the fluidity and organization of mitochondrial membrane lipids of the brown adipose tissue of...

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 167, 505-518 (1375)

The Fluidity and Organization of Mitochondrial Membrane Lipids of

the Brown Adipose Tissue of Cold-Adapted Rats and Hamsters as

Determined by Nitroxide Spin Probes’

BARBARA CANNON’

Department of Biochemistry, Chelsea College, London S W3 6LX, Engkznd

CARL F. POLNASZEK AND KEITH W. BUTLER

Division of Biological Sciences, National Research Council of Canada, Ottawa, Canada KlA OR6

L. E. GijRAN ERIKSSON

Department of Biophysics, University of Stockholm, S-10405 Stockholm 50, Sweden

AND

IAN C. P. SMITH

Division of Biological Sciences, National Research Council of Canada, Ottawa, Canada KlA OR6

Received September 3, 1974

A detailed study of lipid fluidity and organization in the mitochondria of the brown adipose tissue from warm- and cold-adapted rats (nonhibernators) and hamsters (hibernators) is made in order to delineate any relationship between lipid properties and the ability to lower body temperature after cold-adaptation. Complete phospholipid analyses are presented; the data are very similar for cold- and warm-adapted rats, and for cold- and warm-adapted hamsters, but the rat lipids have a higher degree of unsaturation than those of the hamsters. Spin probe analogs of stearic acid and cholestane were used to investigate at the molecular level the fluidity and order of the mitochondrial lipids. Studies were made on intact mitochondria, and in liposomes and oriented multibilayers of extracted lipids. In no case was evidence found for a phase transition in the lipids, or for a relationship between the lipid fluidity in brown adipose tissue mitochondria and the ability to survive at lowered body temperatures. The spin probes generally had a decreased mobility in mitochondria relative to extracted lipids. The electron spin resonance spectra were analyzed to include order- and time-dependent phenomena by a recent stochastic method. The results show that more approximate analyses for order parameters and correlation times can yield incorrect conclusions. As segmental motion decreases in rate, order parameters will be overest.imated. Decreasing rates of pseudoisotropic motion lead to incorrect estimates of rotational correlation times. Either of the above can result in the inference of an artifactual phase transition in the lipids.

Brown adipose tissue has a well-docu- tal mammals, in certain cold-adapted ro- mented function as a thermogenic organ in dents and in hibernators (l-6). The heat is a large number of mammalian species. It derived from fatty acid oxidation in the functions as a.n extra heat source in neona- mitochondria (7-9). It is probable that the

high rates of respiration observed in the 1 Issued as NRRC Publication Number 14501. tissue undergoing thermogenesis are made 2 Permanent address: Wenner-Gren Institute, Uni- possible by an uncoupling of this respira-

versity of Stockholm, S-11345 Stockholm, Sweden. tion from the constraints of ATP synthesis Visiting Research Officer, NRRC, 1974. (7-9).

505 Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.

506 CANNON ET AL.

A notable characteristic of animals which hibernate, and which provides a definition of such animals, is that during the hibernation period the body temperature drops close to 0°C (10-11). In this respect, therefore, hibernators, while normally behaving as homeothermic animals, have the ability to function as poikilotherms. Homeothermic animals may re- ceive irreparable tissue damage should their body temperature drop to below about 20°C (10, 11). In recent years it has been determined that the membranes of cells and subcellular organelles of poikilothermic animals differ from those of homeotherms with respect to the fatty acid composition of the membrane phospholipids. Poikilotherms show a higher degree of unsaturation than homeotherms (12). In addition to this they do not show any discontinuity in activation energy when Arrhenius plots are made of the rate of a membrane-bound enzyme activity as a function of temperature. Homeothermic animals exhibit discontinuities, with higher activation energies at temperatures below the discontinuity than at those above (13). With the use of nitroxide- labeled stearic acid spin probes, a corre- spondence has been observed between the temperatures at which discontinuities are found and those at which abrupt changes are seen in the estimated rotational correlation times of the spin probes (14). The conclusion has been that the membrane lipids of homeothermic animals undergo a phase change at the temperature of the discontinuity, this, at least in part, as a consequence of the higher degree of satura- tion of the phospholipid fatty acids. The membrane lipids of poikilothermic animals, however, remain in the more fluid, liquid-crystalline phase over the whole temperature range studied (14).

It was expected that the mitochondria of cold-adapted hamsters which readily hibernate should show characteristics similar to those from poikilothermic animals. This was found to be so when studying respiratory activity as a function of temperature in isolated heart mitochondria (15). Con- trasting behaviour was found in studies of mitochondria isolated from the brown adipose tissue. In the high energy state (16),

respiratory activity and permeability to monovalent anions showed discontinuities in Arrhenius plots, with markedly lower activation energies at temperatures below the discontinuities (15).

The present investigation has been per- formed in an attempt to evaluate the effect of membrane fluidity and organization on the activities of several mitochondrial membrane functions in brown adipose tissue. The spin probe method has proven to be sensitive to fluidity and order in lipid systems and biological membranes (17). Therefore, a variety of lipid spin probes were used to investigate different areas of the membranes, and comparisons were made between warm- and cold-adapted hibernators (hamsters) and warm- and cold-adapted non-hibernators (rats). The paper makes a critical assessment of earlier types of spin probe analysis, using the approach described recently by Polnaszek (18). It also applies the stochastic theory of Freed et al. (17, 19-21) to a biological system for the first time. No evidence is found for a lipid phase transition in any of the samples investigated, although using less rigorous methods of spectral analysis such a transition might have been inferred in some cases. All samples showed great similarity with respect to temperature dependence of spin probe motion. Cold adaptation induces no striking alteration in the membrane phospholipid fatty acid composition in either hamster or rat brown adipose tissue mitochondria as compared with the warm-adapted controls. The hamster and rat samples, however, differ from one another in the percentages of C,,:, and C,,:, fatty acids.

MATERIALS AND METHODS

Mitochondria were isolated from pooled inter- scapular, cervical and axillary brown adipose tissue excised from either rats or golden hamsters. The animals designated as cold-acclimatized had been kept for at least 21 days at 4°C f&l”) with food and water ad lib&m. The control warm-acclimatized animals were maintained at room temperature. The rats used in the latter case weighed ca. 70 g. The animals were decapitated and the tissue rapidly excised and placed into 0.25 M sucrose at 0°C. The mitochondria were then prepared as previously described for the mitocbondria of rat brown adipose tissue (22). Protein was determined by the biuret

BROWN FAT MITOCHONDRIAL LIPID SPIN PROBE STUDY 507

method (23) and the mitochondria stored at about 50 mg mitochondrial protein/ml on ice.

Mitochondrial lipids were extracted according to the method of Bligh and Dyer (24), after initial addition of methanol. Solvents used were bubbled with N, prior to use, and the extractions were per- formed on ice under an atmosphere of N,. The CHCl, phase was filtered, concentrated and stored under N, at -20°C.

The spin probes (Synvar, Palo Alto, CA) were the N-oxyl-4’,4’-dimethyloxazolidine derivatives of 5- and 1Bketostearic acid, here called 5- and 12-doxyl- stearic acid, and the methyl ester of the above 5-doxyl acid, i.e., methyl 5-doxyl-stearate. The analogous derivative of cholestane-3-one (3-doxyl cholestane) was synthesized by the method of Keana et al. (25). The stearic acid spin probes were dissolved in 100 mM

K-TESS pH 7.4 when used in experiments with intact mitochondria and were then used at a final concentration of 4 x 1(lm5 M, being equivalent to 5 x 10e9 mol/mg mitochondrial protein. The spin probe to mitochondrial phospholipid ratio was then approxi- mately 1:50 to L:lOO. Rotenone was added in ethanol to a final concentration of 10 PM. The final mitochondrial concentration was 8 mg/ml, dilution having been made with 100 mM KCI, 20 mM K-TES, pH 7.4. The samples were agitated and stored on ice. Aliquots were removed and each was used to monitor no more than three temperatures.

The following procedure was employed for experiments using liplosomes. A sample of extracted mitochondrial lipid in CHCl, was mixed with the spin probe in CHCl, at concentrations equivalent to the above, evaporated to dryness under Nz and pumped in uacuo overnight. Subsequently, 100 mM KCl, 20 mM K-TES, pH: 7.4 was added, Nz blown over the surface, the tube capped and the sample vigorously agitated, followed by sonication for ca. 10 min in an ice-cooled 40W water bath sonicator (Heat Systems, Plainview, NY). The samples were stored on ice.

To form planar multibilayers a chloroform solution of the mitochondrial lipid plus spin probe (ratio as above) was evaporated in a large es? cell for aqueous samples (J. F. Scanlon, Whittier, CA) with a stream of wet nitrogen, followed by drying in uacuo for at least 2 h. The films were hydrated in 100 mM KCI, 20 mM K-TES, pH 7.4 for a minimum of 20 min and the cells were then drained.

Electron spin resonance spectra of all samples were taken on a Varian E-9 spectrometer equipped with an 6K 620i computer and a Varian temperature control- ler. For the large sample cells a home-built sample holder (K. W. Butler, unpublished) was necessary in variable temperature experiments.

‘Abbreviations used: TES, N-tris (hydroxyme- thyllmethyl-2-amineethane sulphonic acid: esr, electron spin resonance; DEGS, diethylene glycol succi- nate.

Thin layer chromatography of the lipid extracts was done on precoated silica gel G plates (Merck), developed in CHCl,:CH,OH:NH, (30%) (65:25:4). Visualisation was with rhodamine 6G. The individual phospholipid classes were eluted from the silica gel with CHCl,:CH,OH 1:1, followed by CHJOH alone. A fraction of the eluant was evaporated to dryness in uacuo, hydrolysed in 0.5N methanolic KOH until the residue went into solution, cooled and methylated with 14% boron trifluoride in methanol. The methylated derivatives were extracted by shaking with water and petroleum ether (30”~60°C). The petroleum ether phase was evaporated to dryness in uacuo, dissolved in a suitable volume of CHCl, for injection into a Victoreen series 4000 gas chromatograph with a col- umn of 10% DEGS3 at 165°C. Phospholipid phos- phorus was determined by the method of King (26).

Apparent correlation times for isotropic rotation of the spin probes were calculated using the esr motional narrowing formalisms (27-29) from the spectra of methyl 5-doxyl-stearate in liposomes at temperatures above 15°C and from the spectra of the cholestane spin probe in multibilayers oriented parallel to the magnetic field of the spectrometer. The peak-to-peak linewidth of the centre line was measured and the E and C terms in Eq. (I) were calculated (18) from the relative amplitudes of the three principal resonances (27-29).

AH,,, = A + Bm + Cm2 (1)

where AH,,, is the width of the hyperfine line due to the mth component of the ‘“N nuclear spin (m = *LO),

A = AH, (2)

B= T [($)* - ($)‘I (31

c= $5 [($)” + (L$ -21 (4)

where the I,,, are the peak-to-peak amplitudes of the first derivative resonances. Hence,

7 = -1.18 X lo-9B (51

or

T = 1.31 x lo-9c (‘3

using the g and hyperfine parameters specified in the caption to Fig. 8. The spectra of 5-doxyl-stearic acid in either liposomes or mitochondria were analyzed by the usual order parameter formalism (30, 31) which assumes rapid motion of ordered spin probes in a vesicle. A detailed discussion of analyses of these types of spectra, and their limitations, is given by Polnaszek (18) and therefore will not be described here.

508 CANNON ET AL.

The partition of the probe between the lipid and aqueous phases was determined from the spectra of 12-doxyl-stearic acid. The concentration of the probe in the aqueous phase was determined at each temperature by comparison of the absolute amplitude of the high field liquid line in the experimental spectra with the absolute amplitude of the high field line of a standard consisting of 12-doxyl-stearic acid dissolved in the same buffer used in the experiment. The total spin probe concentration responsible for the experimental spectra was either calculated from the amount of probe added or measured by double integration of the standard and experimental spectra. In either case the percentage of probe in aqueous solution is given by

absolute amplitude of free probe spin concentration

resonance standard X x 100.

absolute ampli- total spin concen- tude of corre- tration in sample

sponding standard

Qualitatively similar results were obtained by mea- suring the area under the high field liquid line and the total area of the spectrum by planimetry.

RESULTS AND DISCUSSION

Phospholipid Composition of the Mitochondria From Brown Adipose Tissue

The phospholipid and fatty acid composition of brown adipose tissue mitochondria from cold- and warm-adapted hamsters and rats are shown in Table I. Virtu- ally no differences due to cold adaptation of either hamsters or rats are found in total fatty acids, neither are there any changes in distribution of fatty acid residues within the individual phospholipid classes. These results imply that the fatty acid composition of membrane phospholipids is primar- ily influenced by diet and genetics in these animals, and not by environmental temperature. They are very similar to results obtained from mitochondria of other tis- sues obtained from animals kept at normal temperatures (32-34). This is particularly important for hamsters, which, once cold- adapted, can lower their body temperature to almost 0°C. There appears, therefore, to be little influence of fatty acid composition in the mitochondria of brown adipose tissue on the ability of halzsters to survive at lowered body temperature. There was a significantly decreased percentage of Cz,:, and C,,:, and concomitantly increased per-

centage of C&, in the hamsters when compared with the rats. This change would be expected to give less fluid lipids in hamsters than in rats, a conclusion borne out in the esr results (uide infra). This conclusion is of particular significance for discussions involving the role of membrane lipid fluidity in survival at low body temperatures (12-14). A relatively decreased lipid fluidity in the mitochondria of the brown adipose tissue of hamsters cannot obviously be disadvantageous for hibernation.

Methyl 5-Doxyl-stearate in Liposomes

Liposomes were prepared from mitochondrial lipid from the brown adipose tissue of cold- and warm-adapted rats and hamsters. The esr spectra obtained with the methyl ester of 5doxyl-stearic acid in these liposomes above ca. 15°C allowed calculation of correlation times via Kivel- son’s method (27), Eqs. (5) and (6). At temperatures at or below 15°C the observed spectra (Fig. la) were no longer composed of three narrow lines and may be indicative either of motion sufficiently slow that the motional narrowing formalism is no longer valid or that the ordering has be- come sufficiently large to affect the observed spectra significantly. (Note the ap- pearance of other components at the high and low field extremities of the spectrum in Fig. la.) The calculation of correlation times using standard formulas (27-29) is only valid assuming rapid isotropic motion of the spin probe where the system under study is not imposing order on the probe. Spectra obtained above ca. 15°C resem- bled those expected for isotropic motion in that they had three distinct lines and gave correlation times from the B term, Eq. (5), in the range 1.0-3.8 x lo-’ s (Fig. 2a-d). It is common practice currently to measure arbitrary motional parameters even from spectra such as Figure la. Such spectra are clearly due to motion of an intermediate rate and/or partial ordering (17-21), (uide infra). To make our point, correlation times were calculated from the spectra taken below 15”C, and are included in the Arrhenius plot in Fig. 2. The apparent


TABLE I FATIY ACID COMPOSITIOND OF THE PHOSPHOLIPIDS OF MITOCHONDRIA OF BROWN ADIPOSE TISSUE

Cold-adapted rats

Phosphatidyl choline Phosphatidyl ethanolamine Cardiolipin

c1,:o cm, c,m cm, cm cm3 cm:, c,,:, %JJ

8.8 0.7 10.3 5.3 10.6 0.7 5.7 - 41.7 3.5 0.4 12.6 4.2 6.7 1.9 11.0 6.4 46.3 2.3 0.2 1.7 2.2 4.6 0.3 0.8 - 11.9

Total 14.6 1.3 24.6 11.6 21.9 2.9 17.5 6.4

Warm-adapted rats cm c,,:, Cl.,, Cl.:, C,.:, Cl83 Cm:, cm. %P

Phosphatidyl choline 9.7 0.7 7.1 6.0 10.5 1.1 4.9 - 39.7 Phosphatidyl ethanolamine 4.5 0.7 14.3 7.8 6.0 1.2 11.1 3.9 49.1 Cardiolipin 1.3 0.1 1.7 2.0 5.4 0.3 0.6 - 11.2

Total 15.5 1.5 23.1 15.8 21.9 2.6 16.6 3.9

Cold-adapted hamsters c,m CM1 C,.:, Cl,.? c,,:, C,,:, cm:, C226 %P

Phosphatidyl choline 9.8 1.6 4.6 9.8 10.0 2.3 1.1 - 38.9 Phosphatidyl ethanolamine 2.4 1.4 15.4 15.0 10.9 0.8 6.9 0.9 53.1 Cardiolipin 0.9 0.4 0.5 2.0 4.0 0.3 - - 8.0

Total 13.1 3.4 20.5 26.8 24.9 3.4 8.0 0.9

Warm-adapted hamsters C,,:, Cl,,, Cl,:, cm:, Cl&? c1s:s C,,:, cm. %P

Phosphatidyl choline 9.9 1.0 5.2 8.6 11.5 1.4 1.1 - 38.5 Phosphatidyl ethanolamine 3.5 0.9 16.3 16.1 11.6 3.1 3.5 - 54.5 Cardiolipin’ 0.9 - 1.7 0.9 1.6 0.2 0.4 - 7.0

Total 14.3 1.9 23.2 25.6 24.7 4.7 5.0 -

*The estimated accuracy of these determinations is +l%. bin the cardiolipin fraction an extra peak was observed between those due to C1,:, and C,,,, accounting for

1.5 weight %.

discontinuity at 15°C is clearly due to breakdown of the approximations made in calculating correlation times, which would obscure changes due to a phase transition in the lipids. Consideration of the variation of the order parameter of 5-doxyl stearic acid with temperature (uide infra) demon- strates that in fact no phase transition occurs.

We must emphasize that since the correlation times calculated from B in Eq. (5) were not equal to those calculated from C in Eq. (6) (Fig. 2) it must be concluded that the motion of the probe at all temperatures studied is either anisotropic and/or

the system is ordered (see Polnaszek (18) for explanation). Another indication that the methyl 5-doxyl-stearate is ordered or rotating anisotropically can be obtained from the asymmetry of the line shapes of the esr spectra, even those at high temperatures (18). The asymmetry of a first- derivative esr resonance is defined as the ratio of the positive and negative derivative amplitudes. It is, therefore, not possible to ascribe any quantitative significance to the correlation times calculated from Eqs. (5) and (6) since the primar>f assumptions of rapid isotropic motion and no ordering are not upheld. The values can consequently

510 CANNON ET AL.

FIG. 1. (a) Experimental esr spectrum taken at 155°C of methyl 5-doxyl-stearate in liposomes of mitochondrial lipids from the brown adipose tissue of warm-adapted hamsters. (b) Theoretical esr spectrum for a spin probe in a vesicle calculated by the stochastic method (18). The input magnetic parameters are g, = 2.0087, g, = 2.0061, g, = 2.0027, a, = 6.5G, a, = 5.5G, a, = 32.OG. The resonance field, B,, for the centre line of a spectrum for a system possessing no order and fast motion is 3300G. The rotationally invariant peak to peak line-width is given as T,-' = T,: + T;: (3cos*fl - 1)/2 where 0 is the angle between the magnetic field and the nitroxide z-axis. For this spectrum T;: = LOG and T,l = LOG. The input order parameter S = 0.43. The correlation time for segmental motion of the chain is 5 x lo-” s and that for the overall rotat,ion of the entire chain is 3 x lO-‘s. This spectrum, and all others are simulated for Brownian rotational diffusion. (See text and Ref. 18.)

have at best, only qualitative significance, and at worst can lead to erroneous indica- tions of phase transitions.

Thus, from the analysis of the methyl Sdoxyl-stearate correlation time data, one cannot conclude that there is a phase transition in the region of the membrane probed by the nitroxide moiety in any of the samples. However, the esr spectra indicate that the rat lipids are more fluid than the hamster lipids, with the lipids from cold-adapted rats exhibiting a greater fluidity than those from warm rats.

At temperatures below ca. 15”C, as noted above, one should not use the motional narrowing formulas. However, one could not analyze the spectra using the order parameter formalism because the apparent degree of order is too low. Thus, the stochastic method (17, 19-21) was used for the simulation of vesicle spectra (18) including correlation times and an order parameter (35). In these stimulations the rapid segmental motion of the nitroxide moiety occurs simultaneously with the slow overall motion of the fatty acid ester. Because methyl 5-doxyl-stearate is not anchored at the polar interface, this overall motion is expected to contribute to the esr spectrum. When the rapid internal

, , . i I, 30 32 34

l/TAO3 36 32 34 36

l/T.103

FIG. 2. Arrhenius plots of correlation times of methyl 5-doxyl-stearate in liposomes of mitochondrial lipids from the brown adipose tissue of (a) cold-adapted rats; (b) warm-adapted rats; (c) cold-adapted hamsters; (d) warm-adapted hamsters. O--O correlation times calculated from the B term of Eq. (5). x-x correlation times calculated from the C term of Eq. (6). The solid lines in all the Arrhenius plots represent least square fits to the results.


segmental motion is much faster than the overall motion of the long chain, one can treat the motions separately and obtain correlation times for each motion. A typi- cal experimental spectrum for methyl 5- doxyl-stearate in vesicles of the lipids from warm-adapted hamsters at 15.5% is shown in Fig. la. Simulation of spectra similar to the above could be obtained when segmental motion of the fatty acid probe was sixty times faster than the overall motion of the long chain axis with an order parameter of 0.43 (Fig. lb). It should be noted that it is impossible to simulate the spectrum of Fig. la without inclusion of order, if the principle axis of rotation is ta.ken as the stearate chain axis.

It was also found possible to simulate spectra resembling those for higher temperatures (i.e., spectra with three distinct lines) merely by altering the input data for the spectrum simulated in Fig. lb for 15.5% Close fits for the experimental spectra at 28°C could be obtained by either reducing the order parameter from 0.43 to O-09, or by increasing the rates of rotational motion while maintaining the other parameters constant. Thus no unique solution could be obtained to allow explanation of the higher temperature spectra although decreasing the order parameter is more satisfactory. At the high temperatures, there was less asymmetry of the individual esr lines with the rat lipids than with the hamster lipids; this is an indication that the spin probe is less ordered in

the rat lipids (18). No differences between rat and hamster lipids were seen, however, in spectra taken at temperatures below 34°C. Very small differences in asymmetry of the line shapes existed between the cold- vs warm-adapted animals. The esr spectra of the lipids from cold-adapted hamster showed the greatest line shape asymmetry, again indicating a higher degree of order and/or anisotropy of the spin probe motion. From the small differences in the observed asymmetry in the outer hyperfine lines at the higher temperatures, and from the observation that the degree of order varies only slightly between systems (see 5-doxyl- stearic acid results), it must be concluded that there are measurable differences in the rates of motion.

5-Doxyl-stearic Acid in Liposomes

The spin probe 5-doxyl-stearic acid was used in liposomes prepared from the lipids of cold- and warm-adapted hamsters and rats. The esr spectra obtained were of a type readily analyzable by the order parameter (S) formalism (30, 31). This method assumes that the rate of segmental motion of the spin probe is sufficiently rapid, and the rotational motion of the vesicle sufficiently slow, as not to influence the spectrum. No discontinuity was observed in plots of log S vs l/T for any of the systems investigated, yielding no evidence for a lipid phase transition (Fig. 3a-d). In addition, the graphs in all four cases were virtually superimposable, implying a con-

T 30 32 32 34 36

l/TAO3 34 36

lITJO

FIG. 3. Variation of order parameter with l/T for 5-doxyl-stearic acid in liposomes of mitochondrial lipids of brown adipose tissue of (a) cold-adapted rats; (b) warm-adapted rats; (c) cold- adapted hamsters; and (d) warm-adapted hamsters.

512 CANNON ET AL.

siderable similarity in lipid properties in the region probed. At low temperature (2°C) a high degree of order was found (S = 0.78), and even at the highest temperatures the order parameter remained substantial (S = 0.53). Figure 4a and b shows experimental and simulated spectra for liposomes at 2°C from warm-adapted hamsters. The spectrum was simulated for rapid motion by the stochastic approach used for methyl 5-doxyl-stearate, with the inclusion of correlational times for spin probe and overall rotation (35). Since this spectrum corresponded to the experimental spectrum at the lowest measured temperature when motion would be expected to be slowest, it can be concluded that the order parameter formalism is valid for analysis of liposome spectra for the 5-doxyl-stearic acid probe in all systems studied here. The order parameters are greater in the case of the 5-doxyl-stearic acid probe compared with those required to simulate the esr spectra of methyl 5-doxyl-stearate at all temperatures. This is presumably because the methyl ester of the carboxylic acid is not anchored at the polar interface and freer transverse motion of the probe is allowed. Some order was found for methyl 5-doxyl-stearate (uide supra) but it was not

FIG. 4. (a) Experimental esr spectrum taken at 2°C of 5-doxyl-stearic acid in liposomes of mitochondrial lipids from the brown adipose tissue of warm- adapted hamsters. (b) Theoretical esr spectrum simulated with the stochastic method using input parameters to correspond to the order parameter formalism. Input magnetic parameters are g, = 2.0093, g, = 2.0055,g, = 2.0027,a, = 5G,a, = 7G,a, = 32G,B, = 3300G, S = 0.74, T;.: = 1.5G, T;;; = l.OG. The correlation time for segmental motion is 2 x IO-” s and that for the overall motion is 5 x 10e8 s.

large enough to allow extraction of order parameters directly from the spectra. Using the methyl ester it was only possible to conclude with certainty that there was no phase transition above 15°C. For the free acid, however, it could be concluded that no phase transition occurred over the whole temperature range studied.

5Doxyl-stearic Acid in Mitochondria

Since a discontinuity in an Arrhenius plot for membrane-bound enzyme activity had been observed in intact brown adipose tissue mitochondria (15) and yet no discontinuity had been encountered in the spin probe study of liposomes, spin probe motion in the intact mitochondria was investigated. A possibility existed that the en- zymatic discontinuity was a consequence of a gross change in lipid-protein interac- tions rather than of a lipid phase transition.

With the 5-doxyl-stearic acid probe in mitochondria, esr spectra were obtained which were qualitatively similar to those for liposomes and it was therefore possible to apply the order parameter analysis. The results are shown in Fig. 5a-d. No discontinuity was found for any of the mitochondrial samples and the curves were again almost superimposable. The order parameter at high temperature was almost the same as that for liposomes, although at low temperatures a significantly higher order parameter was calculated for mitochondria (S = 0.86 vs. 0.78 for liposomes). A spectrum demonstrating the highest order parameter obtained for cold-adapted hamsters is shown in Fig. 6a. Various explana- tions exist for the differences in order parameter between liposomes and mitochondria observed at low temperature. If the order parameter analysis is valid for all temperatures it could be concluded that genuine differences do exist between liposomes and intact mitochondria. Alterna- tively, the analysis of the low temperature mitochondrial spectra may be invalid since the peak arrowed in Fig. 6a is normally expected to be above the base-line. This effect could be explained as exchange broadening, but when the experiments were repeated at fourfold lower spin probe


::b/:d/: i , , . :

30 32 lITJO

34 36 32 l/TAO3

34 36

FIG. ii. Variation of order parameter with l/Tfor 5-doxylstearic acid in intact mitochondria isolated from the brown adipose tissue of (a) cold-adapted rats; (b) warm-adapted rats; (c) cold- adapted hamsters; (d) warm-adapted hamsters. In (c) x-x is at a 4-fold lower spin-probe concentration.

concentration, identical results were obtained (Fig. 5~). Another possibility is that the motion has slowed down to such an extent that the order parameter formalism can no longer be used. In addition, the effect may he due to natural line broadening from unresolved proton splittings.

Using recently developed methods of esr spectra simulation (17, 19-21) one can hope to resol.ve this question. It was shown that intermediate rates of rotation of the spin probe can affect the observed order parameter when the molecular ordering becomes appreciable, and that the observed order parameter increases as the rate of rotation of t.he spin probe decreases from the fast motion limit for a constant input order parameter (35). A spectrum simulated with an input order parameter of 0.74 is shown in Fig. 6b. The similarity to the experimental spectra in the region of interest should be particularly noted. In Fig. 6c a spectrum simulated where the rate of rotation of the spin probe is sufficiently rapid for the order parameter formalism to hold, but with a large intrinsic line width of 2.5G and an input order parameter of 0.90 is shown. It can be seen that the peak under discussion again falls below the base-line although the agree- ment in the other parts of the spectrum was not as good as for the case of the simulation with slower motion shown in Fig. 6b. It shlould also be noted that such large intrinsic line widths are not to be

FIG. 6. (a) Experimental esr spectrum taken at 2°C of 5-doxyl-stearic acid in intact mitochondria isolated from the brown adipose tissue of cold-adapted hamsters. (b) Theoretical esr spectrum simulated with the same input parameters as in Fig. 4b, except that 2’;; = l.OG, T& = O.OG and the correlation time for nitroxide rotation is 1O-9 s. (c) Theo- retical esr spectrum simulated with the same input parameters as in Fig. 4b, except that S = 0.90, T;.’ = 2.5G. T& = O.OG and the correlation time for nitroxide rotation is lo- I0 s.

expected for rapid rotation (36). The spectra observed experimentally are, therefore, probably the result of the effects of decreasing rate of motion. Because this effect is expected to change the observed order parameter gradually with decreasing rate of rotation of the spin probe, one should not expect to see a discontinuity in the order parameter curve. A change of the slope of the entire curve for mitochondria would

514 CANNON ET AL.

occur such that the slope approaches that of the liposomes at the higher temperatures. The input correlation time for the simulated liposome spectrum in Fig. 4b was 2 x 10-l’ s whereas that for the simulated mitochondrial spectrum in Fig. 6b was lo-’ s.

Another indication of the decreased mobility of the spin probe in mitochondria relative to liposomes can be obtained by measurement of the apparent isotropic splitting from the esr spectra of the ordered probes (30, 31, 35). For liposomes it remains constant at all temperatures, whereas for mitochondria it increases by 0.5 i 0.1 G over the range of temperatures studied. It has been shown that an increase in the apparent isotropic splitting is indicative of reduced molecular motion (35). Thus it can be concluded that in the mitochondrial membranes the rate of rotation of the spin probe is slower than in the corresponding liposomes. This reduced rate may be due to the presence of the proteins in the mitochondrial membranes. Alternatively it may be due to the spin probe being present in different environments in the two systems. It has not been unequivocally demon- strated whether the spin probe dissolves in the inner and/or outer mitochondrial membrane. However, it is believed that free fatty acids can readily penetrate the outer membrane, and since this membrane only accounts for about 5% of the total membrane area in brown adipose tissue mitochondria (T. Barnard, personal communi- cation), it appears that the spin probe must be present predominantly in the inner membrane. This does not resolve though whether the possible lipid heteroge- neity in the mitochondrial membrane is the same as in the liposomes, and this may be a contributing factor to the reduced rate of spin probe rotation in the mitochondria.

12-Doxyl-stearic Acid in Liposomes

In order to probe another region of the membranes, 12-doxyl-stearic acid was employed. Motion could be studied in regions considerably farther removed from the polar head group than with the 5-doxyl derivatives. The 12-doxyl-stearic acid is

somewhat more soluble in an aqueous envi- ronment than the 5-doxyl compound, and it partitions between the lipid and the bathing medium (40). This partitioning results in a complex spectrum composed of subspectra of the spin probe in both environments (Fig. 7a). If a phase transition occurs within the lipids it is expected that one lipid phase will demonstrate a greater affinity for the spin probe than the other (40, 41). Measurement, therefore, of the concentration of free probe in the aqueous phase as a function of temperature would yield a discontinuous plot when plotted against inverse absolute temperature.

Using procedures described in the methods section, the percentage of free probe was calculated at each temperature for liposomes from the four systems under investigation. There was no discontinuity in the temperature response of any of the samples, suggesting that also in the region of the membrane investigated by this probe no phase transition occurs over the range of temperature studied. The relative amount of free probe was small in all cases, but increasing the spin probe concentration twofold increased only slightly the percentage of free probe. The curves for the two concentrations were almost parallel

FIG. 7. Experimental esr spectra taken at 20°C of 12-doxyl-stearic acid in (a) liposomes of mitochondrial lipids from the brown adipose tissue of cold- adapted hamsters; (b) intact mitochondria from the brown adipose tissue of cold-adapted hamsters. The spectra are a composite of a spectrum of three narrow lines due to the spin probe in aqueous medium and a spectrum of three broad lines due to the spin probe in lipid.


and only slightly shifted from one another. Decreasing the lipid to water ratio increased the percentage of free probe considerably, but again no transition was observed. The amount of bound probe increased with increasing temperature, demonstrating a greater probe solubility in lipid at higher temperature.

12-Doxyl-stearic Acid in Mitochondria

The motion of the 12-doxyl-stearic acid probe in intact mitochondria was also studied (Fig. 7b). After allowance for the presence of the spectrum of narrow lines due to the free probe, it is evident that the resonances of the bound probe are considerably broader in the spectra of the mitochondria relative to those in the corresponding liposomes. Thus, the rate of motion at the 12-position in the mitochondria is reduced relative to that in the liposomes, as in the case of the 5-doxyl-stearic acid. The 12- doxyl-stearic acid probe was degraded rapidly in mitochondria at temperatures above 30°C during the course of the experiment. The signal-to-noise ratios of the esr spectra of the unbound component were decreased sufficiently to make the analysis in terms of amount of probe free in solution very difficult. Only a very small amount of probe remained free and this decreased with increasimg temperature. It was not possible to conclude, however, if the temperature dependence of the partition coef- ficients was linear or discontinuous.

3-Doxyl-cholestane in Multi&layer Films

The spin probe 3-doxyl-cholestane was used in multibilayer films of lipids from all samples. Films were used instead of liposomes because the esr spectra of films have greater information content than those of liposomes where the resonances are very broad (17, 42., 43). This is not unexpected because the overall motion of the cholestane spin probe is reduced considerably compared to the stearic acid spin probes (see spectral simulations) and the esr spectra of spin probes with slow motion of the long axis i:n liposomes are insensitive to changes in the ordering and rate of rotation about the long axis (18). The spectra were taken with thle surface plane of the film

parallel and perpendicular to the applied magnetic field. The spectra obtained in the perpendicular orientation were characteristic of slow molecular motion (Fig. 8b). However, in the parallel orientation, the spectra allowed estimation of correlation times in a manner similar to that used for methyl 5-doxyl-stearate (Fig. Ba). Further- more, for slow motions of the 3-doxyl-cholestane long axis, one can use the spin jump method applied recently by Mailer et al. (37), to calculate correlation times for the motion about the cholestane long axis when the surface plane of the film is oriented parallel to the field. The appro- priate formulas for 7, the correlation time for rotation about the long axis, are

7 = 2.82 x lo-‘OB (7)

-r = 2.25 x 10-‘°C. (8)

U IOG

FIG. 8. (a and b) Experimental esr spectra taken at 6°C of 3-doxyl-cholestane in planar multibilayers of the mitochondrial lipids from the brown adipose tissue of cold-adapted rats. (a) Spectrum obtained with the surface plane of the film oriented parallel to the applied magnetic field. (b) Spectrum obtained with the surface plane of the film oriented perpendicular to the applied magnetic field. (c) Theoretical esr spectrum simulated using the stochastic method for an ordered film. Input magnetic parameters are a,. = 32.OG, uy. = 6.OG, a,. = 6.OG, g,, = 2.0021, g,, = 2.0089, g,. = 2.0062 where the z’-axis is taken to be the principal axis of rotation and of orientation of the steroid spin probe. This Y-axis corresponds to the nitroxide y-axis. Other input parameters are S = 0.30, B, = 33OOG, T<.’ = 2.OG, T,i = -1.OG. The correlation time for rotation about the steroid long axis is 1.5 x 10-O s and that for rotation of the long axis is 3.6 x 10-e s.

516 CANNON ET AL.

where the T’S are defined to agree with the usual conventions (27, 28, 38). These formulas are applicable for a nitroxide rotating about its molecular y-axis for experiments done at 9.5 GHz. Plots of log 7 us 1/T showed no meaningful discontinuities, and therefore provide no evidence for a phase transition in any of the systems studied.

The spectra obtained with the sample in the perpendicular orientation, as noted above, and as shown in Fig. Bb, are not readily analysable in terms of rapid isotropic motion or the order parameter formalism. However, using stochastic line- shape theory as developed by Freed et al. (17, 19-21), spectra can be simulated which are exact at all correlation times. This theory has not previously been applied to biological systems. The formalism includes anisotropic rotation and the ordering distribution of the probes (21, 36). Cost precludes simulation of the spectra at all temperatures, but analysis has been carried out at the highest and lowest temperatures.

At the highest temperature the spectra obtained with the sample in the perpendicular and parallel orientations are very similar to one another, demonstrating a lack of order in the system (Fig. 9a and b). Thus the order parameter contributes very little to the overall line shape. The correlation time for rotation around the long axis of the spin probe was taken from the spin jump analysis, Eqs. 7 and 8, as described by Mailer et al. (37), and the order parameter chosen was very small but greater than zero (S = 0.03). Therefore, the rate of rotation of the steroid long axis is the sensitive parameter which can be determined from the spectra (Fig. 9c). At the lowest temperature the order parameter was increased since the spectra obtained with the sample in the perpendicular and parallel orientations were no longer similar (Figure 8a and b). Using the correlation time for the rotation about the long axis (axial correlation time) determined by the spin jump method, and the ratio of the correlation times for axial rotation and of the long axis rotation determined from the high temperature spectrum (it has been

FIG. 9. (a and b) Experimental esr spectra taken at 45°C of 3-doxyl-cholestane in planar multibilayers of mitochondrial lipids from the brown adipose tissue of cold-adapted hamsters. (a) Spectrum obtained with the surface plane of the film oriented parallel to the applied magnetic field. (b) Spectrum obtained with the surface plane of the film oriented perpendicular to the applied magnetic field. (c) Theoretical esr spectrum simulated with the same input parameters as in Fig. 8c, except that S = 0.03, 2”;: = 1.9G. The correlation time for rotation about the steroid long axis is 0.4 x low9 s and that for rotation of the long axis is 1Om8 s.

found that the parameter N = R,, JR,, where RI, is the axial rotational rate and R, that of the long axis, depends only on the molecular geometry and should be in- dependent of temperature and of the ordering of the system for a variety of nitroxide spin probes (38, 39)), the order parameter was adjusted to give the best fit for the spectrum at 6°C (Fig. 8~). Even at this temperature the order parameter is low (S = 0.30). The low ordering is indicative of the fact that 3-doxyl-cholestane is chemically different from the principal lipids of the films and is probably not easily accommodated in the bilayer structure, whereas the stearic acid spin probes are chemically similar to the lipid region of the systems being studied and would be expected to fit into the structure and be ordered by it. This model gives a good fit to the experimental spectra indicating that the anisotropy of the motion is mainly determined by the probe geometry rather than the ordering of the system. Because the stochastic method has not yet been extended to calculate spectra for the parallel orientations of multibilayer films one cannot be certain that this model is


unique. The cholestane probe has been found to have condensing properties similar to those of cholesterol (44), and to be a sensitive indicator of the influence of cholesterol on lipid organization and fluidity (40, 42, 43). Mitochondria, however, usu- ally contain only very low levels of cholesterol (15, 33, 34).

Because we did not simulate spectra at all temperatures for the perpendicular orientation, we can say little about possible phase transitions from these spectra except that the degree of anisotropy of the probe rotation does not change over the entire temperature range. This is consistent with the results of the spin-jump analysis of the spectra taken at the parallel orientation where there is no indication of a phase transition.

CONCLUSION

Cold adaptation of either rats or hamsters produces no striking alteration in the fatty acid composition of the phospholipids from the mitochondria of the brown adipose tissue. However, there is a decreased degree of unsaturation in the hamster lipids compared with the rat lipids. The hamster lipids are significantly more satu- rated than those of mitochondria obtained from other animals kept at normal temperatures. Lipid spin probes indicate that the degree of order is slightly lower, and the fluidity greater, in the lipids of rats relative to those of hamsters. Simulation of spin probe electron spin resonance spectra using complex contemporary techniques indicates that caution must be taken in extracting order parameters and correlation times by more approximate methods; in some cases artifactual transitions can appear in plots of order parameter or correlation time versus reciprocal temperature. No evidence for a lipid phase transition was obtained with any of the four spin probes in intact mitochondria, liposomes, or oriented multibilayers of extracted lipids. Discontinuities in Arrhenius plots of mitochondrial enzyme activity observed in earlier studies of brown adipose tissue are therefore not due to lipid phase transitions. The fluidity of the lipids in intact mitochondria is considerably lower than in

liposomes of extracted lipids, presumably due to protein-lipid interaction. There is apparently no connection between the fluidity of lipids in the mitochondria of brown adipose tissue and the ability of hamsters to lower their body temperature on cold-adaptation.

ACKNOWLEDGMENTS

This work was supported by grants from the Science Research Council (England), the Swedish Natural Science Research Council, the Wallenberg Foundation Jubilee Fund, and the National Research Council of Canada. We are grateful to Professor A. Ehrenberg for advice and encouragement and to Mr. R. Cyr for lipid analyses.

REFERENCES

1. SMITH, R. E., ANDHOCK, R. J. (1963) Science 140, 199-200.

2. DAWKINS, M. J. R., AND HULL, D. (1964) J. Physiol. (London) 172, 216-238.

3. JOEL, C. D. (1965) in Handbook of Physiology, Section 5, Adipose Tissue (Renold, A. E., and Cahill, G. F., ed.), pp. 59-85, American Physio- logical Society, Washington.

4. SMITH, R. E., AND HORWIT~, B. A. (1969) Physiol. Reu. 49, 330-425.

5. LINDBERG, O., ed. (1970) Brown Adipose Tissue, Elsevier, New York.

6. JANSKY, L. (1973) Biol. Reu. 48, 85-132. 7. SMITH, R. E., ROBERTS, J. C., AND HVITELMAN, K.

J. (1966) Science 154, 653-654. 8. LINDBERG, O., DE PIERRE, J., RYLANDER, E., AND

AFZELIUS, B. A. (1967) J. Cell Bid. 34,293-310. 9. PRUSINER, S. B., CANNON, B., TE MAY CHING, AND

LINDBERG, 0. (1968) Eur. J. Biochem. 7,51-57. 10. KAYSER, C. (1957) Annu. Rev. Physiol. 19,83-120. 11. LYMAN, C. P., AND BLINKS, D. C. (1959) J. Cell

Comp. Physiol. 54, 53-63. 12. RICHARDSON, T., AND TAPPEL, A. L. (1962) J. Cell

Biol. 13, 43-53. 13. LYONS, J. M., AND RAISON, J. K. (1970) Comp.

Biochem. Physiol. 37,405-411. 14. RAISON, J. K., LYONS, J. M., MEHLHORN, R. J.,

AND KEITH, A. D. (1971) J. Biol. Chem. 246, 4036-4040.

15. CANNON, B., AND POLNASZEK, C. F. (1975) in De- pressed Metabolism and Cold Thermogenesis (Mussacchia, X. J., and Jansky, L., eds.), in press.

16. CHANCE, B., AND WILLIAMS, G. R. (1956) J. Biol. Chem. 217, 383-451.

17. BERLINER, L. J., ed. (1975) Spin Labeling Theory and Applications, Academic Press, New York, in press.

518 CANNON ET AL.

18. POLNASZEK, C. F. (1975) Quart. Reu. Biophys. in press.

19. FREED, J. H., BRUNO, G. V., ANDPOLNASZEK, C. F. (1971) J. Phys. Chem. 75, 3385-3399.

20. FREED, J. H. (1972) Annu. Reu. Phys. Chem. 23, 265-310.

21. POLNASZEK, C. F., BRUNO, G. V., ANDFREED, J. H. (1973) J. Chem. Phys. 58, 3185-3199.

22. HITIXLMAN, K. J., LINDBERG, O., AND CANNON, B. (1969) Eur. J. Biochem. 11, 183-192.

23. GORNALL, A. G., BARDAWILL, C. J., AND DAVID, M. M. (1949) J. Biol. Chem. 177, 751-766.

24. BLIGH, E. G., AND DYER, W. J. (1959) Can. J. Biochem. Physiol. 37. 911-917.

25. KEANA, J. F. W., KEANA, S. B., AND BEETHAM, D. (1967) J. Amer. Chem. Sot. 89, 3055-3056.

26. KING, E. J. (1932) Biochem. J. 26, 292-297. 27. KIVELSON, D. (1960) J. Chem. Phys. 33,

1094-1106. 28. FREED, J. H., AND FRAENKEL, G. K. (1963) J.

Chem. Phys. 39.326-348. 29. MCCONNELL, H. M. (1956) J. Chen. Phys. 25,

709-711. 30. SEELIG, J. (1970) J. Amer. Chem. Sot. 92,

3881-3887. 31. HUBBELL, W. L., AND MCCONNELL, H. M. (1971) J.

Amer. Chem. Sot. 93, 314-326. 32. CHAFFEE, R. R. J., PLATNER, W. S., PA~ON, J., AND

JENNY, C. (1967) Proc. Sot. Exp. Biol. Med. 127, 102-106.

33. FLEISCHER, S., ROUSER, G., FLEISCHER, B., CASU,

A., AND KRITCHEVSKY, G. (1967) J. Lipid Res. 8, 170-180.

34. FIEHN, W., PETER, J. B., MEAD, J. F., AND GAN- ELEPANO, M. (1971) J. Biol. Chem. 246, 5617-5620.

35. MASON, R. P., POLNASZEK, C. F., ANDFREED, J. H. (1974) J. Phys. Chem. 78, 1324-1329.

36. POLNASZEK, C. F. (1974) Doctoral Dissertation, Cornell University.

37. MAILER, C., TAYLOR, C. P. S., SCHREIER-MUCCILLO, S., AND SMITH, I. C. P. (1974) Arch. Biochem. Biophys.163,671-678.

38. GOLDMAN, S. A., BRUNO, G. V., POLNASZEK, C. F., AND FREED, J. H. (1972) J. Chem. Phys. 56, 716-735.

39. KUZNETSOV, A. N., VOLKOV, A. Y., LIVSHITS, V. A., AND MIRZOIAN, A. T. (1974) Chem. Phys. Lett. 26,369-372.

40. BUTLER, K. W., TA~RIE, N. H., AND SMITH, I. C. P. (1974) Biochim. Biophys. Acta. 363, 351-360.

41. KLEEMAN, W., AND MCCONNELL, H. M. (1974) Biochim. Biophys. Acta 345, 220-230.

42. SMITH, I. C. P. (1971) Chimia 25, 349-360. 43. SMITH, I. C. P., AND BUTLER, K. W. (1975) in Spin

Labeling Theory and Applications (Berliner, L. J., ed.), Chapter 11, Academic Press, New York, (in press).

44. CADENHEAD, D. A., DEMCHAK, R. J., AND MULLER- LANDAU, F. (1972) Ann. N.Y. Acad. Sci. 195, 218-223.

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