Mitochondria of brown adipose tissue

In the mitochondria of brown adipose tissue, very little ATP is synthesized, and most of the energy liberated in the electron transport system is converted to heat. 

The rate of electron transfer in isolated mitochondria from brown adipose tissue, in the absence of ADP and phosphate, is high indicating that the mitochondria are uncoupled (see Appendix 9.9). 

The activity of the ATP synthase is low in comparison with that in mitochondria from other tissues i.e. mitochondria in brown adipose tissue can generate very little ATP. 

This review is updated to autumn 1983. For other reviews of brown adipose tissue mitochondria see Refs. 7-9, for a review on the integration of mitochondrial function in the brown fat cell see Ref. 3, and for the function of brown adipose tissue as such, see e.g., Refs. 4, 10-12. 

With our present understanding, the thermogenic qualities of brown adipose tissue mitochondria are a consequence of the existence in the mitochondrial inner membrane of a polypeptide, thermogenin, uniquely [13-15] found in brown adipose tissue. (For technical and historical reasons, thermogenin is also known under several other names, such as the GDP-binding protein, the 32000 protein, the purine-nucleotide-binding protein (NbP), the uncoupling protein (UCP), the proton conductance pathway, etc.)... 

When mitochondria are isolated and tested in media which only contain osmotic support (sucrose or KCl) and a buffer (and Mg, Pj and EDTA, if necessary), they respire rapidly on substrates such as succinate or glycerol-3-phosphate (citrate, 2-oxoglutarate and malate are poor substrates in brown fat mitochondria from most species, as the substrate permeases are poorly developed [16]). This rapid respiration is seen in Fig. 10.2. This observation was initially made even before the thermogenic function of brown adipose tissue was known [17]. When R. Em. Smith had established that heat production was the function of the tissue [18], an intrinsic uncoupled state of the mitochondria [19,20] could be understood as the means of heat production, the intensity of which would be limited only by substrate supply [21]. However, Horwitz et al. [21a] found that addition of the artificial uncoupler DNP could potentiate the respiration of the tissue, and the conclusion had to be that the mitochondria — although uncoupled when isolated — were coupled in situ. 

It may thus be envisaged, that for the genersil understanding of the regulation of the biogenesis of mitochondria, research on thermogenin may — like the metabolism of brown adipose tissue itself — be inefficient, but never futile. 

Figure 5.23 Structure of brown adipose tissue as seen by transmission electron microscopy. FG = Fat globule MIT = mitochondria. Reproduced with kind permission of Dr M. Ashwell.

Thiolester hydrolases are present in most tissues and cell compartments. High concentrations are found in liver microsomes and in brown adipose tissue mitochondria and peroxisomes. Several acyl-CoA hydrolases have shown a close relationship to the nonspecific carboxylesterases EC 3.1.1.1. Thus, palmitoyl-CoA hydrolase purified from rat liver microsomes was found to be identical to esterase pI 6.2I6A (ES4 type). An acyl-CoA hydrolase was isolated that showed high similarity to esterase pI 6.1 [74a] [129] [130]. These few examples are further illustrations of the unsatisfying situation of the traditional classification of esterases. 

In order to provide heat when it is required to maintain or increase body temperature, a mechanism must exist for the regulation of the activity of this uncoupling protein. The mechanism has been established by following the principles described in Chapter 3. The properties of the uncoupling protein are studied using mitochondria isolated from brown adipose tissue in vitro (as described in Appendix 9.9). 

The properties are as follows, (i) The activity of the protein (i.e. the inward transport of protons) is inhibited by ATP. (ii) The activity of the protein is increased by the presence of long-chain fatty acids, since they relieve the ATP inhibition, (iii) When mitochondria, isolated from brown adipose tissue, are incubated in the presence of fatty acids, there is a sharp increase in the rates of electron transfer, substrate utilisation and oxygen consumption, whereas the rate of ATP generation remains low. These studies indicate that the rate of proton transport, by the uncoupling protein, depends on the balance between the concentrations of ATP and fatty acids, (iv) In adipocytes isolated from brown adipose tissue, the rate of oxygen consumption (i.e. electron transfer) is increased in the presence of catecholamines. 

The first defect, described in 1962 is, in fact, one of the rarest (Luft s syndrome). It arises from the uncoupling of mitochondria. The resting metabolic rate is markedly raised, there is profuse sweating, fever and generalised muscle weakness. The mitochondria of these patients have an increased permeability, not so much to protons, as in brown adipose tissue mitochondria, but to cations, such as Ca, the entry of which similarly dissipates the proton motive force. 

Brown fat is a form of adipose tissue found under the skin on the backs of many young animals. Mitochondria from this tissue have a P/O ratio of less than 1 for ATP synthesis arising from oxidation of NADH. What may be the physiological function of brown fat tissue ... 

Only in three tissues — in two plant tissues (the Arum lily flower and the skunk cabbage spadix) and in mammalian brown adipose tissue — has a direct thermogenic role of mitochondria been substantiated. 

Within the last decade we have obtained a tentative concept of the molecular basis for this mammalian mitochondrial thermogenesis, and we know that in contrast to the thermogenic plant mitochondria, substrate oxidation in brown adipose tissue mitochondria is basically energy conserving, with proton extrusion occurring [5], with respiratory control, and with an ability, in principle, to capture the chemical energy in the form of ATP. 

In this review we have not attempted to provide a historical account of the development of this concept (for such an account see Ref. 6), but rather attempted to show how this concept has enabled a series of apparently disparate observations on brown adipose tissue mitochondria to be unified. 

The uncoupled state of traditionally isolated and tested brown adipose tissue mitochondria... 

Fig. 10.6. The effect of respiration and membrane potential (Ai )) on Cl permeation in brown adipose tissue mitochondria. When brown fat mitochondria were incubated in KCl in the presence of the ionophore, nigericin, they swelled (A, B). If a respiratory substrate (here G-3-P glycerol-3-phosphate) was added to the expanded mitochondria, they contracted, and this contraction ceased immediately and swelling was reintroduced if azide (NaNj) and an uncoupler (FCCP) were added (Fig. A). The passive halide ion permeability can be inhibited by GDP (cf.. Fig. 10.5), but respiration-driven contraction in KCl-expanded mitochondria was only partially inhibited by the presence of GDP (Fig. B) if again azide and uncoupler were added during the contraction, the mitochondria did not swell, indicating that the thermogenin channel was closed by GDP. This behaviour can partly be explained by the fact that the Cl permeation is driven by the membrane potential. Indeed, when, under similar conditions, the rate of contraction was plotted as a function of the membrane potential, it was seen that the rate was membrane potential dependent. It should, however, he noted that at low membrane potentials GDP nearly totally abolished the Cl permeation but when the membrane potential was increased above 30 mV, the inhibitory effect of GDP was apparently partially lost. The basis for this phenomenon is not understood it is not even known if there is a lower affinity of thermogenin for GDP in the energized membrane, as measurements of GDP affinities always refer to the non-energized situation. (Adapted from Nicholls et al. [27] (A, B) and Nicholls [94] (C).)...

Fig. 10.10. Determination of thermogenin amount in brown adipose tissue mitochondria by the enzyme-linked immunosorbent assay (ELISA) system. The amount of thermogenin was determined as elsewhere described (Cannon et al. [13] Sundin et al. [40] Hansen et al. [56]) in an assay system based on the competition between absorbed and added thermogenin for rabbit on/r-rat-thermogenin antibodies. The interaction was followed with a sheep onri-rabbit-IgG antibody conjugated to alkaline phosphatase. The reaction was linearized as indicated (abs 0 is the absorbance developed in the absence of competing thermogenin). It is seen that this assay can detect less than 0.25 fig thermogenin, i.e., the content in less than 10 fig of mitochondria. It is also seen that the thermogenin content of rat brown fat mitochondria is approximately doubled after a 24 h cold stress. (Our unpublished observations.)...

In conclusion it would seem that GDP-binding is an adequate way of determining thermogenin concentration in brown fat mitochondria. It should however be added that for physiological studies, this is not the only relevant parameter. As brown adipose tissue hypertrophies when stimulated, increases in thermogenin content per animal are often markedly greater than increases in thermogenin concentration in... 

Fig. 10.11. The effect of osmolarity on the apparent number of GDP binding sites in brown adipose tissue mitochondria. The number of GDP-binding sites was measured in mitochondria from control and cold-exposed (24 h at 4°C) rats as earlier described (Sundin and Cannon [37]), but in media with the indicated concentrations of sucrose. Note that, if iso-osmotic sucrose is used (250 mM), a low GDP binding can be observed, especially in control rats. This may be related to the condensation phenomenon discussed in section 2.4. However, if 100 mM sucrose is used, the mitochondria swell, and the full number of binding sites is determined. (Our unpublished observations.)...

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