Tudor Barnard

Changes in interscapular brown adipose tissue of rat during perinatal and early postnatal development and after cold acclimation: IV. Morphometric investigation of mitochondrial membrane alterations

Abstract Some changes in the contribution of inner and outer mitochondrial membranes to mitochondrial structure have been quantified during the development and after cold acclimation of rat brown adipose tissue. The results show that the mitochondria are structurally (as well as enzymatically) most developed for oxidative capacity two to three weeks after birth of the animal and also after cold acclimation in the adult. By the fifth postnatal week, mitochondrial structure has begun to involute, mainly as the result of a decreased average mitochondrial volume. Unlike the inner membrane changes, outer membrane involution is not structurally affected by cold acclimation. This investigation has also confirmed that the disappearance of large matrix granules (type II inclusions) in the mitochondria takes place during a period of rapid increase in the amount of inner membrane, both in individual mitochondria and (together with parallel compositional studies) in the interscapular fat pad. Further circumstantial evidence is thus available, associating the type II inclusion phenomenon with an intense formation of new inner membrane.

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.

The ultrastructural differentiation of brown adipose tissue in the rat

The development of interscapular brown adipose tissue has been observed in rats from 6 days ante partum to some days after birth. The most obvious alterations prior to birth were increases in cell volume, triglycerides, and glycogen stores as well as considerable growth of the chondriome. There was a marked differentiation of mitochondrial structure, including the formation of large intramitochondrial dense granules. At birth there was a rapid decrease in amount of glycogen and temporarily a depletion of stored triglycerides, simultaneously with increased numbers of autophagic vacuoles. The large intramitochondrial dense granules disappeared and chondriome growth was rapid during this time. In the days after birth the chondriome volume in the cells appeared to increase still further, and the triglyceride droplet fractional volume in the tissue also continued to grow. Evidence for the hypothesis that capillary endothelial cells are a major type of stem cell for adipocytes in brown adipose tissue, is discussed.

Preparation of Biological Material For X‐Ray Microanalysis of Diffusible Elements

The appearance of ultrathin, dry‐cut cryosections of brown adipose tissue and liver was found to be strongly dependent upon adequate freeze‐drying. If freeze‐drying was inadequate, diffusion of substances could be demonstrated and freezing damage was not apparent. Diffusion was manifested as an electron dense film over structural features such as triglyceride droplet profiles and the edges of the section; when this film was thick, X‐ray signals for P, S, Cl, K and Ca could be detected from it, in different proportions to those found in the section. The frequency and intensity of diffusion were lowered by decreasing the temperature in the cryochamber from about 200 K ± 5 K to 163 K ±5 K by forced evaporation of liquid N2 using an extra heater. The lowest incidence of diffusion was obtained in conjunction with this device, by leaving the sections in the cryochamber for at least 2 h during drying, either over a drying agent or under moderate vacuum. Such sections showed a narrow zone (A) at the edge that lacked obvious ice‐crystal damage, a thicker zone (B) of moderate ice‐crystal damage and the bulk of the interior (zone C) severely damaged by freezing. Qualitatively different, reproducible X‐ray spectra could be obtained from ultrastructural features even in areas showing some signs of diffusion.

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.

Morphology and biochemical properties of perirenal adipose tissue from lamb (Ovis aries). A comparison with brown adipose tissue

Abstract 1. 1. Perirenal adipose tissue from lamb resembles brown adipose tissue from other species in many respects: morphological appearance; double sympathetic innervation; mitochondrial energy conservation; pattern of fatty acid oxidation. 2. 2. The tissue differs significantly from brown adipose tissue in having a high mitochondrial ATP synthetic capacity; elementary particles on the mitochondrial inner membrane; possessing the specific mitochondrial ATPase inhibitor protein. 3. 3. The simple delineation of a definition for brown adipose tissue is not possible. 4. 4. The tissue appears useful in studying the postulated interconvertibility of brown and white adipose tissue.

PHOSPHORUS STATUS AND CYTOPLASMIC STRUCTURES IN SCENEDESMUS (CHLOROPHYCEAE) UNDER DIFFERENT METABOLIC REGIMES1

Phosphorus deficiency affects the anatomy of the unicellular green alga Scenedesmus obtusiusculus Chad. and influences the distribution of other inorganic elements in the cell in addition to phosphorus. Scenedesmus was grown under standard conditions with or without phosphorus. Cells were then cultured with phosphorus under conditions favouring glycolysis, respiration, or photophosphorylation for 2 h or photosynthesis for up to 8 h. The dominating features of phosphorus starvation, were loss of phosphorus and coions from polyphosphate bodies, accumulation of starch, decrease in the volume density of ribosomes both in the chloroplast and cytoplasm, and an increase in wall thickness. Under conditions favoring photosynthesis the mass fraction for phosphorus is low after 1 h, exceptionally high by 2 h, and diminishes by 8 h. High amounts of phosphorus are also regained under conditions favoring glycolysis and photophosphorylation but not respiration. After 2 h under photosynthetic conditions the volume densities of the chloroplast, cytoplasmic ribosomes, the vacuole, and the mitochondrion increased over controls. By 8 h the relative volume of the single ramified mitochondrion had decreased slightly and recognizable segments of it were sequestered within the vacuome. The autophagic nature of the vacuole was further evidenced by the presence of ribosomes and whorls of lamellae within it. Serial sections showed that all polyphosphate granules and sequestered materials were located within a continuous vacuolar cisterna.

Ultrastructural effects of the high molecular weight cryoprotectants Dextran and polyvinyl pyrrolidone on liver and brown adipose tissue in vitro

The ultrastructural changes caused by incubation of rat liver and brown fat in buffered solutions of high molecular weight cryoprotectives (Dextran and polyvinyl pyrrolidone) at high concentrations (up to 25% w/v) have been examined. Under appropriate conditions of incubation rather small qualitative changes were found. Hepatocytes showed some signs of plasmolysis. Evidence for the endocytosis of Dextran and polyvinyl pyrrolidone was obtained. Cryoultramicrotomy of unfixed, quench‐frozen specimens after the same incubation procedures showed good cutting properties and only slight ice‐crystal damage. Several of the larger tissue compartments were recognizable in such sections. Although high molecular weight cryoprotectants do penetrate cells by endocytosis (and these effects require further functional evaluation), the present observations provide further support for the idea that such compounds significantly improve the quench‐freezing of biological specimens and offer a practical way forward for the preparation of material for X‐ray microanalysis of diffusible elements.

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.