C. Adán

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.

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.

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.

Effect of adrenalectomy on the slimming activity of liposome-carried oleoyl-estrone in the rat

OBJECTIVE: To determine the extent of glucocorticoid counter-regulatory control in the slimming action of oleoyl-estrone.DESIGN: Control and adrenalectomized rats were subjected to a seven-day treatment with 3.5 μmol/kg/d oleoyl-estrone in liposomes injected i.v. continuously by implanted osmotic minipumps.SUBJECTS: Sham-operated control and adrenalectomized lean Zucker rats.MEASUREMENTS: Body weight and food intake; plasma glucose, urea, insulin, leptin and corticosterone; liver glycogen.RESULTS: Treatment with oleoyl-estrone resulted in decreases in body weight and in food intake, as well as in circulating glucose, insulin and leptin. Combined adrenalectomy and oleoyl-estrone treatment resulted in a loss of almost 15% body weight in only seven days, with a severe drop in circulating glucose and insulin, almost total disappearance of plasma leptin and liver glycogen and a 3-fold rise in circulating urea. Food intake decreased sharply, which resulted in the exhaustion of energy reserves.CONCLUSION: The results presented here, strongly support the hypothesis that glucocorticoids play an important role in the modulation of oleoyl-estrone-induced imbalance of energy intake and expenditure. The large effect of oleoyl-estrone on glucose, glycogen- and protein-derived (urea levels) energy in adrenalectomized rats, provides more evidence for the assumed protective role of glucocorticoids against the oleoyl-estrone-induced net loss of energy reserves. The results also show the powerful destabilizing effects of unchecked oleoyl-estrone on energy balance.

Short-term treatment with estrone oleate in liposomes (Merlin-2) does not affect the expression of the ob gene in Zucker obese rats

Young female Zucker fa/fa rats of 370-430 g were implanted with osmotic minipumps releasing 3.5 μmol/dayúkg of estrone oleate in liposomes (Merlin-2) into the bloodstream for up to 14 days. Merlin-2 induced a sustained loss of appetite, and a decrease in body weight of 3.5%, which contrasts with the 8.2% increase in controls during the period studied. Plasma insulin, glucose and urea decreased, and liver glycogen increased with Merlin-2 treatment. Plasma ACTH and corticosterone increased to a maximum at the end of the experiment. The expression of the ob gene in adipose tissue was unchanged, and plasma leptin levels were also unchanged by treatment. Estrone levels increased more than 1500-fold, and estrone oleate rose 100-fold during treatment. The fact that estrone oleate had no effect on the leptin levels or expression in obese rats, in contrast with the marked inhibition observed in the lean suggests that the functionality of the leptin receptor is essential for estrone oleate inhibition of the ob gene. This also suggests that leptin may control ob gene expression in white adipose tissue and that estrone oleate may activate this process. The slimming effect of estrone oleate is, thus, not directly dependent on leptin, since both normoleptinemic and hyperleptinemic animals lose fat following treatment nor are the effects on appetite and energy expenditure mediated by leptin. However, leptin levels and the expression of the ob gene are directly linked with estrone oleate function. A possible involvement of leptin in estrone oleate action is postulated. The results support the participation of estrone oleate in the control of body weight and hint at the complexity of its regulation by leptin and glucocorticoids.

Effect of food deprivation on rat plasma estrone fatty acid esters

The present study was devised to determine whether the circulating levels of estrone fatty esters are modified by 6–48 h starvation in the rat, in parallel to changes in fat reserves, as a test to check the plausibility of its function as a ponderostat signal in the mammal. Food deprivation resulted in a decrease in glucose and triacylglycerols, rapid disappearance of liver glycogen and increases in fatty acids and, especially, 3‐hydroxybutyrate. Insulin and leptin decreased, corticosterone and free estrone increased from 6 h onwards and total estrone levels were maintained. Starvation reduced the lipid content of the rat by 25.6%. Plasma esterified estrone levels decreased more slowly, by 13% in 48 h, but its circulating mass decreased in the same proportion as the total lipid content of the rat. The small change in circulating estrone fatty esters is consistent with the postulated role of oleoyl‐estrone as a medium‐term ponderostat signal.

Lactate-bicarbonate interrelationship during exercise and recovery in lean and obese Zucker rats

OBJECTIVE: To determine the relationship between muscle-derived lactate at fatigue and earlier onset of fatigue in the obese rat subjected to intense exercise. DESIGN: Rats were subjected to a short, intense exercise protocol on a treadmill. Blood was drawn from hind leg vein and artery during exercise and up to 1 h afterwards. Assuming an exercise respiratory quotient of 1.0, the extra carbon dioxide released was computed and assumed to be displaced by equimolar amounts of lactic acid produced by the rat during exercise. SUBJECTS: Conscious female adult Zucker lean and Zucker obese rats. MEASUREMENTS: Oxygen consumption and carbon dioxide release. Lactate and bicarbonate levels in hind leg venous and arterial blood; balances were estimated by measuring blood flow with fluorescent microspheres. Lactate levels in periovaric white adipose tissue were also measured. RESULTS: Muscle released, during exercise and post exercise roughly 2.3 mmol lactate in lean rats and 2.6 mmol in obese ones. Of these amounts, hind leg lactate release accounted for 0.40 mmol in lean rats and only 0.11 in the obese ones, which showed a release of acid (mainly lactate) elsewhere in the rats totalling about 19.9 mmol CO2 in lean rats and 4.4% in the obese ones; that is both hind quarters accounted for only 17.2% of all lactate produced in the lean rats and 4.4% in the obese ones. The amount of lactate produced by the rats was roughly similar. White adipose tissue lactate levels (in the basal state and after exercise) were much higher than could be expected from blood sources alone, indicating an active production of lactate. CONCLUSION: Fatigue appears earlier in the obese rats than in lean ones because of loss of buffering ability caused by massive extra-muscular glycolysis (probably in adipose tissue) and lactate production triggered by exercise-induced adrenergic stimulation.

Treadmill chamber for studies of respiratory gas exchange in the rat during exercise

A treadmill for studying gas exchange in small mammals during exercise is presented. The system consists of a motor-driven running mat enclosed in a gastight chamber that receives a measured flow of air from a compressed air cylinder. The gas flow and temperature, pressure and instantaneous gas composition of the chamber (oxygen, carbon dioxide and water) are measured continuously and the data are computed to include the effects on chamber atmosphere of the rat activity, either running or at rest. The system is completed with a shock delivery grid that stimulates the rat to run. The calculations are based on the changes in the composition of the gas in the chamber (constantly stirred by a small electric fan) induced by the rat instead of relying on the alterations induced in the outflowing gas. The consumption of oxygen, and production of carbon dioxide and water by the rat are computed in real time, giving a very fast response to physiological change induced by exercise. The chamber is custom-made from an aluminium block and a plexiglass lid; all other components are available commercially. The system, as described, allows for a detailed analysis of respiratory gas (and water) exchange by rats under varying exercise conditions, there is practically no time lag between changes in respiratory gases and the detection of these changes, and the buffering effect of the chamber size is practically eliminated because of the calculation approach used.

Effect of cold-exposure on rat organ blood flows

Rat tissue blood flows and heart output were determined in adult Wistar rats under up to two hours of cold (4 degrees C) exposure, using radioactive 46Sc microspheres. Circulating glucose, lactate and triacylglycerol levels were also determined. Glucose concentrations increased with cold exposure in spite of the drainage of substrates induced by the activation of thermogenesis. Plasma triacylglycerol levels agree with a high involvement of fats in the sustenance of heat production. Cold-exposure had an immediate effect decreasing skin circulation, but increased that of muscle and brown adipose tissue. Kidney and intestine blood flows were maintained. In liver, blood flow increased progressively with cold-exposure. White adipose tissue showed--at first--low blood flow, but increased in parallel to that of liver. The data presented show a distribution of the blood in the body of the cold-exposed rat in which thermogenic responsibilities and supply of blood are evenly distributed throughout. The importance of haemodynamic changes in brown adipose tissue was considerable but the increased share of muscle blood flow suggests that it may have a global role in maintaining thermal homeostasis.