Maria del Mar Grasa

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.

Effect of oral oleoyl-estrone on adipose tissue composition in male rats.

OBJECTIVE: To determine whether the oral administration of oleoyl-estrone has similar mass-decreasing effects on the main different sites of white adipose tissue (WAT).DESIGN: Adult male Zucker lean rats were given a daily oral gavage of oleoyl-estrone (OE, 10 µmol/kg) in 0.2 ml of sunflower oil for 10 days, and were compared with controls receiving only the oil. The mass of the main WAT sites: subcutaneous, epididymal, mesenteric, retroperitoneal, gluteal, perirenal and interscapular, as well as perirenal and interscapular brown adipose tissue (BAT), were dissected and studied.MEASUREMENTS: The tissue weight, DNA, protein, lipid and total cholesterol content, together with the levels of leptin and acyl-estrone in the larger WAT and BAT masses, were measured.RESULTS: The weights of WAT depots were correlated with body weight but those of BAT were not. Cell size was maximal for epididymal and mesenteric and minimal for subcutaneous and retroperitoneal WAT and BAT. Differences were detected in DNA, and in protein and lipid content between distinct WAT sites. OE treatment tended to decrease cell number and cell size in WAT; only small differences in composition were found between WAT locations inside the visceral cavity and those outside. Decreases in lipid content were maximal in mesenteric fat. Leptin and acyl-estrone content were fairly uniform at the different WAT sites, except for high concentrations in gluteal WAT. OE induced a greater decrease in leptin and acyl-estrone than in DNA and lipids; changes in these hormones were fairly parallel in all sites.CONCLUSIONS: In general, the differences in composition between visceral and peripheral subcutaneous WAT and their responses to OE were less marked than the individual differences observed between specific sites, regardless of location. WAT sites are fairly diverse in composition, but their response to OE treatment was uniform. OE decreased the weight of WAT through reduction of both cell numbers and size; but did not change the mass or composition of BAT significantly. The effects of OE are more marked in the hormonal signals (leptin and acyl-estrone) from the tissue than in its composition and mass.

Oral oleoyl-estrone induces the rapid loss of body fat in Zucker lean rats fed a hyperlipidic diet.

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 μmol/kg of fatty-acyl esters of estrone. This diet was supplemented with additional oleoyl-estrone to produce diets with 2.5 μmol/kg (diet OE2.5), 4.4 μmol/kg (diet OE4.4), and 33.3 μmol/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.

Effect of oral oleoyl-estrone treatment on plasma lipoproteins and tissue lipase activities of Zucker lean and obese female rats

Objective: To study the effect of oral oleoyl-estrone on the plasma lipoprotein profile and tissue lipase activities in order to determine the handling of circulating lipids by adipose tissue, liver and muscle of obese female rats.Design: Lean (Fa/?) and obese (fa/fa) female Zucker rats treated for 10 days with a daily gavage of 0.2 ml sunflower oil containing 0 (controls) or 10 µmol/kg of oleoyl-estrone. After sacrifice, samples of tissues and plasma were taken.Measurements: Plasma lipoprotein classes and composition; lipoprotein lipase and hepatic lipase activities in plasma, liver, skeletal muscle and periovaric and mesenteric white adipose tissue (WAT).Results: Oleoyl-estrone decreased plasma cholesterol (mainly in HDLs: 76%) of lean rats, but dramatically decreased all lipid classes in obese rats, in which chylomicra and VLDL lost most of their triacylglycerols (95 and 81%, respectively). Hepatic lipase activity decreased markedly with oleoyl-estrone in all groups, both in plasma (79% lean, 100% obese) and liver (62% in both groups). Lipoprotein lipase activity was largely unchanged by oleoyl-estrone in lean rats, but in the obese it decreased in WAT (82% in periovaric, and 49% in mesenteric), and increased in plasma (×4) and in skeletal muscle (×5); liver levels showed no change.Conclusions: The shift observed in obese rats from a decrease in liver and WAT lipoprotein lipase and hepatic lipase activities to an increase in muscle lipoprotein lipase is coincident with the hypolipemic effect of oleoyl-estrone, especially in obese rats, and indicates that muscle is a key site for the disposal of endogenous fat mobilized due to oleoyl-estrone treatment.

Semiquantitative RT-PCR measurement of gene expression in rat tissues including a correction for varying cell size and number

BackgroundCurrent methodology of gene expression analysis limits the possibilities of comparison between cells/tissues of organs in which cell size and/or number changes as a consequence of the study (e.g. starvation). A method relating the abundance of specific mRNA copies per cell may allow direct comparison or different organs and/or changing physiological conditions.MethodsWith a number of selected genes, we analysed the relationship of the number of bases and the fluorescence recorded at a present level using cDNA standards. A lineal relationship was found between the final number of bases and the length of the transcript. The constants of this equation and those of the relationship between fluorescence and number of bases in cDNA were determined and a general equation linking the length of the transcript and the initial number of copies of mRNA was deduced for a given pre-established fluorescence setting. This allowed the calculation of the concentration of the corresponding mRNAs per g of tissue. The inclusion of tissue RNA and the DNA content per cell, allowed the calculation of the mRNA copies per cell.ResultsThe application of this procedure to six genes: Arbp, cyclophilin, ChREBP, T4 deiodinase 2, acetyl-CoA carboxylase 1 and IRS-1, in liver and retroperitoneal adipose tissue of food-restricted rats allowed precise measures of their changes irrespective of the shrinking of the tissue, the loss of cells or changes in cell size, factors that deeply complicate the comparison between changing tissue conditions. The percentage results obtained with the present methods were essentially the same obtained with the delta-delta procedure and with individual cDNA standard curve quantitative RT-PCR estimation.ConclusionThe method presented allows the comparison (i.e. as copies of mRNA per cell) between different genes and tissues, establishing the degree of abundance of the different molecular species tested.

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.

Effect of the slimming agent oleoyl-estrone in liposomes on the body weight of Zucker obese rats

OBJECTIVE: To determine whether the mechanisms by which estrone acyl-esters carried by lipoproteins induce the loss of body fat can affect Zucker fa/fa rats, since they are hyperphagic and could not eliminate excess energy through thermogenesis, two aspects essential for the slimming effect of oleoyl-estrone in normal rats. DESIGN: The rats were infused for 28 d (osmotic minipumps) with oleoyl-estrone in liposomes (Merlin-2) at a dose of 3.5 mmol/day·kg. SUBJECTS: Lean (L) and obese (O) Zucker rats. MEASUREMENTS: Body weight changes. Oxygen consumption, body composition (water, lipid, protein), nitrogen balance, plasma chemistry. RESULTS: Treatment resulted in loss of body weight: 12.0 % (28 g) L, 9.4 % (34 g) O, mainly due to fat: 37.5 % (10.8 g) L, 11.7 % (15.5 g) O and water, preventing further increases in body weight and fat storage. Untreated rats increased their body weight: 10.5 % (24 g) L, 32.2 % (101 g) O and lipid stores: 20.3 % (5.9 g) L, 39.8 % (49.0 g) O, making the differences more marked. On day 28, glucose levels were maintained in all groups; in L, triacylglycerols increased and total cholesterol decreased; O showed no changes in plasma composition. In all rats, food intake decreased with treatment, and heat production (oxygen consumption) was unchanged (L) or slightly decreased (O). Energy expenditure per unit of fat-free mass remained unchanged. Protein balance was maintained in all groups; slimming was achieved without loss of body protein. CONCLUSION: Treatment of genetically obese rats with oleoyl-estrone in liposomes (Merlin-2) results in sustained loss of body weight – mainly lipid, sparing protein – for up to 28 d, essentially preventing further increase in body weight and accumulation of lipid and protein. This is achieved through lower food intake and relatively small changes (if any) in energy expenditure.

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.