Olov Lindberg

[8] Mitochondria from brown adipose tissue: Isolation and properties

Publisher Summary Brown adipose tissue (BAT) is heat producing and is specialized for the oxidation of fatty acids. No enzymes characteristic of mitochondria in general appear to be missing in BAT mitochondria, but their relative activities are strikingly different. These mitochondria have been studied to gain insight into the mechanism of thermogenesis and mitochondrial bioenergetics. The rapid proliferation of mitochondria in BAT in connection with birth and cold adaptation renders the tissue suitable for studies of mitochondriogenesis. BAT occurs in most neonatal mammals, in certain cold-adapted adult rodents, and in hibernators. The state of the tissue and its stage of development vary considerably with the physiological state of the experimental animal; care must be exercised in this respect if comparisons are made between species and between research groups. The most commonly used experimental animals are rats, hamsters, and guinea pigs. Mitochondrial preparations have been made from ground squirrels, hedgehogs, young lambs, and warm-adapted Syrian hamsters.

The Brown Fat Cell

Publisher Summary Brown fat cells produce heat by oxidation of the fatty acids. Isolated cells are prepared by collagenase treatment of brown fat from hamsters and rats. The chapter outlines the isolation procedure for the brown fat cells and discusses the properties of the isolated cell suspension. The effect of the incubation conditions on the parameters such as membrane potential, lipolysis, and respiration as an indirect measure of thermogenesis are studied. Brown fat thermogenesis in vivo is stimulated by norepinephrine (NE), liberated from sympathetic nerve endings in the tissue. The hormones studied on brown fat cells can be divided into three major groups—namely, (1) the hormones that exert their effect on the brown fat cell itself; (2) the hormones that act solely by releasing stored catecholamines, within the tissue or in impure cell preparations; and (3) the hormones that only affect the tissue indirectly in the intact animal. The fatty acids released by hormone-sensitive lipase activity may be exported either as such, or activated to acyl coenzyme A (acyl-CoA) in the cell. The acetyl coenzyme A (acetyl-CoA), produced by mitochondrial β-oxidation, is further metabolized by different mitochondrial matrix enzymes. The chapter also discusses the mechanism of heat production in the brown fat cell.

Changes in interscapular brown adipose tissue of the rat during perinatal and early post-natal development and after cold acclimation—II. Mitochondrial changes

Abstract 1. 1. Changes in some biochemical parameters of mitochondria isolated from interscapular brown adipose tissue in different development stages and after cold acclimation of rats have been studied. 2. 2. These parameters varied in a regular pattern. The highest values were found at 5 days post partum and after cold acclimation. 3. 3. The significance of these changes with respect to the amount of inner membrane in mitochondria is discussed in relation to mitochondrial differentiation, and also in the context of alterations in respiratory activity of the whole tissue during development.

Ultrastructural changes in the chondriome during perinatal development in brown adipose tissue of rats

Morphometric observations on interscapular brown adipose tissue of pre- and neonatal rats have enabled a quantitative analysis of some changes in mitochondrial size and number. During late prenatal development, the mitochondrial fractional volume and the number of mitochondria in the tissue were both considerably increased. Over the period of birth, there was a 60 % increase in mitochondrial fractional volume, while the number of mitochondria was unchanged. Twenty-four hours later, the mitochondrial fractional volume had decreased slightly, and the number of mitochondria probably increased. When expressed in terms of a “triglyceride droplet-free” adipocyte volume, the changes in the tissue could be interpreted in terms of alterations in the chondriome of the adipocytes. During the prenatal stages, the mean mitochondrial volume grew a little, while the number per cell volume remained constant. However, the volume of tissue occupied by adipocytes increases. In contrast, the large increase in mitochondrial fractional volume over birth was caused mainly by a considerable increase in mean mitochondrial volume. Over the period studied there was no statistically significant increase in the number of mitochondria per “triglyceride droplet-free” adipocyte volume. Mitochondrial ultrastructure changed considerably during these stages. In addition to the changes in volume mentioned above, there was also an unquantified increase, at least up to 1 day ante partum (a.p.), and probably up to the day of birth, in the amount of cristae per mitochondrial profile. Also, the size of the intramitochondrial dense granules increased to a maximum mean value of 1230 A at 1 day a.p. By a few hours after birth, the size of the inclusions had slightly decreased and by 1 day postpartum, no large intramitochondrial granules were seen. The significance of these results for a hypothesis relating the intramitochondrial dense granules to formation of inner membrane is discussed.

Changes in interscapular brown adipose tissue of the rat during perinatal and early postnatal development and after cold acclimation. I. Activities of some respiratory enzymes in the tissue.

Abstract 1. 1. Specific activities of succinic dehydrogenase, α-glycerophosphate dehydrogenase and cytochrome oxidase were measured in homogenates of interscapular brown adipose tissue from developing and cold-acclimated rats. These activities changed in similar patterns, reaching maximal levels at 17 days post partum and after cold acclimation. 2. 2. The total respiratory capacity of interscapular brown adipose tissue, related to body weight, was calculated for each enzyme and stage studied. They were all greatest at 5 days post partum and after cold acclimation. 3. 3. The results are interpreted to imply that brown adipose tissue has maximal in vitro functional capacity around the fifth day of postnatal life and after cold acclimation.

A cytochemical investigation into the distribution of cytochrome oxidase activity within the mitochondria of brown adipose tissue from the prenatal rat

Using 3,3′-diaminobenzidine as substrate, the localization of cytochrome oxidase activity has been studied in mitochondria of brown adipose tissue from late prenatal rats. The reaction demonstrated cytochrome c oxidase since it was stimulated by exogenous cytochrome c, inhibited by KCN, and was insensitive to antimycin. The reaction product was found in the cristal and intermembrane spaces of unfixed, isolated mitochondria after as little as 2 minutes of incubation. Even after 45 minutes of incubation, reaction product was not clearly localized to the type II inclusions. This negative result was not altered by the presence of exogenous cytochrome c or by 2,4-dinitrophenol in the incubation medium. In fixed tissue, 30–40% of the mitochondrial profiles contained cristae oriented longitudinally. These had no observably different cytochrome oxidase activity compared to cristae oriented transversely. The results are discussed. It is concluded that they support the hypothesis that type II inclusions are composed of incomplete inner membrane lacking some membrane-bound respiratory enzymes.

Monoamine oxidase and cytochrome oxidase activities in brown adipose tissue of the rat during development

Abstract 1. 1. Monoamine oxidase (MAO) and cytochrome c oxidase (COX) activities were measured simultaneously in both homogenatc and isolated mitochondria of rat interscapular brown adipose tissue (ISBAT) during development and after cold acclimation. 2. 2. The MAO activity of ISBAT is less than one-tenth that-of adult rat liver (in both homogenates and mitochondria). 3. 3. A considerable part of MAO is localized extramitochondrially m ISBAT; an extraadipocyte location seems most probable. 4. 4. A close relationship was found between MAO and the physiological activity of brown fat, which might also be correlated with the importance of norepinephrine (NE) in stimulating thermogenesis.

Metabolic control in isolated brown fat cells

Experiments with brown fat cell preparations from the adult hamsters are described. The mitochondria of brown adipose tissue were shown to have a classical electron transport system. The basal respiration of brown fat cells was demonstrated to be coupled to oxidative phosphorylation. Evidence is presented for partial uncoupling of oxidative phosphorylation as a mechanism for controlling respiration during norepinephrine stimulation. Exogenously added fatty acids were found to mimick the norepinephrine stimulation of respiration. Norepinephrine and cyclic AMP were shown to have no effect on brown fat mitochondria. Experiments with labeled oleate showed that the triglyceride reesterification cycle does not control respiration in brown adipose tissue.