Practical aspects of indirect calorimetry in laboratory animals.

Oxidation of the energetic substrates by the body is associated with oxygen consumption, carbon dioxide production, and heat release specific to the nature of the energetic substrates being oxidized. Therefore, measurement of respiratory exchanges (indirect calorimetry) is a powerful method to investigate heat production of a living organism. In this article, we review the elementary principles of indirect calorimetry and describe the operating principle of the two most typical devices used to perform indirect measurements of energy expenditure in the laboratory animal: the closed-circuit and the open-circuit. We then discuss some practical aspects of the day-to-day use of these devices: respective advantages and limitations of each technique, data processing, calibration, correction for body-size, and computation of the energy expended for activity. In the second part, we review some of the standard formulas of indirect calorimetry that offer the possibility to obtain more precise information such as the rate of oxidation of carbohydrates (CHO), lipids and proteins if some hypotheses are made on the intensity of lipogenic, ketogenic, and gluconeogenic processes. Finally, a practical example of the measurement of energetic cost of activity and thermic effect of food in the rat is given.

Hindlimb immobilization applied to 21-day-old mdx mice prevents the occurrence of muscle degeneration.

Dystrophin-deficient skeletal muscles of mdx mice undergo their first rounds of degeneration-regeneration at the age of 14-28 days. This feature is thought to result from an increase in motor activity at weaning. In this study, we hypothesize that if the muscle is prevented from contracting, it will avoid the degenerative changes that normally occur. For this purpose, we developed a procedure of mechanical hindlimb immobilization in 3-wk-old mice to restrain soleus (Sol) and extensor digitorum longus (EDL) muscles in the stretched or shortened position. After a 14-day period of immobilization, the striking feature was the low percentage of regenerated (centronucleated) myofibers in Sol and EDL muscles, regardless of the length at which they were fixed, compared with those on the contralateral side (stretched Sol: 8.4 +/- 6.5 vs. 46.6 +/- 10.3%, P = 0.0008; shortened Sol: 1.2 +/- 1.6 vs. 50.4 +/- 16.4%, P = 0.0008; stretched EDL: 05 +/- 0.5 vs. 32.9 +/- 17.5%, P = 0. 002; shortened EDL: 3.3 +/- 3.1 vs. 34.7 +/- 11.1%, P = 0.002). Total numbers of myofibers did not change with immobilization. This study shows that limb immobilization prevents the occurrence of the first round of myofiber necrosis in mdx mice and suggests that muscle contractions play a role in the skeletal muscle degeneration of dystrophin-deficient mdx mouse muscles.

Chronic vascular catheterization in the mouse.

A method is described for stress-free periodic sampling of blood and/or intravenous infusions in the normal awake and behaving mouse. The surgical procedure consists of the permanent implantation of a Silastic-rubber catheter in the right external jugular vein. The free end of the catheter is then pulled subcutaneously up to the skull and fixed on the skull with a form of dental acrylic that sticks firmly on the bone and thus does not require anchor screws in the skull. This technique allowed us to perform glucose tolerance tests in the mouse, and to follow the time course of blood glucose levels in individuals.

Effect of intraperitoneal injection of glucose on glucose oxidation and energy expenditure in the mdx mouse model of duchenne muscular dystrophy.

Previous studies suggesting that glucose metabolism could be impaired in the skeletal muscles of the mdx mouse led us to study the metabolic response to i.p. injection of either glucose or glucose and insulin in the free-moving mdx mouse. In the first study, changes in blood glucose and plasma insulin levels were measured in mice with chronic venous cannulae. In the second study, the thermogenic response to glucose and the changes induced on glucose oxidation (Gox) and lipid oxidation (Lox) were assessed by indirect calorimetry. The experiments showed that insulin response, as well as whole body glucose uptake, were normal in mdx mice. Addition of exogenous insulin abolished the increase in blood glucose level similarly in mdx and control mice. The thermogenic response to glucose was identical in mdx and control mice but, when insulin was injected with glucose, it increased significantly in mdx mice. Exogenous glucose increased Gox less and decreased Lox less in mdx than in control mice. Addition of exogenous insulin reduced the difference between mdx and control mice but affected Gox and Lox less in mdx than in control mice. Key words Duchenne muscular dystrophy middle dot mdx Mouse middle dot Glucose tolerance test middle dot Respiratory quotient middle dot Energy metabolism middle dot Indirect calorimetry

Components of energy expenditure in the mdx mouse model of Duchenne muscular dystrophy.

Previous observations showing that basal heat production rates and glucose metabolism were reduced in mdx mouse skeletal muscles incubated in vitro led us to study the components of total energy expenditure by open-circuit indirect calorimetry in the intact, free-moving mdx mouse. Our purpose was to verify if the mdx mouse exhibited whole-body alterations in energy metabolism. The results revealed that total and basal energy expenditure, as well as spontaneous activity, energetic cost of activity, and, therefore, energy expended in relation to activity were not significantly different in C57B1/10 (control) and in dystrophic (mdx) mice. In contrast, the thermic effect of food was 32% larger in mdx than in control mice and was accompanied by significant differences in post-prandial glucose and lipid oxidation. The present in vivo study could not show a direct demonstration that impaired glucose metabolism by skeletal muscles participated in this phenomenon. However, since post-prandial glucose metabolism by skeletal muscles contributes a significant part of the thermic effect of food, the present data are in line with previous studies in vitro that show that mdx mouse skeletal muscles probably suffer an impaired control of their energy metabolism.

Effects of treadmill exercise and high-fat feeding on muscle degeneration in mdx mice at the time of weaning.

1. Dystrophin-deficient hindlimb muscles of mdx mice undergo necrosis at the time of weaning when the motor activity of the mice greatly increases and muscle energy metabolism becomes more dependent on insulin and carbohydrates. 2. We have attempted to determine if the onset of myofibre necrosis in mdx mice at the time of weaning is related to the development of motor activity and/or the change in diet. 3. Fourteen-day-old mdx mice were divided into two groups after weaning. One group was trained to run on a treadmill and the other group was kept on a high-fat diet. Muscle necrosis was assessed histologically in the soleus and extensor digitorum longus muscles of mice in both experiments. 4. Keeping mice on a high-fat milk diet from the time of weaning up to 42 days of age did not influence the occurrence of necrosis in the soleus and extensor digitorum longus muscles of the mdx pups. In contrast, treadmill exercise greatly increased necrosis in both muscles. 5. We conclude that an increase in motor activity exacerbates the degeneration of hindlimb muscles of mdx mice at the time of weaning.