Cristina Cabot

Oleoyl-estrone treatment affects the ponderostat setting differently in lean and obese Zucker rats

OBJECTIVE: To determine whether the slimming effects of treatment with oleoyl-estrone (OE) in liposomes of normal and obese rats are permanent, or disappear as soon as the treatment with the drug ceased. This study was devised to gain further knowledge on the postulated role of OE as a ponderostat signal, evaluating whether (in addition) it can lower the ponderostat setting of the rat.DESIGN: The rats were infused for 14 d (using osmotic minipumps) with oleoyl-estrone in liposomes at a dose of 3.5 μmol/kg· ·d, and were studied up to one month after the treatment ceased.SUBJECTS: Young adult lean controls (CL) or treated (TL) and obese controls (CO) or treated (TO) Zucker rats.MEASUREMENTS: Energy balance, blood glucose, liver glycogen, plasma insulin, leptin corticosterone, ACTH and estrone (free and total) concentrations, and expression of the OB gene in white adipose tissue (WAT).RESULTS: The loss of body weight caused by OE was recovered quickly in the TO, which gained weight at the same rate as the CO. TL rats, however remained at the low weight attained for one month after the treatment ceased. However, no differences were observed in calculated energy expenditure (EE) between the TL and TC rats once treatment had stopped. In TL and TO rats, liver glycogen concentrations decreased to normal shortly after treatment ceased, and leptin expression and concentrations remained normal and unchanged after the end of OE treatment. In TO rats, plasma glucose, insulin and leptin were lower than in the CO. Total estrone concentrations decreased rapidly in TL rats and more slowly in the TO, and free estrone followed a similar pattern.CONCLUSION: Continuous infusion of liposomes loaded with OE resulted in a decreased energy intake (EI), maintenance of EE and the utilization of body fat reserves in lean and obese rats alike. This process ended in obese rats as soon as the infusion ceased, so that even when the levels of free and total estrone in plasma remained high, there was a marked (and relatively fast) shift toward the basal situation, which translated into an increase in EI, maintenance of estimated EE and a marked buildup of energy stores. In lean rats, the effects of OE on leptin concentrations and OB gene expression persisted after infusion ended.

Structural determinants of oleoyl-estrone slimming effects.

Female adult 9-week old Wistar rats were implanted with osmotic minipumps releasing for 14 days a liposome suspension (controls) loaded with oleoyl-estrone or other compounds of the Merlin series: estrone, estradiol, oleoyl-estradiol, oleoyl-DHEA, stearoyl-estrone, palmitoyl-estrone, oleoyl-diethylstilbestrol (DES), estrone oleoyl-ether and oleoyl-3-methoxy-estrone. All compounds were given at the same dose of 3.5 micromol/day x kg for 14 days. The effects on body weight and food intake were recorded. In the case of estrone esters, the body composition and nitrogen balance were also determined. The chronic administration of oleoyl-estrone in liposomes to rats lowers food intake, maintaining energy consumption, thus inducing the active utilization of internal stores and, consequently, the loss of body weight. This loss is mainly due to a decrease in fat, with lower proportional losses of water and a limited consumption of body protein. Free estrone had no effects on body weight, but estradiol did induce a decrease in body weight, similar to that of oleoyl-estradiol. Oleoyl-DHEA had no significant effect on body weight nor in food intake. Oleoyl-DES mimicked fairly well the effects of oleoyl-estrone, both affecting food intake and body weight. There was a relative lack of effects of estrone oleoyl-ether and of oleoyl-3-methoxy-estrone. The effects of oleoyl-estrone were in part mimicked by stearoyl- and palmitoyl-estrone, but their activity on a molar basis was lower, which suggests that the fatty acid moiety significantly influences the activity of the estrone ester as a slimming agent. The differences observed in the appetite suppression and overall slimming power of the stearoyl and palmitoyl-estrone clearly indicate that the sites of action of the physiological agonist oleoyl-estrone are at least two; the shape of the molecule, thus, may elicit a different degree of response of the systems controlled by oleoyl-estrone levels. From this interaction a series of global effects are elicited, such as appetite suppression and the loss of body (fat) weight, the latter in part (but not only) due to decreased food intake. The results shown here also suggest that the overall configuration of fatty acyl-estrone is more constrictive for its function as slimming agent than for its role as appetite suppressant, which hints to different target organs or sites of action endowed with receptors showing different degrees of fulfilling the structural constrictions of the agonist molecule.

Oleoyl-estrone does not have direct estrogenic effects on rats.

The estrogenic effects of oleoyl-estrone (OE) administration, either though continuous i.v. infusion with osmotic minipumps or administered by daily oral gavage, were studied. Binding of OE to human recombinant purified alpha receptors was negligible, and that of estrone (E1) was only a fraction of 17beta-estradiol (E2) binding. Intravenous--but not oral--OE administration resulted in marked increases of both E1 and E2 in rat plasma, but oral OE did not induce significant changes in either plasma hormone in Wistar or Zucker rats. The weight of uteri and ovaries increased with time of administration in Zucker rats treated with i.v. OE, but inguinal mammary gland proliferation between subcutaneous adipose tissue was even more marked. Oral administration of OE, however, did not increase either uterine weight or mammary gland proliferation, even at doses (10 micromol/kg x d) higher than those given i.v. (3.5 micromol/kg x d). The results indicate that i.v. administration of OE resulted in limited estrogenic effects mainly due to the high accumulation of E1 giving rise to significant increases in E2. On the other hand, oral administration of OE, even at higher daily doses, did not increase the circulating levels of either estrogen and, therefore, there were no significant effects on mammary gland proliferation or uterine weight. The oral administration of OE as a slimming drug, then, do not result in estrogenic side effects over a wide range of daily doses.

Oral gavage of oleoyl-oestrone has a stronger effect on body weight in male Zucker obese rats than in female.

This study was carried out to determine the effect of sex and oral administration of oleoyl‐oestrone on body weight of 12‐week‐old female and male Zucker obese (fa/fa) rats initially weighing 350–380 g and 405–420 g, respectively. The rats were maintained in standard conditions and given a daily oral gavage of 0.2 ml oleoyl‐oestrone dissolved in sunflower oil at a dose of 10 μmol/kg/day for 10 days, and their body weight and food intake was monitored. They were then killed, and their carcass composition (water, lipid, protein and total energy), liver lipids and glycogen and plasma chemistry, insulin, free and total oestrone were measured. Oral administration of oleoyl‐oestrone via gavage resulted in significant losses of fat, energy and–ultimately–weight. Treatment with oleoyl‐oestrone decreased food intake; the energy expenditure was kept close to that of controls at the expense of internal fat stores. Nevertheless, body protein and plasma metabolite homeostasis were preserved. The slimming effects were more marked in males than in females. Treatment increased circulating acyl‐oestrone and reduced to normal levels the high insulin observed in controls. Treatment of genetically obese rats with a daily oral gavage of oleoyl‐oestrone resulted in the loss of fat reserves with little modification of other metabolic parameters, except for lower plasma glucose and insulin levels. The results suggest that oleoyl‐oestrone, in addition to its slimming effects may be effective as an antidiabetic agent in type 2 diabetes.

Zucker obese rats are insensitive to the CRH-increasing effect of oleoyl-estrone

Adult female Zucker lean and obese rats were treated for 14 days with 3.5 nm/kg oleoyl-estrone (OE) in liposomes (Merlin-2) through continuous i.v. injection with osmotic minipumps. Rat wt. and food intake were measured daily. On days 0, 3, 6, 10, and 14, groups of rats were killed and their hypothalamic nuclei [lateral preoptic (LPO), median preoptic (MPO), paraventricular (PVN), ventromedial (VMH), and arcuate (ARC)] were dissected, homogenized, and used for the measurement of corticosterone-releasing hormone (CRH) by radioimmunoassay. The OE treatment decreased food intake by 67.4% in lean and 62.6% in obese rats (means for 14 days). Body wt. decreased steadily in lean and obese rats, the gap between controls and treated rats becoming 11.5% of initial body wt. in the lean and 12.4% in the obese. The levels of CRH in the ARC nucleus were at least 10-fold higher than in the other nuclei. No changes in CRH were observed in any of the nuclei of obese rats, with levels up to day 6 similar to those of lean rats. In the lean rats, the LPO and ARC nuclei showed peaks on day 10, while the MPO showed no changes and the PVN and VMH nuclei showed a progressive increase, to a maximum at the end of the study (day 14). This contrasted with the peak of plasma adrenocorticotropic hormone (ACTH) and corticosterone (day 6 in lean and day 14 in obese rats). There was a definite lack of correlation between the plasma levels of these two hormones and the levels of CRH in the hypothalamic nuclei, and between the latter and the decreases in appetite in the rats. The loss of appetite induced by OE is not necessarily mediated by CRH, because the obese rats show an intense decrease in voluntary food intake but their hypothalamic nuclei CRH levels do not change at all. Hypothalamic nuclei CRH does not, necessarily, mediate the rise in glucocorticoids induced by OE treatment, because this is observed in lean and obese rats, lean rats increases being mismatched with those of hypothalamic CRH. The OE induced changes in hypothalamic CRH require a fully functional leptinergic pathway, because it is not observed in Zucker fa/fa rats lacking a working leptin receptor. This--indirectly--shows that leptin is needed for its synthesis or modulation.

Plasma acyl-estrone levels are altered in obese women.

A group of obese women (BMI>27 kg/m2; N=73) was studied together with lean controls (BMI <27 kg/m2; N=25). Three groups were defined by the compliance with: BMI lower than 27 kg/m2, glycaemia lower than 5.5 mM and insulinaemia lower than 0.2 nM (controls, group 1, N=19). The subjects with BMI>27 kg/m2, glucose >5.5 mM and insulin >0.2 nM constituted group 3 (N=41), and those with BMI>27 with glycaemia and/or insulinaemia lower than the limits set constituted group 2 (N=32). The women in group 3 had higher fat content, BMI and fat-free mass than those in group 2 and the controls. There were no changes in most plasma parameters, such as free estrone and β-estradiol. Leptin levels were higher in groups 2 and 3 than in controls. In controls, leptin and acyl-estrone levels were well correlated with BMI and fat content; this correlation was not found in groups 2 and 3 for acyl-estrone, although it was found for leptin. Acyl-estrone levels were lower than expected in most obese women when compared to those of controls, suggesting an altered availability or function of this hormone. In obese women, acyl-estrone levels –and probably function– are lower than expected, contrasting with maintained leptin-BMI correlations. The role of insulin in the control of body weight, perhaps through acyl estrone-mediated effects, should be re-evaluated.

Short-term oral oleoyl-estrone treatment increases plasma cholesterol turnover in the rat.

OBJECTIVE:Oral treatment with oleoyl-estrone induces the loss of body fat and improvement of insulin resistance. Since cholesterol levels are deeply affected by oleoyl-estrone, we investigated here whether short-term treatment affected cholesterol turnover and overall metabolite changes.DESIGN:Wistar female rats received a single oral dose of 10 μmol/kg oleoyl-estrone in 0.2 ml of sunflower oil. Groups of animals were killed at timed intervals and blood samples were taken. In a second experiment series, rats had implanted carotid and jugular cannulas and were given a single gavage of oleoyl-estrone. These rats were used for the measurement of the cholesterol turnover rate.MEASUREMENTS:Body weight change and food intake: Glucose, total and HDL-cholesterol, triacylglycerols, 3-hydroxybutyrate, nonesterified fatty acids, insulin, HOMA score in the rats of the first series. Cholesterol: Cholesterol pool changes and cholesterol turnover rates in the rats of the second series.RESULTS:OE induced early effects, decreasing food intake, cholesterol and HDL-cholesterol levels, and increasing insulin sensitivity (HOMA score). OE also increased cholesteryl-ester turnover, and decreased circulating total cholesterol, especially esterified cholesterol pools.CONCLUSIONS:The role of early changes in insulin sensitivity induced by oral OE cannot explain per se the deep changes in cholesterol handling, essentially a consequence of accelerated lipoprotein turnover. However, the increase in cholesteryl-ester turnover observed with OE treatment may be, at least in part, a consequence of the decrease in insulin resistance. The compounded effect of increased insulin sensitivity and accelerated lipoprotein turnover may help explain the early and marked hypocholesterolaemic effects of OE.

Corticosteroid-binding globulin synthesis and distribution in rat white adipose tissue.

Corticosterone binding (CB) capacity was determined in visceral and subcutaneous white adipose tissue (WAT), as well as in plasma of lean Zucker rats. Perfusion of rats with saline eliminated most liver and kidney corticosterone binding but did not affect CB in WAT. The cytosol extracts of isolated cells, however, did not bind corticosterone in detectable amounts. By means of a RT-PCR procedure it was found that corticosterone-binding globulin (CBG) was expressed in WAT. By immunohistochemical detection in WAT sections, CBG was seen in a thin layer surrounding the cells near the plasma membrane. These data suggest that the CBG layer surrounding the cells may act as a protective barrier limiting the access of glucocorticoids to adipocytes.

In the rat, estrone sulphate is the main serum metabolite of oral oleoyl-estrone

Two different oral doses of oleoyl-estrone: 1 and 10 nmol/g a day were given once to male Wistar rats. the serum levels of free estrone, estrone sulphate, estradiol, and acyl-estrone were measured at intervals up to 72 h after the gavage. Oleoyl-estrone was rapidly absorbed; with the 1 nmol/g dose no changes were observed in plasma acyl-estrone but levels increased dramatically with 10 nmol/g, peaking at 6 h; high acyl-estrone levels were maintained up to 24 h, returning to normalcy at 48 h. With the 10 nmol/g dose, free estrone at most doubled its levels but estrone sulphate concentrations rose by one order of magnitude; in both cases, the increases soon (2 h) reached a plateau that was maintained for almost two days. Estradiol levels remained unchanged except for a transient peak at 2 h at the 10 nmol/g dose. The relationship between free estrone and its sulphate was linear, and those of estrone and estrone sulphate versus acyl-estrone showed the existence of an upper serum concentration limit for both molecules. The results hint at estrone sulphate being an important metabolite of oleoyl-estrone disposal, confirm the limited estrogenic response to oleoyl-estrone administration and agree with a rapid absorption and disposal of oleoyl-estrone, nevertheless maintaining high circulating levels of the ester for a time after its oral administration.

Oleoyl-estrone does not alter hypothalamic neuropeptide Y in zucker lean and obese rats

Female Zucker lean and obese rats were treated for 14 days with 3.5 micromol/kg oleoyl-estrone (OE) in liposomes (Merlin-2). After 0, 3, 6, 10, and 14 days of treatment, the rats were killed and hypothalamic nuclei (lateral preoptic, median preoptic, paraventricular, ventromedial and arcuate) were used for neuropeptide Y (NPY) radioimmunoassay. In 14 days, OE decreased food intake by 26% in lean and 38% in obese rats and energy expenditure by 6% in lean and 47% in obese rats; the body weight gap between controls and treated rats becoming -17.8% of initial b.wt. in the lean and -13.6% in the obese rats. Obese rats showed higher NPY levels in all the nuclei than the lean rats. Despite a negative energy balance and decreased food intake, there were practically no changes in NPY with OE treatment. The results indicate that oleoyl-estrone does not act through NPY in its control of either food intake or thermogenesis in lean and genetically obese rats.