STUDIES OF THE MITOCHONDRIAL ENERGY-TRANSFER SYSTEM OF BROWN ADIPOSE TISSUE

An investigation of the mechanisms of norepinephrine action and heat production in brown adipose tissue from newborn rabbits has been carried out. Data obtained with the use of biochemical techniques has been correlated with morphological data from electron microscopy. Norepinephrine was found to stimulate the respiration of brown fat in vitro. Inhibitors of glycolysis abolish this effect, whereas inhibitors of oxidative phosphorylation do not, at least not to the same extent. Brown fat is readily permeable to added Krebs cycle intermediates. Substrate level phosphorylation, but no electron transport-coupled phosphorylation, could be demonstrated in isolated mitochondria. It is suggested that the rate of fatty acid oxidation is limited by the availability of phosphate acceptor systems which break down ATP formed at the substrate level and thus provide ADP for further substrate level phosphorylation. The theory of respiratory control by the action of reesterification of fatty acids is discussed in the light of these findings. Under the electron microscope, brown fat mitochondria are characterized by their large size, tightly packed cristae, and by the different types of granules in the matrix. No elementary particles are seen when the mitochondria are examined by the negative-staining technique. The absence of electron transport-coupled phosphorylation together with the apparent absence of elementary particles seems to be of particular significance.     

Intramitochondrial filamentous bodies in the thick limb of henle of the rat kidney

"Intramitochondrial filamentous bodies" (IMFB) were occasionally found within the matrix of some mitochondria of the thick limb of Henle of the rat kidney, but not elsewhere in the tubular system. Three types were recognized: type I, an accumulation of filaments 55 A thick; type II, a bundle of parallel filaments having the same thickness as those of type I and regular spacing, 87 A apart, from center to center; and type III, consisting of type II with regular light bands of 280 A periodicity and a helical border of prismatic tubular cristae. In addition to these, electron-opaque masses showing variable and faint substructures were found in the matrix of mitochondria. It is suggested that all these IMFB may originate from mitochondrial cristae and that type II IMFB may be an intermediate developmental form between type I and type III. After uranyl acetate staining, IMFB and the membranes of prismatic tubular cristae showed highly increased electron opacity. The literature has been reviewed for reports of intramitochondrial filamentous inclusions in various types of cells. These inclusions have been classified according to their structural characteristics and the localization in the mitochondria and compared with IMFB reported herein.     

FINE STRUCTURE CHANGES DURING FUNCTION OF THE DIGESTIVE GLAND OF VENUS'S-FLYTRAP

Changes in the structure of the digestive gland cells of Venus's-flytrap during the digestive process have been studied with light and electron microscopy. Large vacuolar lipid-protein inclusions break up and become smaller; however, they never completely disappear during the entire 7-10-day cycle. Dictyosomes in the resting digestive gland are associated with small, inconspicuous vesicles, whereas during the digestive cycle two types of prominent vesicles are observed on the peripheral tubules. Changes in plastid fine structure are complex and involve the disappearance of lipid globules and the tubular complex, followed by the formation of microtubules on the thylakoids and cisternae on the outer plastid membrane. Mitochondrial fine structure changes from the small cristae and light matrix of the resting state to large cristae and a very dense matrix representative of a change to a state where phosphorylation is tightly coupled to electron transport. Pronounced changes which occur in the cell envelope (cell wall and membrane taken together) are apparently associated with secretion of the digestive fluid. Numerous other changes are observed such as polysome formation, multivesicular body formation, mitochondria division, and changes which can be attributed in general to elevated cell activity.     

STRUCTURAL CHANGES IN ISOLATED LIVER MITOCHONDRIA OF RATS DURING ESSENTIAL FATTY ACID DEFICIENCY

Liver mitochondria isolated in 0.44 M sucrose from rats deficient in essential fatty acids (EFA) oxidized citrate, succinate, α-ketoglutarate, glutamate, and pyruvate at a faster rate than did mitochondria isolated from normal rats; however, the oxidation of malate, caprylate, and β-hydroxybutyrate was not significantly increased. The mitochondria from deficient rats exhibited an increased ATPase activity and extensive structural damage as revealed by electron microscope examination of thin sections. An increase in citrate oxidation and ATPase activity, together with some structural damage, could be demonstrated as early as the 4^th week in rats on a fat-free diet. Saturated fat in the diet did not prevent the change in mitochondrial structure but accelerated its appearance. Both the biochemical and structural defects could be reversed within three weeks after feeding deficient rats a source of EFA. In the presence of a phosphate acceptor the effect of EFA deficiency on substrate oxidation was largely eliminated. A trend toward a reduced efficiency of oxidative phosphorylation was noted in mitochondria from EFA-deficient rats, but significant uncoupling was found only in the case of citrate, β-hydroxybutyrate, and glutamate in the presence of malonate. Together with the increased ATPase activity, the uncoupling of phosphorylation could account for the poor respiratory control found with the deficient preparation. However, EFA deficiency was without effect on the respiration of liver slices, which supports the belief that the observed changes in oxidation and phosphorylation are an artifact resulting from damage sustained by the deficient mitochondria during their isolation.     

CORTISONE-INDUCED ALTERATIONS IN MITOCHONDRIAL FUNCTION AND STRUCTURE

The effects of cortisone treatment on oxygen consumption, oxidative phosphorylation, and fine structure of rat liver mitochondria have been studied. Male rats weighing 125 g were treated for 6 days with 5 mg of cortisone acetate or isotonic saline. On the 7th day, sections of liver were excised and processed for light and electron microscopy. Mitochondrial respiration and oxidative phosphorylation were studied with mitochondria isolated from these livers. Cortisone treatment is responsible for a 14–40% decrease in the amount of oxygen consumed per mg of mitochondrial protein when succinate, α-ketoglutarate, or β-hydroxybutyrate are used as substrates, or with ascorbate and N,N,N¹,N¹-tetramethyl p-phenylenediamine as electron donors. In addition, oxidative phosphorylation is uncoupled with a lowering of the P:O ratios. Randomly selected liver cells have been analyzed by quantitative morphometric techniques. The average mitochondrial volume is increased fourfold in the peripheral and midzonal regions with a commensurate decrease in the number of mitochondria per cell. These alterations are present throughout the hepatic lobule, but are most marked in midzonal cells. The total mitochondrial volume per cell and the per cent of the total cytoplasmic volume occupied by mitochondria remains relatively unaltered, as does the total amount of cristae surface per cell. While the mitochondria are enlarged, they are not "swollen." The relationships between the steroid hormone treatment and the alterations in mitochondrial function and structure are discussed.     

Effects of tetrazolium salts on oxidative phosphorylation in rat-liver mitochondria

1. The effects of five different tetrazolium salts on oxidative phosphorylation in rat-liver mitochondria have been investigated. 2. In all cases the mitochondria were uncoupled by very low concentrations of the tetrazolium salts. Further, the transition from a system just exhibiting respiratory control to one in which the mitochondria were totally uncoupled has been shown to occur over very small concentration ranges of the tetrazolium salts. 3. The effectiveness of the five tetrazolium salts as uncoupling agents is discussed in the light of their standard electrode potentials and effectiveness as electron acceptors in dehydrogenase-linked reactions.     

Mitochondrial superoxide and aging: uncoupling-protein activity and superoxide production

Mitochondria are a major source of superoxide, formed by the one-electron reduction of oxygen during electron transport. Superoxide initiates oxidative damage to phospholipids, proteins and nucleic acids. This damage may be a major cause of degenerative disease and aging. In isolated mitochondria, superoxide production on the matrix side of the membrane is particularly high during reversed electron transport to complex I driven by oxidation of succinate or glycerol 3-phosphate. Reversed electron transport and superoxide production from complex I are very sensitive to proton motive force, and can be strongly decreased by mild uncoupling of oxidative phosphorylation. Both matrix superoxide and the lipid peroxidation product 4-hydroxy-trans-2-nonenal can activate uncoupling through endogenous UCPs (uncoupling proteins). We suggest that superoxide releases iron from aconitase, leading to a cascade of lipid peroxidation and the release of molecules such as hydroxy-nonenal that covalently modify and activate the proton conductance of UCPs and other proteins. A function of the UCPs may be to cause mild uncoupling in response to matrix superoxide and other oxidants, leading to lowered proton motive force and decreased superoxide production. This simple feedback loop would constitute a self-limiting cycle to protect against excessive superoxide production, leading to protection against aging, but at the cost of a small elevation of respiration and basal metabolic rate.     

Succinate dehydrogenase localization in the mitochondria of the renal tubules of cold- and warm-blooded vertebrates

Using electron microscopic histochemical technique, studies have been made on the activity of succinic dehydrogenase in the kidneys of the cod Gadus morrhua and dog. It was shown that chelate granules indicating localization of the enzyme in the mitochondria of nephronal cells, concentrate mainly in two zones -- between the membranes and inside the cristae. This distribution of the enzyme implies the presence of two pools of succinic dehydrogenase in the mitochondria which are utilized at different stages of oxidative phosphorylation. Succinic dehydrogenase content of the cristae is lower in cod than in dog.     

Ultrastructural study of the yeast-mycelium transition in Histoplasma capsulatum. II. Changes at 34 degrees C

The ultrastructural changes which occur during the mycelium to yeast transition in Histoplasma capsulatum induced by a temperature shift from 25 degrees C to 34 degrees C are described and compared to those observed after a temperature shift from 25 degrees C to 37 degrees C. 24 hours after the temperature shift to 34 degrees C only 8% of the cells are lysed. However, many mitochondria have lost their characteristic elongated form and have become rounded. Vesicular cristae which are no longer oriented parallel to the long axis of the mitochondria are also observed. In contrast a temperature shift from 25 degrees C to 37 degrees C induces lysis of 70% of the cells; mitochondria are rarely observed in the remaining cells. These ultrastructural changes can be correlated with the uncoupling of oxidative phosphorylation and the production of heat shock proteins.     

Electron microscopy morphology of the mitochondrial network in human cancer

Mitochondria have been implicated in the process of carcinogenesis, which includes alterations of cellular metabolism and cell death pathways. The aim of this review is to describe and analyze the electron microscopy morphology of the mitochondrial network in human cancer. The structural mitochondrial alterations in human tumors are heterogeneous and not specific for any neoplasm. These findings could be representing an altered structural and functional mitochondrial network. The mitochondria in cancer cells, independently of histogenesis, predominantly are seen with lucent-swelling matrix associated with disarrangement and distortion of cristae and partial or total cristolysis and with condensed configuration in minor scale. Mitochondrial changes are associated with mitochondrial-DNA mutations, tumoral microenvironment conditions and mitochondrial fusion–fission disequilibrium. Functionally, the structural alterations suppose the presence of hypoxia-tolerant and hypoxia-sensitive cancer cells. Possibly, hypoxia-tolerant cells are related with mitochondrial condensed appearance and are competent to produce adequate amount of ATP by mitochondrial respiration. Hypoxia-sensitive cells are linked with lucent-swelling and cristolysis mitochondria profile and have an inefficient or null oxidative phosphorylation, which consequently use the glycolytic pathway to generate energy. Additionally, mitochondrial fragmentation is associated with apoptosis; however, alterations in the mitochondrial network are linked with the reduction in sensitivity to apoptosis induces and/or pro-apoptotic conditions. Pharmacological approaches designed to act on both glycolysis and oxidative phosphorylation can be considered as a new approach to selectively kill cancer cells.     

The fine structure of mitochondria and the mitochondrial cloud during oogenesis on the sea anemone Actinia

The appearance and arrangement of the mitochondria during all stages of oocyte growth in the sea anemone Actinia fragacea (Cnidaria: Anthozoa) have been examined by electron microscopy. In small oocytes, the mitochondria are generally squat, with a dense matrix and numerous cristae, although a proportion may show an unusual arrangement of prismatic cristae. During early oogenesis, the mitochondria tend to be arranged in aggregates rather than randomly scattered, and may be associated with nuage material. With the onset of vitellogenesis, a large mitochondrial aggregate forms next to the nucleus. During early vitellogenesis this aggregate enlarges and comes to resemble the mitochondrial clouds found in some amphibian oocytes. Within the cloud, many mitochondria appear to be highly elongate and irregular in shape. The cloud begins to fragment and disperse midway through vitellogenesis at about the time when cortical granules appear. In fully grown oocytes, some mitochondria may have a much less dense matrix and fewer cristae than the remainder, which may be related to their state of activity.     

Corn Mitochondrial Swelling and Contraction-an Alternate Interpretation

Mitochondria isolated from 3-day-old etiolated corn shoots (Zea mays L.) can be categorized into three separate groups, each group characteristic of the cell type from which the mitochondria were isolated. Phloem sieve tubes and some adjacent parenchyma cells contain mitochondria that have few cristae and little amorphous matrix. Mitochondria from meristematic and undifferentiated cells have more cristae and matrix. Vaculate and differentiated cells have mitochondria with well-developed cristae and abundant matrix. Each mitochondrial type exhibits typical in vitro spontaneous swelling and substrate-induced contraction responses. characterized by change or lack of change in cristae size and in density of amorphous material. For the second and third types of mitochondria, swelling and contraction are characterized by a change in degree of cristae size and in matrix density. The first type undergoes few changes upon swelling or contraction. Radical changes of the inner membrane, withdrawal and infolding, are associated with cell differentiation and not with swelling and contraction of isolated corn shoot mitochondria.     

Über hydrophobe Acridinfarbstoffe zur Fluorochromierung von Mitochondrien in lebenden Zellen

Summary The hydrophobic fluorescence dye 10-n-nonyl-acridinium-orange-chloride, NAO, stains specifically the mitochondria of living HeLa-cells. A dye concentration of 1·10^–8 M is sufficient for vital staining and at 5·10^–7 M an incubation time less than 1 min is enough to generate the bright green fluorescence of the mitochondria. The retention of NAO by the mitochondria is longer than 7 days.The dye accumulation is not affected by the ionophores valinomycin, nigericin, gramicidin, the uncoupling agents DNP, CCCP or by ouabain. In contrast to Rh 123 the trans-membrane potential is not the driving force of the NAO accumulation. We assume that NAO is bound to the hydrophobic lipids and proteins in the mitochondrial membranes by hydrophobic interaction.With valinomycin, 500 ng/ml, 10 min, the mitochondria in HeLa-cells swell. Now it is possible to observe some details in the enlarged mitochondria by light microscopy. After vital staining with NAO, 5·10^–7 M, 10 min, the periphery of the swollen mitochondria shows an intense green fluorescence, the inner part is dark. Obviously the dye is bound to the membranes. By electron microscopy it can be shown that the valinomycin treated and NAO stained mitochondria have outer and inner membranes and cristae. They differ from untreated mitochondria mainly in the size.After incubation of the HeLa-cells with relatively high NAO concentrations, 5·10^–6 M, 10 min, the mitochondria show a weak orange fluorescence. It is generated by the dimers D of NAO. Therefore the dye concentration in the mitochondrial membranes is locally very high and causes dye dimerisation. The weak orange fluorescence is instable and disappeares within a few seconds. Instead we observe a green fluorescence with growing intensity that is generated by the monomers M of NAO. The intensity has its maximum value after a few seconds. Using low NAO concentrations for incubation, 1·10^–7 M, 10 min, we observe only the green fluorescence with increasing intensity. In this case the orange fluorescence is too weak for observation (concentration quenching). It can be shown by experiments and quantum mechanics that the orange fluorescence is assigned to an optical forbidden, the green fluorescence to an allowed electronic transition of D or M respectively. Our results indicate a dissoziation of D in 2 M by irradiation of the mitochondria under the fluorescence microscope.The intensity changes of the orange and the green fluorescence of bound D and M by irradiation has been measured in living cells with a microspectrophotometer. The experimental data agree quantitatively with a first-order reaction mechanism for the dissoziation of D in 2 M by irradiation. There is some evidence for energy transfer between dimers at higher NAO concentration.The oxygen consumption of HeLa-cell suspensions has been measured electrochemically at various NAO concentrations and incubation times with an oxygen electrode. Up to 5·10^–7 M NAO, 10 min, the respiratory activity is not affected. After that we observe an increasing inhibition of the oxygen consumption with growing NAO concentration and incubation time. At 5·10^–6 M, 30 min, the inhibition is 40% relative to the untreated cells.The ultrastructure of the mitochondria in incubated HeLa-cells has been investigated by electron microscopy and compared with untreated cells. Similar to the resiratory experiments there is no difference in ultrastructure up to 5·10^–7 M NAO, 10 min. Then the ultrastructure changes rapidly with increasing NAO concentration and incubation time. At the final stage, 5·10^–6 M, 1 h, the cristae totally or partially disappeared. The outer and inner membranes are still visible. Obviously the mitochondria without cristae are instable and collapse. They change into liposomes with stacks of four, eight and more membranes on the periphery. They enclose cytoplasm. The genesis of the liposomes is discussed in some detail.These experiments show that the dye NAO is accumulated at the inner mitochondrial membrane and the cristae. It blocks the enzymes of the oxydative phosphorylation in the inner membranes and affects the self-organization of the cristae. NAO is specifically bound to the membranes of the mitochondria. Neither by fluorescence microscopy nor by electron microscopy we observe binding of NAO to the membranes of the nuclei.     

Water movement from intracristal spaces in isolated liver mitochondria

When analyzing mitochondria isolated in a sucrose medium that had been embedded for thin sectioning according to one low denaturation embedding technique, large intracristal spaces were present in close to 90% of the mitochondria. The two crista membranes were closely apposed in only 40% of all cristae. When the mitochondria were transferred to an incubation medium, the percentage of mitochondria with intracristal spaces was reduced to 40%. About 90% of all cristae were lacking any space separating the two crista membranes. The presence of inorganic phosphate in the medium was required for the closing of the intracristal spaces. The percentage of cristae lacking an intracristal space remained the same after addition of substrate for respiration (state 4) and of ADP (state 3). Inhibition or uncoupling of respiration led to an increase in the percentage of intracristal spaces, showing that oxidative phosphorylation is required to maintain the crista membranes closely apposed. The appearance and disappearance of the intracristal spaces was an indication of water movements across the crista membranes. The mean volume of the mitochondria increased 33% when they were transferred from the sucrose medium to the incubation medium, showing that the removal of water from the cristae was not caused by a passive osmotic effect. Addition of substrate made the volume decrease by 28%. After further addition of ADP, the volume decreased another 23%. No change in volume was associated with inhibition or uncoupling of respiration. The observations revealed that water can move into or out of the cristae independently of water movement out from the entire mitochondrion. Therefore, the water moving out from or into the cristae is translocated across the cristae membrane. The observations are interpreted to reveal the presence of a mechanism that actively prevents water from accumulating in the crista membrane. This mechanism allows for a low water activity to be maintained within the membrane. The variations in the frequency of intracristal spaces occurred without any simultaneous changes in the width of the space appearing between the two surface membranes after isolation of the mitochondria. The observations, therefore, do not agree with the concept that there is an outer compartment that communicates freely with intracristal spaces.     

Chloride-dependent uncoupling mediated by oligomycin in rat liver mitochondria

In rat liver mitochondria suspended in KCl medium and containing a low concentration of a K(+)-specific cationophore (valinomycin or Triton X-100), oligomycin was shown to induce uncoupling of oxidative phosphorylation, stimulation of adenosine triphosphatase activity, release of the respiratory control, decrease of energy-dependent changes in the fluorescence of the dye 8-anilino-1-naphthalenesulphonic acid and rapid swelling of mitochondria. Oligomycin caused none of the above effects when Br(-) or NO(3) (-) was substituted for Cl(-) as the major anionic species or when Na(+) replaced the K(+). The same concentration of oligomycin that caused uncoupling and swelling slightly improved energy-conserving reactions when the cationophores were omitted. In the presence of KSCN, valinomycin or Triton X-100 by itself caused uncoupling and swelling which was not further enhanced by oligomycin. On the basis of the above results it is suggested that the energy dissipation resulting from the concerted action of the cationophores and oligomycin is connected with the simultaneous transport of K(+) and its counter ion and that oligomycin plays its role in the uncoupling by facilitating the permeation of Cl(-) through the cristae membrane of the mitochondria.     

Mitochondrial oxidative stress and inflammation: an slalom to obesity and insulin resistance

Mitochondria, in addition to energy transformation, play a role in important metabolic tasks such as apoptosis, cellular proliferation, heme/steroid synthesis as well as in the cellular redox state regulation. The mitochondrial phosphorylation process is very efficient, but a small percentage of electrons may prematurely reduce oxygen forming toxic free radicals potentially impairing the mitochondria function. Furthermore, under certain conditions, protons can reenter the mitochondrial matrix through different uncoupling proteins (UCPs), affecting the control of free radicals production by mitochondria. Disorders of the mitochondrial electron transport chain, overgeneration of reactive oxygen species (ROS) and lipoperoxides or impairments in antioxidant defenses have been reported in situations of obesity and type-2 diabetes. On the other hand, obesity has been associated to a low degree pro-inflammatory state, in which impairments in the oxidative stress and antioxidant mechanism could be involved. Indeed, reactive oxygen species have been attributed a causal role in multiple forms of insulin resistance. The scientific evidence highlights the importance of investigating the relationships between oxidative stress and inflammation with obesity/diabetes onset and underlines the need to study in mitochondria from different tissues, the interactions of such factors either as a cause or consequence of obesity and insulin resistance.     

Cold exposure differently influences mitochondrial energy efficiency in rat liver and skeletal muscle

This study deals with mitochondrial energy efficiency in liver and skeletal muscle mitochondria in 15 days cold exposed rats. Cold exposure strongly increases the sensitivity to uncoupling by added palmitate of skeletal muscle but not liver mitochondria, while mitochondrial energy coupling in the absence of fatty acids is only slightly affected by cold in liver and skeletal muscle. In addition, uncoupling protein 3 content does not follow changes in skeletal muscle mitochondrial coupling. It is therefore concluded that skeletal muscle could play a direct thermogenic role based on fatty acid-induced mild uncoupling of mitochondrial oxidative phosphorylation.     

Influence of CSP 310 and CSP 310-like proteins from cereals on mitochondrial energetic activity and lipid peroxidation in vitro and in vivo

Background  

The development of chilling and freezing injury symptoms in plants is known to frequently coincide with peroxidation of free fatty acids. Mitochondria are one of the major sources of reactive oxygen species during cold stress. Recently it has been suggested that uncoupling of oxidation and phosphorylation in mitochondria during oxidative stress can decrease ROS formation by mitochondrial respiratory chain generation. At the same time, it is known that plant uncoupling mitochondrial protein (PUMP) and other UCP-like proteins are not the only uncoupling system in plant mitochondria. All plants have cyanide-resistant oxidase (AOX) whose activation causes an uncoupling of respiration and oxidative phosphorylation. Recently it has been found that in cereals, cold stress protein CSP 310 exists, and that this causes uncoupling of oxidation and phosphorylation in mitochondria.     

An animal model to study human muscular diseases involving mitochondrial oxidative phosphorylation

Mitochondria are producing most of the energy needed for many cellular functions by a process named oxidative phosphorylation (OXPHOS). It is now well recognized that mitochondrial dysfunctions are involved in several pathologies or degenerative processes, including cardiovascular diseases, diabetes, and aging. Animal models are currently used to try to understand the role of mitochondria in human diseases but a major problem is that mitochondria from different species and tissues are variable in terms of regulation. Analysis of mitochondrial function in three species of planarian flatworms (Tricladia, Platyhelminthes) shows that they share a very rare characteristic with human mitochondria: a strong control of oxidative phosphorylation by the phosphorylation system. The ratio of coupled OXPHOS over maximal electron transport capacity after uncoupling (electron transport system; ETS) well below 1.0 indicates that the phosphorylation system is limiting the rate of OXPHOS. The OXPHOS/ETS ratios are 0.62?±?0.06 in Dugesia tigrina, 0.63?±?0.05 in D. dorotocephala and 0.62?±?0.05 in Procotyla fluviatilis, comparable to the value measured in human muscles. To our knowledge, no other animal model displays this peculiarity. This new model offers a venue in which to test the phosphorylation system as a potential therapeutic control point within humans.