Energy dissipation in the brown fatty tissue of warm-blooded animals

The uncoupling mechanism of respiration and phosphorylation in brown adipose tissue as well as factors affecting this process are discussed. An assumption has been suggested and substantiated experimentally that the uncoupling of respiration and phosphorylation in liver mitochondria caused by vitamin K alimentary deficit may have mechanism similar to the unique uncoupling mechanism of brown fat mitochondria.     

The bioenergetics of brown fat mitochondria from UCP1-ablated mice. Ucp1 is not involved in fatty acid-induced de-energization ("uncoupling").

The bioenergetics of brown fat mitochondria isolated from UCP1-ablated mice were investigated. The mitochondria had lost the high GDP-binding capacity normally found in brown fat mitochondria, and they were innately in an energized state, in contrast to wild-type mitochondria. GDP, which led to energization of wild-type mitochondria, was without effect on the brown fat mitochondria from UCP1-ablated mice. The absence of thermogenic function did not result in reintroduction of high ATP synthase activity. Remarkably and unexpectedly, the mitochondria from UCP1-ablated mice were as sensitive to the de-energizing ("uncoupling") effect of free fatty acids as were UCP1-containing mitochondria. Therefore, the de-energizing effect of free fatty acids does not appear to be mediated via UCP1, and free fatty acids would not seem to be the intracellular physiological activator involved in mediation of the thermogenic signal from the adrenergic receptor to UCP1. In the UCP1-ablated mice, Ucp2 mRNA levels in brown adipose tissue were 14-fold higher and Ucp3 mRNA levels were marginally lower than in wild-type. The Ucp2 and Ucp3 mRNA levels were therefore among the highest found in any tissue. These high mRNA levels did not confer on the isolated mitochondria any properties associated with de-energization. Thus, the mere observation of a high level of Ucp2 or Ucp3 mRNA in a tissue cannot be taken as an indication that mitochondria isolated from that tissue will display innate de-energization or thermogenesis.     

Brown adipose tissue mitochondria: recoupling caused by substrate level phosphorylation and extramitochondrial adenosine phosphates.

1. Uncoupled oxidative phosphorylation in isolated guinea pig brown-adipose-tissue mitochondria is reflected by a low phosphorylation state of adenosine phosphates in the mitochondrial matrix and in the extramitochondrial space during oxidation of succinate or glycerol 1-phosphate in the presence of serum albumin and 100 muM ADP. Recoupling of respiration and phosphorylation in the mitochondria is indicatdd by a dramatic increase in the phosphorylation state of adenine nucleotides in both compartments, when substrates inducing substrate level phosphorylation are respired. In this case ATP/ADP ratios in the extramitochondrial compartment are 10-15 times higher than in the mitochondrial matrix. 2. Recoupling mediated by substrate level phosphorylation depends on the presence of extramitochondrial adenosine phosphate and on intact adenine nucleotide translocation. In the presence of substrate level phosphorylation the amount of extramitochondrial ADP required to restore energy coupling can be extremely low (20 muM ADP or 10 nmol ADP/mg mitochondrial protein respectively). If substrate level phosphorylation is prevented by rotenone or in the presence of atractyloside, 20-50 times higher amounts of extramitochondrial adenine nucleotides are necessary to cause coupled oxidative phosphorylation. The recoupling effect of ATP is significantly stronger than that of ADP. 3. GDP (100 muM) causes a rapid increase of the ATP/ADP ratio in both compartments which is independent of substrate level phosphorylation as well as of the extramitochondrial adenosine phosphate concentration and the adenine nucleotide carrier. 4. The amount of extramitochondrial adenosine phosphate in guinea pig brown-adipose-tissue (18 nmol/mg mitochondrial protein or 2.5 mM respectively) would suffice for recoupling of oxidative phosphorylation mediated by substrate level phosphorylation under conditions in vitro; this suggests that substrate level phosphorylation is of essential importance in brown fat in vivo with respect to energy conditions in the tissue during different states of thermogenesis.     

MITOCHONDRIAL CHANGES IN THE LIVER OF ESSENTIAL FATTY ACID-DEFICIENT MICE

Livers of mice on diets deficient in essential fatty acids (EFA) have been studied by light and electron microscopy. The most conspicuous changes occur in the mitochondria. In light microscopy the mitochondria appear very much enlarged in the periportal region of the lobule. In electron micrographs they have additional cristae, sometimes very abundant. ranged in stacks in the central cavity. The matrix may be more electron-opaque than normal. This is in contrast with the enlarged mitochondria appearing under other experimental conditions, where the cristae are reduced in number and the matrix is less electron-opaque. It is known that there is an uncoupling of oxidative phosphorylation in EFA-deficient mitochondria. As a hypothesis it is proposed that the uncoupling may be due to a molecular defect caused by the absence of EFA in the structure that determines the spatial relationship between the electron transport chain and oxidative phosphorylation. It is further tentatively suggested that the changes in mitochondria may be attributed to lack of ATP. The possibility is discussed that the mitochondrial changes are ineffective attempts at compensation for this lack.     

UCP1 and defense against oxidative stress. 4-Hydroxy-2-nonenal effects on brown fat mitochondria are uncoupling protein 1-independent

Uncoupling proteins have been ascribed a role in defense against oxidative stress, particularly by being activated by products of oxidative stress such as 4-hydroxy-2-nonenal (HNE). We have investigated here the ability of HNE to activate UCP1. Using brown fat mitochondria from UCP1+/+ and UCP1-/- mice to allow for identification of UCP1-dependent effects, we found that HNE could neither (re)activate purine nucleotide-inhibited UCP1, nor induce additional activation of innately active UCP1. The aldehyde nonenal had a (re)activating effect only if converted to the corresponding fatty acid by aldehyde dehydrogenase; the presence of a carboxyl group was thus an absolute requirement for (re)activation. The UCP1-dependent proton leak was not increased by HNE but HNE changed basal proton leak characteristics in a UCP1-independent manner. In agreement with the in vitro results, we found, as compared with UCP1+/+ mice, no increase in HNE/protein adducts in brown fat mitochondria isolated from UCP1-/- mice, irrespective of whether they were adapted to thermoneutral temperature (30 degrees C) or to the cold (4 degrees C). The absence of oxidative damage in UCP1-/- mitochondria was not due to enhanced activity of antioxidant enzymes. Thus, HNE did not affect UCP1 activity, and UCP1 would appear not to be physiologically involved in defense against oxidative stress. Additionally, it was concluded that at least in brown adipose tissue, conditions of high mitochondrial membrane potential, high oxygen tension, and high substrate supply do not necessarily lead to increased oxidative damage.     

Regulation of the uncoupling protein in brown adipose tissue

Presumptive evidence suggests that the brown fat mitochondrial uncoupling protein, thermogenin, is involved in the mechanism of stimulation of respiration by norepinephrine in the intact tissue. Conflicting data have been reported which suggest involvement of either adenine nucleotides, or fatty acids, or long chain acyl-CoA, or protons in the physiological regulation. We measured the electrical potential gradient across the mitochondrial membrane (delta psi m) in control cells and in cells stimulated with norepinephrine, using the accumulation of lipophilic cation, tetraphenylphosphonium, as an indicator of the potential gradient. The value of delta psi m in the cells in the control state is 116 mV, and in the hormonally stimulated state it is 56.6 mV. This supports the view that the protein is involved in the mechanism of hormone action. Other studies were designed to distinguish between the effects of fatty acids and ATP levels on the uncoupling protein in isolated mitochondria and in the adipocytes. ATP levels and fatty acid levels inside intact cells were independently varied using oligomycin or external fatty acids. Their effect on thermogenin was monitored as the capacity of the cells for reverse electron transport from durohydroquinone. The results suggest that ATP modulates the activity of thermogenin, while fatty acids can alter the relationship between ATP and thermogenin activity such that the protein appears to be activated at a higher cellular ATP level in the presence of fatty acids than in their absence.     

Fatty acid circuit as a physiological mechanism of uncoupling of oxidative phosphorylation.

Free fatty acids, natural uncouplers of oxidative phosphorylation, are shown to differ from artificial ones in that they fail to increase conductance of phospholipid bilayers which are permeable for the protonated form of fatty acids but impermeable for their anionic form. Recent studies have revealed that uncoupling by fatty acids in mitochondria is mediated by the ATP/ADP antiporter and, in brown fat, by thermogenin which is structurally very similar to the antiporter. It is suggested that both the ATP/ADP antiporter and thermogenin facilitate translocation of the fatty anions through the mitochondrial membrane.     

Organization and regulation of fatty acid oxidation in mitochondria of brown adipose tissue

In mitochondria from brown adipose tissue no GTP-dependent oleate synthetase could be detected. The ATP-dependent acyl-CoA synthetase is probably the only enzyme involved in oleate oxidation.

In the absence of added carnitine, oleate oxidation proceeds only in the presence of malate and of phosphate, and only for a few minutes. This is apparently due to the very low ATP/AMP ratio which is unfavourable for the synthetase activity. The substrate level phosphorylation supports continuous oleate oxidation by dramatically increasing the ATP/AMP ratio. This effect probably depends on the transphosphorylation of GTP with AMP.

In the presence of added carnitine, oleate oxidation is stimulated by phosphate and is inhibited by atractyloside. These effects are probably due to a direct action of these substances on the ATP-dependent synthetase.     

Quantification of fatty acid activation of the uncoupling protein in brown adipocytes and mitochondria from the guinea-pig

Brown adipocytes from cold-adapted guinea-pigs (C-cells) are more sensitive to uncoupling by exogenous palmitate than are cells from warm-adapted animals (W-cells) with much less uncoupling protein. Half-maximal respiratory stimulation of C-cells requires 80 nM free palmitate. Noradrenaline-stimulated lipolysis is not rate-limiting for the respiration of either C-cells or W-cells. Half-maximal stimulation of fatty acid oxidation by mitochondria from warm-adapted guinea-pigs (W-mitochondria) and cold-adapted guinea-pigs (C-mitochondria) both require 12 nM free palmitate. Palmitate uncouples C-mitochondria much more readily than M-mitochondria, paralleling its action on the adipocytes. The uncoupling is partially saturable, about 100 nM free palmitate being required for half-maximal response of C-mitochondria. W- and C-mitochondria show identical binding characteristics for palmitate. The respiratory increase of mitochondria is calculated as a function of bound palmitate. After correcting for the residual uncoupling protein present in W-mitochondria, palmitate is estimated to be almost ineffective as an uncoupler of brown fat mitochondria in the absence of the protein. It is concluded that fatty acids display characteristics required of a necessary and sufficient physiological activator of the uncoupling protein.     

Influence of mitochondrial membrane fatty acid composition on proton leak and H2O2 production in liver

Mitochondrial membrane fatty acid composition has been proposed to play a role in determining mitochondrial proton leak rate. The purpose of this study was to determine if feeding rats diets with different fatty acid sources produces changes in liver proton leak and H(2)O(2) production. Six-month-old male FBNF(1) rats were fed diets with a primary fat source of either corn or fish oil for a 6-month period. As expected, diet manipulations produced substantial differences in mitochondrial fatty acid composition. These changes were most striking for 20:4n6 and 22:6n3. However, proton leak and phosphorylation kinetics as well as lipid and protein oxidative damage were not different (P > 0.10) between fish and corn oil groups. Metabolic control analysis, however, did show that control of both substrate oxidation and phosphorylation was shifted away from substrate oxidation reactions to increased control by phosphorylation reactions in fish versus corn oil groups. Increased mitochondrial H(2)O(2) production was observed in corn versus fish oil-fed rats when mitochondria were respiring on succinate alone or on either succinate or pyruvate/malate in the presence of antimycin A. These results show that mitochondrial H(2)O(2) production and the regulation of oxidative phosphorylation are altered in liver mitochondria from rats consuming diets with either fish or corn oil as the primary lipid source.     

Alkylsulfonates activate the uncoupling protein UCP1: implications for the transport mechanism

Fatty acids activate the uncoupling protein UCP1 by a still controversial mechanism. Two models have been put forward where the fatty acid operates as either substrate ("fatty acid cycling hypothesis") or prosthetic group ("proton buffering model"). Two sets of experiments that should help to discriminate between the two hypothetical mechanisms are presented. We show that undecanosulfonate activates UCP1 in respiring mitochondria under conditions identical to those required for the activation by fatty acids. Since alkylsulfonates cannot cross the lipid bilayer, these experiments rule out the fatty acid cycling hypothesis as the mechanism of uncoupling. We also demonstrate that without added nucleotides and upon careful removal of endogenous fatty acids, brown adipose tissue (BAT) mitochondria from cold-adapted hamsters respire at the full uncoupled rate. Addition of nucleotides lower the respiratory rate tenfold. The high activity observed in the absence of the two regulatory ligands is an indication that UCP1 displays an intrinsic proton conductance that is fatty acid-independent. We propose that the fatty acid uncoupling mediated by other members of the mitochondrial transporter family probably involves a carrier to pore transition and therefore has little in common with the activation of UCP1.     

Barbara Cannon's Data on the UCP1-Ablated Mice: “Non-Cannonical” Point of View

The data of Cannon and co-workers on UCP1-ablated mice are interpreted assuming that UCP2 and UCP3 are involved in thermoregulation as fatty acid-dependent uncouplers although they are not sufficient, in the absence of UCP1, for long term maintenance of normal body temperature of mice after sudden and strong decrease in the ambient temperature. I would like to suggest that in brown fat of control mice, UCP1 is present in an amount higher than UCP2 and 3 and, therefore, is able to cause (a) some fatty acid-mediated decrease in proton motive force in resting state and, hence, (b) oxidation of CoQH2 to CoQ which is shown by Klingenberg and coworkers to be cofactor for UCPs. This results in strong uncoupling and thermogenesis mediated by UCP1, 2 and 3. In the UCP1-ablated mice, activity of UCP2 and 3 appears to be insufficient to induce CoQH2 oxidation in resting brown fat mitochondria, which results in hypothermia.     

Development of brown fat cells in monolayer culture. II. Ultrastructural characterization of precursors, differentiating adipocytes and their mitochondria

The stroma of mature brown fat has been shown to contain cells which can proliferate and accumulate fat in monolayer cultures, and which have inherent characteristics distinct from those of white fat precursor cells. The purpose of the present investigation was to characterize by electron microscopic analysis these brown fat cells and their subsequent development when they were grown in vitro. By comparison with the existing ultrastructural data on brown fat in situ, it could thus be determined whether or not the precursor cells have the capacity to differentiate in culture. The stromal-vascular fraction isolated from the brown fat of weaned rats was identified as containing adipocyte stem cells, preadipocytes, endothelial cells and a few mature adipocytes. During the first week in culture (i.e., growth phase to confluence), when multilocular fat accumulation occurred, the mitochondria of the preadipocytes developed cristae and matrix granules, as they do in differentiating brown fat in situ. Such granules have been shown to be a sign of intense inner membrane synthetic activity. After confluence, the mitochondria regressed in internal structure and became morphologically more similar to white fat mitochondria. It was concluded that mature brown fat contains precursor cells which can differentiate in vitro. However, this differentiation was incomplete, and the necessity of specific factors for a full mitochondrial development in brown fat is discussed.     

The oxidation of long-chain unsaturated fatty acids by isolated rat liver mitochondria as a function of substrate concentration.

1. The oxidation of linoleate by rat-liver mitochondria has been studied as a function of substrate concentration. The oxidation of other long-chain unsaturated fatty acids shows similar characteristics. 2. At low concentrations, linoleate is readily oxidized in the absence of carnitine. Its rate of activation by the intramitochondrial acyl-CoA synthetase (EC 6.2.1.2) and subsequent oxidation is limited by the availability of intra-mitochondrial ATP. 3. A gradual increase of the linoleate concentration leads to (i) a strong depression of the rate of linoleate oxidation, and (ii) uncoupling of respiratory-chain phosphorylation together with induction of a mitochondrial ATPase activity. At still higher linoleate concentrations this ATPase activity is lowered rather than further stimulated and, concomitantly, the rate of linoleate oxidation increases again. 4. Evidence is presented that the inhibition by linoleate of the ATPase activity occurs at the level of the ATPase complex itself. This oligomycin-like effect of linoleate allows intramitochondrial linoleate activation to take place at the expense of ATP derived from substrate-level phosphorylation. 5.At very high concentrations of linoleate, its detergent action predominates and causes a complete inhibition of respiration as well as an extensive stimulation of an oligomycin-insensitive, Mg2+-dependent ATPase activity. 6. Measurement of the binding of radioactively labelled linoleate by isolated mitochondria shows that, at a given ratio of linoleate to mitochondrial protein, the ratio of bound to added linoleate is dependent on the concentration of the mitochondria.     

Physiological regulation of the transport activity in the uncoupling proteins UCP1 and UCP2

Brown fat is a thermogenic organ that allows newborns and small mammals to maintain a stable body temperature when exposed to cold. The heat generation capacity is based on the uncoupling of respiration from ATP synthesis mediated by the uncoupling protein UCP1. The first studies on the properties of these mitochondria revealed that fatty acid removal was an absolute prerequisite for respiratory control. Thus fatty acids, that are substrate for oxidation, were proposed as regulators of respiration. However, their ability to uncouple all types of mitochondria and the demonstration that several mitochondrial carriers catalyze the translocation of the fatty acid anion have made them unlikely candidates for a specific role in brown fat. Nevertheless, data strongly argue for a physiological function. First, fatty acids mimic the noradrenaline effects on adipocytes. Second, there exists a precise correlation between fatty acid sensitivity and the levels of UCP1. Finally, fatty acids increase the conductance by facilitating proton translocation, a mechanism that is distinct from the fatty acid uncoupling mediated by other mitochondrial carriers. The regulation of UCP1 and UCP2 by retinoids and the lack of effects of fatty acids on UCP2 or UCP3 are starting to set differences among the new uncoupling proteins.     

Effect of chronic ethanol ingestion on fatty acid oxidation by hepatic mitochondria.

To study possible factors in the pathogenesis of the ethanol-induced fatty liver, we investigated the effect of chronic ethanol consumption on the metabolism of fatty acids by isolated hepatic mitochondria. Chronic ethanol consumption resulted in decreased fatty acid oxidation, as evidenced by a reduction in oxygen uptake and CO2 production associated with the oxidation of fatty acids. The State 3 rate of oxygen uptake was depressed to a greater extent than the State 4 or the uncoupler-stimulated rate; the respiratory control ratio was also decreased. Therefore, one site of action of chronic ethanol feeding is on oxidative phosphorylation. The reduction in fatty acid oxidation, in general, is not due to an effect on the activation or translocation of fatty acids into the mitochondria. There was no effect by ethanol feeding on the activity of palmitoyl coenzyme A synthetase, whereas carnitine palmitoyltransferase activity was increased. The use of an artificial system (formazan production) to study beta oxidation in the absence of the electron transport chain is described. In the presence of fluorocitrate, which inhibits citric acid cycle activity, ketogenesis and formazan production were increased by chronic ethanol consumption. Thus beta oxidation to the level of acetyl-CoA is not impaired by chronic ethanol consumption. Total oxidation of fatty acids to CO2 is depressed by chronic ethanol intoxication because of effects on oxidative phosphorylation or the citric acid cycle (or both). Neither nutritional deficiency, cofactor depletion, nor the presence of ethanol in vitro explains these effects. Several of the effects of chronic ethanol consumption on fatty acid oxidation are mimicked by acetaldehyde and acetate, products of ethanol oxidation. Chronic ethanol consumption leads to persistent impairment of mitochondrial oxidation of fatty acids to CO2. However, oxidation of fatty acids to acetyl-CoA is not decreased by chronic ethanol consumption.     

Uncoupling effect of fatty acids on heart muscle mitochondria and submitochondrial particles.

The effect of ATP/ADP-antiporter inhibitors on palmitate-induced uncoupling was studied in heart muscle mitochondria and inside-out submitochondrial particles. In both systems palmitate is found to decrease the respiration-generated membrane potential. In mitochondria, this effect is specifically abolished by carboxyatractylate (CAtr) a non-penetrating inhibitor of antiporter. In submitochondrial particles, CAtr does not abolish the palmitate-induced potential decrease. At the same time, bongkrekic acid, a penetrating inhibitor of the antiporter, suppresses the palmitate effect on the potential both in mitochondria and particles. Palmitoyl-CoA which is known to inhibit the antiporter in mitochondria as well as in particles decreases the palmitate uncoupling efficiency in both these systems. These data are in agreement with the hypothesis that the ATP/ADP-antiporter is involved in the action of free fatty acids as natural uncouplers of oxidative phosphorylation.     

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.     

Pleiotropic Effects of Cavin-1 Deficiency on Lipid Metabolism

Mice and humans lacking caveolae due to gene knock-out or inactivating mutations of cavin-1/PTRF have numerous pathologies including markedly aberrant fuel metabolism, lipodystrophy, and muscular dystrophy. We characterized the physiologic/metabolic profile of cavin-1 knock-out mice and determined that they were lean because of reduced white adipose depots. The knock-out mice were resistant to diet-induced obesity and had abnormal lipid metabolism in the major metabolic organs of white and brown fat and liver. Epididymal white fat cells from cavin-1-null mice were small and insensitive to insulin and β-adrenergic agonists resulting in reduced adipocyte lipid storage and impaired lipid tolerance. At the molecular level, the lipolytic defects in white fat were caused by impaired perilipin phosphorylation, and the reduced triglyceride accumulation was caused by decreased fatty acid uptake and incorporation as well as the virtual absence of insulin-stimulated glucose transport. The livers of cavin-1-null mice were mildly steatotic and did not accumulate more lipid after high-fat feeding. The brown adipose tissues of cavin-1-null mice exhibited decreased mitochondria protein expression, which was restored upon high fat feeding. Taken together, these data suggest that dysfunction in fat, muscle, and liver metabolism in cavin-1-null mice causes a pleiotropic phenotype, one apparently identical to that of humans lacking caveolae in all tissues.     

Uncoupling proteins UCP2 and UCP3 from mitochondria of liver and skeletal muscle of ground squirrel Spermophilus undulatus do not transport pyruvate in contrast to UCP1 from brown adipose tissue

Physiological role of mitochondrial uncoupling proteins UCP2 and UCP3, homologous to UCP1 from brown adipose tissue, is unclear. It was proposed recently that UCP2 and UCP3 are metabolic triggers that switch oxidation of glucose to oxidation of fatty acids, exporting pyruvate from mitochondria. In the present study we tried to verify this hypothesis using ground squirrels (Spermophilus undulatus), since expression of all UCPs in different tissues increases during winter season, and UCP1 is abundant in brown fat. We confirmed the possibility of nonspecific transport of pyruvate through UCP1 in brown fat mitochondria and tried to identify similar transport in liver and skeletal muscle mitochondria where UCP2 and UCP3 are expressed. Transport of pyruvate mediated by UCP1 in mitochondria of brown fat was observed using valinomycin-induced swelling of non-respiring mitochondria in 55 mM potassium pyruvate and was inhibited by GDP. In contrast, mitochondria of liver and skeletal muscles in similar conditions did not exhibit electrogenic transport of pyruvate anions that could be related to functioning of UCP2 and UCP3. At the same time, functioning of pyruvate carrier was detected in these mitochondria by nigericin-induced passive swelling or valinomycin-induced active swelling in potassium pyruvate that was inhibited by α-CHC, a specific inhibitor of the pyruvate carrier. Thus, our results suggest that in contrast to UCP1 of brown fat, UCP2 and UCP3 from intact liver and skeletal muscle mitochondria of winter active ground squirrels are unable to carry out pyruvate transport.