A. Ardévol

Cooling rates of tissue samples during freezing with liquid nitrogen.

The time-course of temperature changes undergone by tissue samples during freezing in liquid nitrogen has been studied with small thermocouple probes. The specific heat of liver, muscle, adipose tissue and blood has been measured with a calorimeter. Most tissues have lower specific heat values than water. The lower thermal conductance of adipose tissue and fat muscle result in much longer freezing times. It may take an inordinate amount of time for tissue samples to freeze when immersed in liquid nitrogen; in addition, not all tissues behave in the same way, so it takes longer for a muscle sample to freeze than for a piece of liver. These differences between tissues are considerable and may affect the outcome of experiments if not taken into account.

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.

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.

Hind leg muscle amino acid balances in cold-exposed rats.

The changes in hind leg tissue (muscle and skin) amono acid pool size and arteriovenous balance were measured in rats subjected to 0–90 min of cold exposure (4°C). Tissue free amino acid pools presented a different composition pattern from protein amino acids. Muscle rapidly reacted to cold exposure by releasing small amounts of some amino acids (alanine, aspartate), with only small changes in pool size during the first 30 min. Amino acid oxidation was very limited during the whole period of cold exposure, since at all times tested there was either nil ammonia efflux or net absorption of ammonia and glutamine; i.e. the muscle was in positive nitrogen balance throughout the period studied. Thus most of the amino acid nitrogen taken up from the blood and not found in the free amino pools must have been incorporated into protein, since it was not oxidized, as shown by the glutamine and ammonia blance. The data on amino acid incorporation into proteins indicate that hind leg protein turnover is rapidly and widely modulated from a low initial setting upon cold exposure to a higher protein synthesis rate immediately afterwards, suggesting that protein turnover may have an important role in short-term events in cold-exposed muscle, in addition to its influence in long-term adaptation.

CARBOHYDRATE HANDLING IN THE HIND LEG MUSCLE OF EXERCISING RATS

The arterio‐venous concentrations of oxygen, glucose and lactate, as well as blood flow and muscle levels of lactate, glucose, hexose‐phosphates and glycogen of rat hind leg muscle were determined under basal conditions and under mild and intense exercise, as well as during post‐exercice recovery. During intense exercise and fatigue glycogen is practically exhausted, providing glycosyl residues to the hexose‐phosphates pool in addition to increased glucose uptake from the blood. The result is the production of huge amounts of lactate, which accumulates in muscle and the skin, and buildup the arterial concentrations. During recovery lactate is slowly disposed of, and the muscle takes up large amounts of glucose which is stored into glycogen, with fully reinstated glucose oxidation. The data shown suggest that the shift from oxidative to mainly anaerobic utilization of glucose is not as streamlined as is usually assumed, since the results found here hint at the wasteful utilization of glycogen‐derived hexose skeletons for other synthetic pathways. Glucose, nevertheless, is of paramount importance as energy staple to sustain untrained intense exercise in the rat

Hind-leg heat losses in cold-exposed rats

Abstract Wistar rats, reared at 22°C, were exposed to 4°C for up to 2 h. During this period, the temperatures of aortic blood (core), thigh muscle and skin were recorded; the blood flow into the skin and muscle of the hind leg was determined with radioactive microspheres; and the arterio-venous balances of oxygen and carbon dioxide, as well as those of lactate, were measured. The data were used to estimate the oxidative and anaerobic energy liberated in the hind leg at the given times, as well as the heat transferred by the blood to the leg tissues. These data were used to calculate the heat balance, and hind leg heat loss during cold-exposure. Despite a doubling in heat production in muscle, this accounted for less than a quarter of all the heat lost by the leg; the difference was made up by a marked increased in the heat carried by the blood, thanks to increased blood flow. It can be concluded that in spite of increased local heat production, hind leg muscle thermogenesis falls short of providing the heat needed to maintain hind leg tissue temperatures under cold-exposure.