups studied, there was a net protein deposition in spite of severe lipid and energy drainage. Amino acid levels agreed with this N-sparing shift, probably a consequence of maintained glucose levels, since amino acids are mainly used as substitute fuels for gluconeogenesis²⁹ in conditions of limited energy (glucose) availability.³⁰ In spite of lowered energy intake, the N losses as urinary urea are (in all groups except OE33) about half the ingested N, and the N gap accounts for a fairly large share of the remaining N, with values that are close to those observed in cafeteria diet-fed rats,²⁵ in which the availability of energy and amino acids was also very high.³¹ In the OE33 group, however, the N gap practically disappeared, in conditions that practically equalled the urinary losses of N with its intake, and there was a profound alteration of the urea cycle amino acid pattern. Nevertheless, despite this compromised situation, the OE33 rats lost only a tiny fraction of their nitrogen, a consequence of well maintained levels of glucose and the availability of other substrates (lipid) as alternative fuels.

In conclusion, treatment of rats with a hyperlipidic diet containing added 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 open the way for its development as an anti-obesity drug.

Acknowledgements

This study was financed by Laboratoris SALVAT, SA. and grants ALI96-1094, BIO98-0316 and 2FD97-0233 of the Government of Spain. Thanks are given to Robin Rycroft from the Language Advisory Service at the University of Barcelona for correction of the text.

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Figure 1  Rate of food ingestion of rats exposed to HL and OE33 diets after a 12 h overnight fast. Each symbol is the mean of two experiments and correspond to the consumption of the diet by two animals in each cage.

Figure 2 Changes in body weight (as a percentage of initial weight) in rats treated with a hyperlipidic diet containing different amounts of added oleoyl-estrone.

Figure 3 Differences in body composition with respect to the HL controls induced by 15 days of treatment with different doses of oleoyl-estrone (logarithmic scale) in a hyperlipidic diet. The values are expressed as percentage of the change in grams (or megajoules in the case of energy) with respect to the HL controls. For comparative purposes, all components are referred to a standard initial weight of 220 g.

Figure 4 Energy balance of rats subjected to 15 days of treatment with different doses of oleoyl-estrone in a hyperlipidic diet. The values presented are means for the whole period studied and are expressed in power units (Watts) vs the dose of oleoyl-estrone ingested (logarithmic scale).

Figure 5 Differences in amino acid concentrations of the plasma of rats treated for 15 days with oleoyl-estrone in hyperipidic diets compared with controls receiving the diet with no oleoyl-estrone added. The height of columns corresponds to the differences in the amino acid values vs those of controls, expressed as percentage of controls. The s.e.m. of individual amino acids are given as crossed lines on top of the columns, the s.e.m. of control data (ie 100%) are presented as vertical lines crossing (±) the baseline. The compound total amino acid value for each group is represented by a thin horizontal line. Statistical significance of the differences vs controls (ANOVA): significant difference (P<0.05) for the indicated amino acid vs controls, the small squares present in the bottom rectangle indicate which amino acids show a significant (P<0.05) effect of oleoyl-estrone dose on the amino acid levels, considering all data.

Table 1 Food and water intake, mass of excreta and oleoyl-estrone intake of rats treated with a hyperlipidic diet supplemented with oleoyl-estrone

Table 2 Plasma parameters of rats treated with an hyperlipidic diet supplemented with oleoyl-estrone

Table 3 Body composition of rats treated with a hyperlipidic diet supplemented with oleoyl-estrone

Table 4 Nitrogen balance or rats treated with a hyperlipidic diet supplemented with oleoyl-estrone

Table 5 Plasma amino acids in rats treated with a hyperlipidic diet

Table 6 Estrone balance in rats treated with a hyperlipidic diet supplemented with oleoyl-estrone