Mitochondria brown fat

Nicholls, D. G., and Rial, E., 1984. Brown fat mitochondria. Trends Biochemical Sciences 9 489—491. 

FIGURE 19-30 Heat generation by uncoupled mitochondria. The uncoupling protein (thermogenin) of brown fat mitochondria, by providing an alternative route for protons to reenter the mitochondrial matrix, causes the energy conserved by proton pumping to be dissipated as heat. 

From the above P/O ratio, it is clear that ATP synthesis in brown fat mitochondria is naturally uncoupled from electron transport. Hence, protons extruded from the mitochondria during electron transport must reenter without concomitant ATP synthesis. The energy released as heat during this reentry may help to keep the young animals warm. Such small organisms have a high surface-to-volume ratio and therefore readily lose heat through convective and radiative processes. 

There is a controversy as to whether a drop in A/tH+ created by net ATP synthesis and by the addition of proton translocator create equal respiratory stimulations this will be discussed in a later section. However, brown fat mitochondria investigated in our laboratory [36,37] show the same relationships between respiratory stimulation and A/tH+ decrease with a proton translocator and the 32000 uncoupling protein... 

Fig. 2.3. Relationship between respiratory rate and hamster brown fat mitochondria when the...

Not all the transporters discussed above are present in aU types of mitochondria the set of activities present in mitochondria depends on the functional needs of the cells from which the mitochondria are isolated. The adenine nucleotide and phosphate transporters are present in all mitochondria thus far studied. This reflects the fact that the major function of mitochondria is the synthesis of ATP. Even in the rare instances (e.g., brown fat mitochondria [55] and mitochondria in anaerobically growing yeast [56]) where the major function is not ATP synthesis, mitochondria normally have active adenine nucleotide transport. The pyruvate transporter also appears to be ubiquitous. The carnitine transporter has been studied in liver [57], heart [35] and sperm [58] and is probably present in all mitochondria which use long-chain fatty acids. 

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. 

Fig. 10.2. The respiratory pattern of isolated brown fat mitochondria. A. When substrate (succinate) is added to brown fat mitochondria (here isolated from cold-acclimated or control guinea-pigs), they respire rapidly. Upon ADP addition the rate is initially increased (normal State 2-3 transition), but the ensuing State 4 rate is lower than State 2. Successive ADP additions result in a successively decreased State 4 rate. Numbers indicate respiratory rates in nmol oxygen-min -mg protein. (Adapted from Pedersen and Flatmark [93] for details see this paper.) B. The specific coupling effect of purine nucleotides (here ADP) can be demonstrated after addition of oligomycin so that the respiratory stimulation due to ATP synthesis is eliminated. Addition of the uncoupler FCCP results in a respiratory rate identical to that prior to ADP, indicating that the ADP effect is on coupling, and not due to inhibition of substrate oxidation. (Adapted from Cannon et al. [23] for details see this paper.)...

Fig. 10.3. The relationship between the proton motive force (pmf) and the respiration (thermogenesis) of brown fat mitochondria the effect of GDP. The membrane potential and proton gradient were determined by ion distribution methods. It is seen that, with increasing concentrations of GDP, the pmf is increased. However, not until the pmf exceeds about 150 mV is the respiration inhibited. (Adapted from Nicholls [5] for details see this paper.)...

When brown fat mitochondria are incubated with isoosmotic solutions of KCl or KBr they swell, provided that valinomycin is added to make permeable. This halide permeability is inhibitable by purine nucleotides, just as is respiration [26]. 

Fig. 10.4. The relationship between respiration (thermogenesis) and Ca control in brown fat mitochondria. uptake was measured with the arsenazo technique. It is seen that when the GDP...

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).)...

The existence of a purine nucleotide binding site on brown fat mitochondria... 

Since it was shown by Cannon et al. [23] that ADP acted from the outside of the mitochondria to induce respiratory control, a specific site of interaction could be envisaged. Such a site was characterized by Nicholls [33] by binding of [ H]GDP. Further, by labelling brown fat mitochondria with [ P]azido-ATP, Heaton et al. [34] demonstrated that — besides the ATP/ADP-translocase at 30 kDa — a specific band with a molecular weight of 32000 was labelled, and this was identical with the GDP-binding site. This protein (i.e., thermogenin) had already been observed by Ricquier and Kader as the only protein the concentration of which was markedly altered in brown fat mitochondria isolated from cold-acclimated animals [35] (Fig. 10.8). 

The ability of brown fat mitochondria to alter their capacity for heat production... 

Fig. 10.8. The effect of cold acclimation on the polypeptide composition of rat brown fat mitochondria. Densitometric tracings of SDS polyacrylamide gels with mitochondrial membranes from control and cold-acclimated rats were superimposed, and the areas where the peak from the cold-acclimated animal exceeded that of the control is indicated in black (for no peak was the inverse true). Only a band at 32 kDa (arrow) was increased by cold acclimation (due to unresolved peaks, the adjacent peaks seem also to be increased, but this is an effect of the base-line broadening of the 32 kDa peak). The 32 kDa peak was later identified with thermogenin. (Adapted from Ricquier and Kad6r [35].)...

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.)...

As the degree-of-activation of mitochondria is said to influence their physical appearance, it may be suggested that some of the observed unmasking phenomena may be due to the actual state of the brown fat mitochondria at the time of isolation, and/or to the incubation procedure, rather than reflecting true changes in the in situ qualities of thermogenin itself. This, however, remains to be investigated. 

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... 

There is no doubt that the addition of free fatty acids to brown fat mitochondria results in a stimulation of respiration. The reason for this response is however not unequivocally clear. As seen in Fig. 10.13, there are at least four possible sites of interaction of free fatty acids with brown fat mitochondria (1) competitively with purine nucleotides on the binding site on thermogenin (2) on another site on thermogenin (3) on another protein site on the membrane or (4) directly with the membrane. 

It has been suggested that free fatty acids in some way stimulate thermogenin by acting on a (putative) site on thermogenin other than the GDP-binding site. The argument for this has until now been rather indirect and simply related to the fact that brown fat mitochondria are more sensitive to free fatty acids as uncouplers than... 

Two other possible sites of interactions of free fatty acids with brown fat mitochondria remain interaction with other proteins (3) or directly with the... 

Fig. 10.13. A sketch of the possible interactions of free fatty acids and their derivatives with brown fat mitochondria. The sketch illustrates some of the candidates for the mediator of thermogenesis (i.e., the substance or process that will activate thermogenin (alt. another site of the mitochondrial membrane) even in the presence of the inhibitory cytosohc nucleotides). Common for the candidates shown here is that they are formed subsequent to the activation of lipolysis of the stored triglycerides (TG) by norepinephrine (NE) via cAMP-dependent processes. The candidates illustrated are free fatty acids (FFA), interacting (1) with the purine-nucleotide binding site on thermogenin, (2) with another site on thermogenin, (3) with another protein than thermogenin, or (4) directly with the membrane, and the acyl-CoAs, interacting (5) specifically with the purine-nucleotide binding site on thermogenin, or (6) unspecifically with the membrane. For discussion, see Section 5.

What remains is the possibility (4) that free fatty acids, also in brown fat mitochondria (under experimental conditions) function as classical uncouplers (i.e., as weak lipophilic acids). The reason for the higher sensitivity to free fatty acids of brown fat than of liver mitochondria — as well as the higher sensitivity of cold-acclimated than of control brown fat mitochondria — may then simply reside in the fact that the more sensitive mitochondria have more mitochondrial inner membrane [83] with which the free fatty acids can interact. This hypothesis has as yet not been experimentally tested. 

Fig. 10.14. The uncoupling effect of free fatty acids and their CoA derivatives on mitochondria. Brown fat mitochondria from co/4-accIimated or control rats, as well as Hver mitochondria, all at a protein concentration of 0.5 mg ml , were studied in the oxygen electrode in a medium containing 0.05% albumin and with 20 mM succinate as substrate. The stimulation of respiration caused by the addition of...

It has been criticized that the effect of acyl-CoA is unspecific and due to a general detergent action. However, acyl-CoA does not induce permeability in parallel with Cl permeability [85], and there are clearly effects of palmitoyl-CoA on brown fat mitochondria at concentrations below those that yield (definitely unspecific ) uncoupling in rat liver mitochondria. Further, the uncoupling effects of free fatty acids are clearly — at low concentrations — better if acyl-CoA formation is allowed to proceed than when it is hampered [81] (Fig. 10.14). 

Brown-Fat Mitochondria Contain an Uncoupler of Oxidative Phosphorylation... 

---