The Department of Biochemistry, Indian Institute of Science, Bangalore 560 012, India ... microsomal redox systems, brown adipose tissue, lipid peroxidation, ...
Molecular and Cellular Biochemistry 92: 147-157, 1990. © 1990 Kluwer Academic Publishers. Printed in the Netherlands.
Original Article
Microsomal redox systems in brown adipose tissue: high lipid peroxidation, low cholesterol biosynthesis and no detectable cytochrome P-450
B. Seshadri Sekhar, C.K. Ramakrishna Kurup and T. Ramasarma
The Department of Biochemistry, Indian Institute of Science, Bangalore 560 012, India Received 6 March 1989; accepted 1 June 1989
Key words." microsomal redox systems, brown adipose tissue, lipid peroxidation, cholesterol biosynthesis, cytochrome P-450 Abstract
The presence of redox systems in microsomes of brown adipose tissue (BAT) in cold exposed rats was investigated and compared with liver. BAT microsomes showed high activity of lipid peroxidation measured both by the formation of malondialdehyde (MDA) and by oxygen uptake. NADH and NADPH dependent cytochrome c reductase activity were present in both BAT and liver microsomes. Aminopyrine demethylase and aniline hydroxylase activities, the characteristic detoxification enzymes in liver microsomes could not be detected in BAT microsomes. BAT minces showed very poor incorporation of [1-14C]acetate and [2-14C] mevalonate in unsaponifiable lipid fraction compared to liver. Biosynthesis of cholesterol and ubiquinone, but not fatty acids, and the activity of 3-hydroxy-3-methyl glutaryl CoA reductase appear to be very low in BAT. Examination of difference spectra showed the presence of only cytochrome b s in BAT microsomes. In addition to the inability to detect the enzyme activities dependent on cytochrome P-450, a protein with the characteristic spectrum, molecular size in SDS-PAGE and interaction with antibodies in double diffusion test, also could not be detected in BAT microsomes. The high activity of lipid peroxidation in microsomes, being associated with large oxygen uptake and oxidation of NADPH, will also contribate to the energy dissipation as heat in BAT, considered important in thermogenesis.
Abbreviations: BAT - Brown Adipose Tissue, MDA - malondialdehyde
Brown Adipose Tissue (BAT 1) is believed to make significant contribution towards heat production under cold and diet-induced non-shivering thermogenic conditions [1-5]. It is proposed that mitochondria of BAT are responsible for the dissipation of some energy in the form of heat, and towards achieving this they are specifically endowed with a nucleotide-binding protein (Mr 32,000) that can discharge electrogenic proton gradient in competition to ATP synthase [6-8]. The brown adipocytes are characterised by the presence of lipid vacuoles
that occupy considerable intracellular volume [5]. Justifiably fatty acids are the major fuel sources for BAT mitochondria for oxidation [9] and also for dehydrogenase-dependent H202 generation [10]. Poly-unsaturated fatty acids of these, have another role in promoting the activity of lipid peroxidation triggered by free iron chelated to nucleotides [11]. This reaction in microsomes produces small rates of formation of malondialdehyde as a degradation product of peroxidation of unsaturated fatty acids but is accompanied by high rates, comparable to
148 that of mitochondrial oxidations, of oxygen uptake and N A D P H oxidation [12, 13]. The question of inadequacy of mitochondria alone explaining the increased cellular oxygen consumption in thermogenic conditions is periodically raised during the last two decades [14-16]. In view of these we have investigated the microsomal redox systems in BAT and compared with liver. The results reported here show that lipid peroxidation, NAD(P)H-cytochrome c reductase and cytochromme b5 occur in BAT microsomes, but cytochrome P-450 and activities dependent on it including cholesterol biosynthesis are low or not detectable.
mal pellets obtained were suspended in 1.15% KC1 solution for lipid peroxidation assays. For other assays the pellets were suspended in 0.25 M sucrose containing 50mM Tris-HCl buffer (pH7.4) and 20% (v/v) glycerol. Microsomal fraction thus prepared from BAT contained most of the activity of the marker enzyme, glucose-6-phosphatase, as in the case of liver. Where mentioned, rats were treated with phenobarbital (80 mg/kg body wt. intraperitoneal) to induce formation of cytochrome P-450, and microsomes from the tissues were similarly prepared.
Enzyme assays Materials and methods
Materials All biochemicals used were purchased from Sigma Chemical Co., St. Louis, MO, U.S.A. Other chemicals were of the purest grades available. [114C]-Acetate (50.3 mCi/mmole) and [2-14C]meval onate (DL) (47.0 mCi/mmole) were purchased from Bhabha Atomic Research Centre, Bombay, India and New England Nuclear Corp., Boston, MA, U.S.A., respectively. Solutions were made in water, double-distilled in an all-quartz apparatus.
Microsomal preparations Male Wistar rats (150-200g) exposed to cold (0-5 ° C) for 30 days were used. Interscapular BAT occurring in small quantity in rats at ambient temperature increased several fold on cold-exposure [10]. The animals were killed by cervical dislocation, BAT and liver were removed, freed of extraneous tissue and homogenized in 1.15% KCI solution (10 vol./g tissue). Livers were perfused with 0.9% NaC1 before excision. Perfusion of BAT was not attempted. The homogenates were centrifuged at 10,000 xg for 10min in a sorvall RC-5B refrigerated centrifuge. The layer of fat at the top was removed and the post mitochondrial supernatant was centrifuged at 100,000 xg for 65 min in a Sorvall OTD 50-B refrigerated centrifuge. The microso-
The activity of lipid peroxidation was measured at 37°C according to Ramasarma et al. [17] both by the formation of malondialdehyde (MDA) as thiobarbituric acid-reacting material, and also by oxygen uptake in a Gilson Oxygraph fitted with a Clark oxygen electrode. The reaction mixture in 0.5 ml contained 50/xM ferrous ammonium sulfate, 50/xM ADP, 300/zM NADPH, Tris (50 mM)-KC1 (150raM) buffer (pH7.4) and 0.2mg microsomal protein. The reaction was carried out for 10 min at 37° C and M D A was measured in the trichloroacetic acid supernatants. Identical reaction mixtures with a total volume of 1.4ml were used for measurement of oxygen uptake, which was obtained with the microsomal preparations only on addition of both N A D P H and Fe 2÷. The rates were negligible when N A D P H or Fe 2÷ was added alone. This oxygen uptake was not sensitive to antimycin A but was abolished by the antioxidant, butylated hydroxytoluene, and thus represents lipid peroxidation activity. The activities are expressed as nmoles of M D A formed or oxygen consumed/min per mg microsomal protein. The activities of NADH-cytochrome c reductase [18] and of NADPH-cytochrome c reductase [19] were measured by increase in absorbance at 550 nm of reduced cytochrome c, and expressed as nmoles cytochrome c reduced/min per mg microsomal protein. The activity of cytochrome P-450 dependent aniline hydroxylase was estimated according to Imai et al. [20]. Thee activity is expressed as
149 nmoles-p-aminophenol formed/min per mg microsomal protein. Using aminopyrine as the substrate, cytochrome P-450 dependent demethylation was determined according to Guengerich [21] and the activity expressed as nmoles formaldehyde produced/min per mg microsomal protein.
Incorporation of [1J4]acetate and [2J~C]mevalonate into unsaponifiable lipids BAT and liver tissues from cold exposed rats were placed in oxygenated Krebs-Ringer phosphate buffer (0.1 M, pH 7.4) and minced with a blade by hand to give lobes of less than 1 mm thickness. These were then washed and amounts of 400mg tissue of these were incubated in 6 ml of the same buffer along with 10/xCi of [1J4C]acetate or 2/xCi of [1-14C]mevalonate for 3 hr at 37 ° C in a reciprocal metabolic shaker under an atomosphere of oxygen. The incorporation of radioactivity was found to increase with time of incubation upto 4hr under these conditions. The reaction was terminated by adding 15ml ethanol. To each flask were added unlabelled cholesterol (50/xg) and lanosterol (50/xg) to prevent any loss of these endogenous labelled compounds and the contents were saponified with K O H (1 ml, 40% w/v) in presence of pyrogallol (lml, 10% w/v) for 30min. The unsaponifiable lipids were extracted with light petroleum (40-60 °) and fractionated on a 5% deactivated alumina column as described by Joshi et al. [22] for the separation of the constituents. Under these conditions the constituents were eluted as follows: light petroleum- hydrocarbons (squalene), 5 % ethyl ether in light petroleum -ubiquinone, and 20% ethyl ether in light petroleum-sterols. Ubiquinone fraction was further purified by tic on silica gel G with the solvent system, 2% (v/v) of acetone in light petroleum. Ubiquinone band detected as a quenching spot in UV light at Rf 0.8, was scraped from the plate and the radioactivity determined. The sterols fraction was further separated on the silica gel G essentially according to Avigan et al. [23] with the solvent system, benzene : ethyl acetate (5:1, v/v). The bands of cholesterol, lanosterol and squalene in reference spots were identified by
exposure to the iodine vapours, and were found at Rfvalues of 0.50, 0.70 and 0.95, respectively. These and l c m portions of other areas in the sample channels were scraped and counted. The residue after extraction of the unsaponifiable lipids was acidified with dil. HC1 to pH 2.0, and after leaving overnight at room temperature, was extracted twice with light petroleum and once with ethyl ether to obtain fatty acids. This was carried out for the samples using [1-14C] acetate in order to ascertain the relative incorporation of the tracer into fatty acids.
Estimations Cholesterol was estimated by the LiebermannBurchard reaction [24]. Ubiquinone concentration was obtained by the absorbance change at 275 nm on reduction of the quinone by sodium borohydride (0.0122 for l nmole/ml solution) [25]. Squalene was measured by a modification of Liebermann-Burchard reaction. Samples having squalene in the range of 502000/zg were taken in i ml chloroform and mixed with 2 ml of a freshly prepared reagent of acetic anhydride: H2SO~ (20:1, v/v) and after 30min standing at room temperature, the absorbance values at 293 nm were taken. Protein was estimated by the method of Lowry et al. [26], Radioactivity was measured in a LKB Rack-beta scintillation counter using a scintillation fluid consisting of 0.5% (w/v) solution of 2,5 diphenyl oxazole in toluene.
Identification of cytochromes Difference spectra of microsomal samples, both reduced against oxidized, and reduced against CO - treated reduced, were recorded in a Hitachi 557 recording spectrophotometer according to the method described by Estabrook and Werringloer [27] for cytochrome P-450, and Matsubara et al. [28] for cytochrome bs.
150
Ouchterlony double diffusion test Microsomal samples were solubilized in a medium containing 50 mM Tris-HCl buffer (pH 7.4), 10 mM EDTA, 10% glycerol, 100mM NaC1, 0.5% deoxycholate, 0.5% Triton X-100 and the supernatants after centrifugation at 100,000 xg for 60 min were placed in outer wells. The double diffusion plates and the medium (pH 7.4) containing 0.9% agarose, 1M glycine 0.08M NaC1, 0.5% Triton X-100 and 15 mM NaN3 were prepared according to Thomas et al. [25]. Antiserum raised against total-protein of microsomes isolated from rats treated with phenobarbital which cross-reacts with native and b,e forms of cytochrome P-450 (gift from Dr. G. Padmanaban of this department) was placed in the centre well. Incubation was carried out for 2 days at 4°C.
N,N,N',N',-tetramethyl ethylene diamine (TEMED), and the separating gel 12% acrylamide 0.4% bisacrylamide, 360mM Tris-HC1 buffer (pH 8.8), 0.1% SDS (w/v), 0.25 mg/ml ammonium persulfate and 0.5/zl/ml of TEMED. The microsomal samples were denatured by heating in a boiling water-bath for 2 min in the sample buffer containing 10% glycerol, 20% SDS and 5% mercaptoethanol (v/v). The gels were run at a constant current of 20mA at room temperature using 125mM Trisglycine buffer (pH 8.8) containing 0.1% SDS. Bromophenol blue was used as the tracking dye. The gels were stained with Coomassie Blue R and destained as described in the method. Densitometric scans at 595 nm of the proteins separated on the gel were taken using a Beckman DU-8B spectrophotometer.
Results
Gel electrophoresis Lipid Peroxidation The electrophoresis was performed essentially according to Laemmli [26] in a Broviga vertical slab gel apparatus with plastic spacers (1.5 mm thick). The stacking gel (1 cm high) contained 3% acrylamide, 0.24% N,N-methylene bis acrylamide, 125 mm Tris-HC1 buffer (pH 6.8), 0.1% SDS (w/v), 0.5mg/ml ammonium persulfate and 1.3txl/ml Table 1. Increase in lipid peroxidation in microsomes of B A T and liver of rats exposed to cold M e a s u r e m e n t Experimental condition
Ambient cold-exposed Ambient cold-exposed
Table 2. Comparison of activities of some microsomal redox enzymes in B A T and liver
nmole/min per m g protein E n z y m e activity BAT
MDA formation Oxygen Uptake
The activity of lipid peroxidation in the microsomes samples was measured both by the formation of MDA and by accompanying oxygen uptake. The rates of oxygen uptake are over 25-fold that of MDA formation and these high rates make lipid peroxidation a reaction of significance for oxygen
1.4+ 6.7 + 57 ± 182 +
Liver 0.4 0.7 7 18
2.0+ 3.9 ± 52 ± 106 +
0.2 0.3 15 12
Control rats were kept at ambient t e m p e r a t u r e and cold exposure was for 30 days at 0-5 ° C. For each sample of B A T in control about 10 rats were used since the a m o u n t of tissue per rat was small (about 100rag). Microsomes were prepared from B A T and liver samples and rates of lipid peroxidation were m e a s u r e d both by M D A formation and oxygen uptake as described in Materials and Methods. T h e values are m e a n + S.D. of six i n d e p e n d e n t determinations.
N A D H - c y t o c h r o m e c reductase N A D P H - c y t o c h r o m e c reductase A m i n o p y r i n e demethylase Aniline hydroxylase
nmole/min per mg protein BAT
Liver
231 ± 14 78 + 3 n.d. n.d.
957 ± 54 68 + 8 8.3 __ 0,8 3.3 _+ 1.2
Microsomes were prepared from B A T and liver obtained from rats exposed to cold (0-5 ° C) for 30 days. B A T from 3 rats was pooled and used for each determination. T h e m e t h o d s for preparation of microsomes and assay of enzymes are described in Materials and Methods. The values arc m e a n _+ S.D. of four i n d e p e n d e n t determinations, n.d. not detected.
151 uptake which is not coupled to energy conservation. The results in Table 1 show that this activity occurs in both BAT and liver at nearly same rates in ambient control animals. It must be mentioned that the amount of sample of BAT is small in such animals and the pooling of tissue from ten rats was necessary for each preparation of microsomes and determination. This tissue proliferated several fold on exposure of rats to cold. BAT from such animals also showed increased specific activities of lipid peroxidation by 3-5 fold, and liver by 2-fold, and the increases are statistically significant (P value < 0.001). In the following experiments, BAT obtained from cold-exposed rats was used.
Table 3. Incorporation of [l-~4C]acetate and [2-14C]mevalonate into unsaponifiable lipids by BAT and liver tissue minces
Fraction
Tracer
Unsaponifiable Acetate lipids Hydrocarbons Ubiquinone Sterols
Fatty acids
Unsapnnifiable Mevalonate lipids Hydrocarbons Ubiquinone Sterols
CPM per g tissue
Some microsomal redox enzyme activities
We next tested for the presence of other redox enzymes, characteristic of hepatic microsomes, in BAT and compared with liver obtained from the same animals exposed to cold. The activities of cytochrome c reductases dependent on NADH and NADPH were found to be present in BAT microsomes (Table 2), implying the presence of the corresponding flavo-proteins that are known to reduce cytochromes b5 and P-450, respectively, in liver microsomes. The two enzymes involved in detoxification mechanisms, aminopyrine demethylase and aniline hydroxylase, gave an interesting contrast between BAT and liver. The activities could not be detected in BAT microsomes even when 10-fold higher concentration of protein concentration, compared to liver, was used in the assay. It may be mentioned that these activities in liver microsomes were little affected in cold-exposed animals, but increased 4-7 fold when such animals were treated with the cytochrome P-450 inducing agent, phenobarbital. But these activities could not be detected in BAT mi-
BAT (%)
Liver (%)
21,460 (100) + 2,500 11,490 (53.5) + 1,125 540 (2.5) + 90 4,300 (20.0) + 425 1,436,030 + 333,990
2,209,910(100) 4- 358,500 29,040 (1.3) 4- 7,890 5,520 (0.3) + 625 1,832,050 (82.9) + 266,450 1,258,140 + 273,330
10,760 (100)
741,700 (100)
0.18
5,160 (48.0) 290 (2.7) 1,665 (15.5)
14,835 (2.0) 1,400 (0.2) 662,540 (89.3)
0.29
Table 4_ Thin layer chromatographic separation of acetate-labelled sterols fractions from BAT
Position in TLC Rf
0.53 BAT and liver samples were obtained from rats exposed to cold (0-5°C) for 30 days. The tissue minces (about 400mg wet wt.) were incubated with 10/xCi of [1-14C]acetate or 2/xCi of [214C]mevalonate for 3 hr at 37 ° C and the radioactivity incorporated into unsaponifiable lipids and fatty acids fraction was measured as described in Materials and Methods section. Values, calculated as CPM per g tissue, are mean + S.D. of four independent determinations for acetate experiment and mean of two for mevalonate experiment. Figures in parentheses are percentage taking total radioactivity in unsaponifiable lipids as 100%.
0.74 0.82
CPM per g tissue
%
1500 + 75 nil 505+25 nil 622+55 nil nil 3924-20 nil nil 170 + 25 620 + 40 nil nil
27 13 16 10 4 16 -
distance upto 1 cm 1- 2 cm 2- 3 c m 3- 4 cm 4- 5cm 5- 6 cm 6- 7 cm 7- 8cm (cholesterol) 8- 9 cm 9-10 cm 10-11 cm (Lanosterol) 11-12 cm 12-13 cm 13-14 cm
Acetate-labelled sterols fractions of BAT from the experiment described in Table III, were fractionated on silica gel TLC and radioactivity measured. Values given as CPM per g tissue, are mean + S.D. of independent determinations of four samples_
152
I J
A = 0,002
,\
7---. f
A
\
/ / J
I
i
400
i
450
]
500
I
400
J
I
450
,
I
["
500
400
,
I
450
,
I
I
5OO
400
,
I
450
,
I
500
W a v e l e n g t h (rim)
Fig. 1. Difference spectra. The spectra A - C were recorded with sample cuvette gased with carbon monoxide against the reference cuvette, while both were in reduced state. The spectra in D were obtained with both cuvettes being gassed with CO and reading the sample cuvette, reduced by adding dithionate, against the reference cuvette. The following were the additions: A-BAT homogenate, 2 mg protein; B-BAT mitochondria, 2 mg protein; C-BAT microsomes, 4 mg protein; liver microsomes, 2 mg protein (broken line): D-BAT microsomes, 4 mg protein; liver microsomes, 1 mg protein (broken line)_
crosomes in any of these animals. These results gave the first clue on the possible absence of cytochrome P-450 in BAT microsomes.
Cholesterol biosynthesis Aniline and aminopyrine are unphysiological sub-
Table5. Concentration of squalene, ubiquinone and cholesterol in BAT and liver Compound
Squalene Ubiquinone Cholesterol
nmoles per g tissue BAT
Liver
280+ 47 227 + 20 2840 + 1280
164+ 40 165 + 7 4160 + 1030
Tissues were obtained from rats exposed to cold (0-5 ° C) for 30 days. For each determination BAT from three rats was pooled. The unsaponifiable lipids were prepared and the compounds were estimated as described in Materials and Methods. The values are mean + S_D_ of three independent determinations.
strates and the absence of their metabolic reactions in an extrahepatic tissue is not sufficient to ~onclude absence of cytochrome P-450 or its reactions. Therefore, the occurrence of metabolism of a P-450-dependent natural substrate was selected for testing. Microsomes contain all the necessary redox enzyme systems to carry out the oxidative reactions needed for conversion of lanosterol (a C-30 sterol) to cholesterol (a C-27 sterol). Of these, the steps involved in the oxidative removal of 14 alphamethyl group are shown to be dependent on cytochrome P-450 [31]. In the absence of this cytochrome the biosynthesis of cholesterol would be affected, and if this were the only defect, lanosterol should accumulate in the tissue. This hypothesis was tested by studying the incorporation of [1-14C] acetate into unsaponifiable lipids, which form the bulk of isoprenoid compounds, and its constituents, hydrocarbons, ubiquinone and sterols. The most unexpected results were obtained in these experiments. Under identical conditions of experiments, BAT showed very poor incorporation of labelled acetate, hardly 10% that of liver, into un-
153
l1
[ A =0.04
[
400
L
i
i
I
I
500 Wavelength (nm)
I
I
600
Fig. 2. Difference spectrum of BAT microsomes. The spectrum of sample cuvette in a reduced state against the reference cuvette, both containing BAT rnicrosomes (4 mg protein) shows the presence of cytochrome b5.
Table 6. Concentration of cytochromes b5 and P-450 in BAT and liver Cytochrome
b5 P-450
pmole/mg microsomal protein BAT
Liver
476 +_ 88 n.d.
495 _+ 69 480 _+ 120
Microsomes prepared from BAT and liver of rats exposed to cold (0-5 ° C) for 30 days were used for the determination of the cytochromes by difference spectra as described in Materials and Methods. BAT from 3 rats was pooled for each preparation of microsomes. The values are mean + S.D. of four independent determinations, n.d. not detected.
saponifiable lipids. Any suspicion of poor uptake of tracer or its metabolic activation was dispelled by the high incorporation obtained into fatty acids fractions in BAT, similar to that in liver (Table 3). Incorporation of labelled acetate into unsaponifiable lipids is nearly of the same order as that into fatty acids in experiments with liver slices and of this about 70% is recovered in sterols fraction [32]. In striking contrast incorporation of labelled acetate into unsaponifiable lipids in BAT was only about 15% of that in fatty acids. Also the sterols fraction accounted for only 20%, whereas the hydrocarbon fraction had 53,5% of the radioactivity in unsaponifiable lipids. This pattern resembled that obtained in livers of cholesterol-fed rats, where a block at the squalene stage occurs [32]. The sterols fraction was further fractionated on TLC. The results in Table 4 show that no unusual accumulation of radioactivity occurred in lanosterol (only 4%), cholesterol itself had only 10%, and other unknown sterol-like polar compounds contained significant labelling. In a similar experiment with liver sterol fraction, cholesterol and lanosterol accounted for 94% and 2%, respectively of the radioactivity, and none at the other spots. Thus the block in biosynthesis of cholesterol is not confined to cytochrome P-450-dependent reactions. Indeed the poor labelling by acetate of unsaponifiable lipids, but not fatty acids, indicated that some step confined to the isoprene biosynthetic pathway is likely to be low in activity. Measurement of the key enzyme in this pathway, 3-hydroxy-3-methyl glutaryl CoA reductase, gave a low activity of about 20 picomoles/min per mg microsomal protein. Another interesting observation was that incorporation of [2-14C] mevalonate was also low in BAT and about the same proportion of that in liver as acetate experiments in most respects (Table 3). These results suggest that the metabolism of mevalonate also is not expressed in BAT to the same extent as in liver. The next question we addressed to was whether concentrations of the isoprenoid compounds arising of this biosynthetic pathway were also low. The results in Table 5 giving the comparative concentrations in BAT and liver show that the ubiquinone and squalene occur at higher concentrations in BAT and that of cholesterol is about
154 70% of that in liver. In the absence of sufficient rates of isoprene biosynthetic pathway, B A T must be maintaining such high concentrations of cholesterol and ubiquinone by drawing them from blood.
Spectral characterization of cytochromes of BATmicrosomes The characterization of hemoproteins of microsomes of B A T by difference spectra was next attempted. The characteristic 450 nm p e a k obtained when C O - t r e a t e d sample was read against the control with both samples in reduced state was not seen with h o m o g e n a t e (A), mitochondria (B), or microsomes (C) of BAT, whereas even with half the concentration, liver microsomes (C, broken line) gave a m a r k e d p e a k (Fig. 1). The p e a k at 420 nm in B A T samples is due to contamination with hemoglobin and this was eliminated in the difference spectra taken according to Matsubara et al. [28] where reduced sample was read against oxidized while both were treated with CO. This showed only a p e a k at 431 nm, characteristic of cytochrome bs, but none at 4 5 0 n m (D). In contrast liver microsomes showed two peaks at 426 nm and 450 nm corresponding to cytochromes b5 and P-450, respectively. The difference spectrum of reduced against oxidized of B A T microsomes (without CO treatment) showed typical spectrum of cytochrome b5 with the two characteristic peaks at 558 nm and 431 nm (Fig.
2). Fig. 3. SDS-PAGE separation of proteins of microsomes of BAT and liver. A- 20 tzg protein of microsomes from liver of phenobarbital-treated rat; B-100/~g protein of microsomes from liver of rat exposed to cold (0.5°, 30 days); C- 200/zg protein of microsomes from BAT of the same cold exposed rat. Absorbance at 595 nm of the coomassie blue stained proteins was also scanned for each gel. The molecular weight markers were also run in the same gel. The position of cytochrome P-450 is marked by broken vertical line indicates its presence in liver microsomes as a major protein, but not in BAT microsomes.
The concentrations of the two cytochromes in microsomes of BAT and liver in cold-exposed rats are given in Table 6.
Gel electrophoresis of BAT microsomal proteins Electrophoresis ( S D S - P A G E ) was carried out on a n u m b e r of samples of BAT and liver microsomes. T h r e e typical ones are shown in Fig. 3. A small concentration of 20 ~g protein of microsomes obtained from livers of phenobarbital treated rats showed a significant band at position about 52 K D and corresponding absorption p e a k at 595 nm in
155
Fig. 4. Ouchterlony immuno double diffusion on agarose gel. The centre well contained 75/zl antiserum prepared for rat liver cytochrome P-450 species. Samples of solubilized protein of microsomes from tissues were placed in the outer wells as follows: l&2 BAT from cold exposed rats; 3&4 liver from cold-exposed rats; 5&6 liver from phenobarbital-treated rats.
the gel scan Fig. 3A. With samples of liver microsomes from cold exposed rats at 5-fold higher concentration showed similar band and peak, marked by a broken line (Fig. 3A and B). Even with 200/xg protein of BAT microsomes, the presence of a protein of molecular size about 50 KD corresponding to cytochrome P-450 was not unambiguous (Fig. 3C). It is also noted the protein pattern of microsomes of BAT was different from that of liver. Some proteins found in liver samples are not seen in BAT. Whereas cytochrome P-450 is a major protein in liver microsomes, three proteins with KD of 46, 32 and 13 are prominent in BAT microsomes. A protein band at the position marked by broken line in Fig. 3C corresponds to the size of 52 KD. But this is unlikely to be related to cytochrome P-450, as judged by Ouchterlony double diffusion test. There was no cross-reacting protein in BAT microsomes with antiserum that can detect native and b, e forms of cytochrome P-450 (Fig. 4). It was further shown that the gels with BAT microsomes did not give positive staining for cytochrome P-450-
dependent peroxidase activity, described by Moore et al. [31], similar to that obtained with liver microsomes. All this evidence supports the view that cytochrome P-450 is either absent or occurs at very low concentration in BAT.
Discussion
The foregoing results show that BAT possess the normal set of redox enzyme system in its endoplasmic reticulum, but not cytochrome P-450 and reactions dependent on it. It is likely that detoxification reactions are unnecessary in this specialized tissue. As expected of the absence of this cytochrome, biosynthesis of cholesterol was very low. BAT is a highly vasculated and innervated tissue and it is hard to separate it from the infiltering white adipose tissue and connective tissue [6]. A trace of another contaminating tissue can account for even the low rates of incorporation of acetate and mevalonate into unsaponifiable lipids observed in these
156 experiments. The defect in biosynthetic pathway of cholesterol is apparently not confined to cytochrome P-450 dependent demethylation of lanosterol, as this sterol, and radioactivity incorporated into it, did not accumulate. Our results also imply that metabolism of mevalonate, but not of acetate, is affected. The two products of isoprene pathway, cholesterol and ubiquinone, occur in BAT at high concentrations [34, 35] and therefore must depend on supply from plasma. The LDL receptors of BAT, and their responses to cold and diet, are likely to play a role in the process of proliferation of this tissue. That lipid peroxidation can occur in the virtual absence of cytochrome P-450 is another interesting finding in these studies. This was indeed foretold by the lack inhibition of lipid peroxidation by carbon monoxide, a potent inhibitor of P-450 dependent electron transfer [36]. Substrates undergoing cytochrome P-450 dependent demethylation are known to inhibit lipid peroxidation implying a competition between the two reactions for electrons [37], but this can occur at the stage of the reductase flavo-protein itself. The role of N A D P H cytochrome c reductase as a necessary component of microsomal lipid peroxidation, an iron-ADP and NADPH-mediated reaction, is now well-established [12, 38]. An antibody raised against the flavoprotein inhibited lipid peroxidation. A reconstituted system composed of purified NADPH-cytochrome c reductase along with microsomal lipids was able to show Fe2+-ADP and N A D P H dependent lipid peroxidation [39]. Reversible changes in the activity of this enzyme in microsomes in thermogenic conditions have been noted in the investigations in our laboratory. We wish to draw the attention to the high rates of oxygen peroxidation. The range of 25-35 was obtained for the ratio of O2/MDA in different experiments, the reducing source for this reduction of oxygen is partly provided by-NADPH, whose oxidation by NADPH-cytochrome c reductase appears to be responsible for keeping iron in the active, reduced-state. It is implicit that the extra oxygen must have been consumed to produce lipid hydroperoxides. Since these reactions involve no conservation, the energy will be released as heat.
Microsomal redox systems will thereby assume a role of considerable significance in cellular thermogenesis in addition to mitochondria and peroxisomes [40].
Acknowledgement Financial assistance from the Indian Council of Medical Research, New Delhi for the proj ect entitled 'Iron-dependent Metabolic Modulation' is acknowledged.
References 1. Smith RE, Horwitz BA: Brown fat and thermogenesis. Physiol Rev 49: 330-425, 1969 2. Flatmark T, Pederson JI: Brown adipose tissue mitochondria. Biochim Biophys Acta 416: 53-103, 1975 3. Foster DO, Frydman ML: Tissue distribution of cold-induced thermogenesis in conscious warm- and cold-acclimated rats reevaluated from changes in tissue blood flow. The dominant role of brown adipose tissue in the replacement of shivering by nonshivering thermogenesis. Can J Physiol Pharmacol 56: 110-122, 1978 4. Lindberg O, Cannon B, Nedergaard J: Thermogenic mitochondria, in 'Mitochondria and Microsomes' (Lee CP et al., eds). Addison-Wiley, Reading, MA, USA 1981, pp 93-119 5. Cannon B, Nedergaard 3: The biochemistry of an inefficient tissue: Brown Adipose Tissue. Essays Biochem 20: 110-163, 1985 6. Ricqier D, Gerrais C, Kader JC, Hemon P: Partial purification by Guanosine-5'-diphosphate-agarose affinity chromatography of the 32000 molecular wt polypeptide from mitochondria of Brown Adipose Tissue. FEBS Lett 101: 35-38, 1979 7. Lin C, Klingenberg M: Characteristics of the isolated purine nucleotide binding protein from brown fat mitochondria. Biochemistry 21: 2950-2956, 1982 8. Nicholls D: Brown Adipose Tissue Mitochondria. Biochim Biophys Acta 549: 1-29, 1979 9. Cannon B, Lindberg O: Mitochondria from Brown Adipose Tissue: Isolation and properties. Methods Enzymol 55F: 65-78, 1979 10. Seshadri Sekhar B, Ramakrishna Kurup CK, Ramasarma T: Generation of hydrogen peroxide by Brown Adipose Tissue Mitochondria. J Bioenergetics Biomemb 19: 397407, 1987 11. Hochstein P, Nordebrand K, Ernster L: ADP-activated lipid peroxidation coupled to TPNH oxidase system of mi-
157
12. 13.
14. 15. 16. 17.
18. 19.
20.
21.
22.
23. 24. 25.
26.
27. 28.
29.
crosomes. Biochem Biophys Res Commun 14: 323-328, 1964 Wills ED: Lipid peroxidation formation in microsomesGeneral considerations. Biochem J 113: 325-332, 1969 Kappus H, Sies H: Toxic effects associated with oxygen metabolism: Redox cycling and lipid peroxidation. Experientia 37: 1233-1241, 1981 de Duve D: Evolution of the peroxisome. Ann NY Acad Sci 168: 369-381, 1969 Howirtz BA: Can J Physiol Pharmacol A54: 45-48, 1976 RamasarmaT: GenerationofHzOzinbiomembranes. Biochim Biophys Acta 694: 69-93, 1982 Ramasarma T, Maukassah Kelly S, Hochstein P: Inhibition of microsomal lipid peroxidation by cytosolic protein in presence of ADP and high concentration of Fe 2÷. Biochim Biophys Acta 796: 243-250, 1984 Strittmatter P: NADH-cytochrome b5 reductase. Methods Enzymol 10: 561-565, 1967 Masters BSS, Williams CH Jr, Kamin H: The preparation and properties of microsomal TPNH- cytochrome c reductase from pig liver. Methods Enzymol 10: 565-573, 1967 Imai Y, Ito A, Sato R: Evidence for biochemically different types of vesicles in the hepatic microsomal fraction. J Biochem (Tokyo) 60: 417-428, 1966 Guengerich FP: In Principles and Methods of Toxicology (Hayes WA, ed). Raven Press, New York, USA, 1982, pp 609-634 Joshi VC, Jayaraman J, Ramasarma T: Incorporation of mevalonic acid-2-C14into ubichromenol and coenzyme Q in rat_ Indian J Exptl Biol 1: 113-123, 1963 Avigan J, Goodman DS, Steinberg D: Studies of cholesterol Biosynthesi. J Biol Chem 238: 1283-1286, 1963 Stadtman TC: Preparation and assay of cholesterol and ergosterol. Methods Enzymol 3: 392-394, 1957 Lester RL, Crane FL: The natural occurrence of coenzyme Q and related compound. J Biol Chem 234: 2169-2174, 1959 Lowry OH, Rosebrough NJ, Farr AL, Randall RJ: Protein measurements with folin phenol reagent. J Biol Chem 193: 265-275, 1951 Estabrook RW, Werringloer J: Estimation of cytochrome P-450. Methods Enzymol 52: 212-220, 1978 Matsubara T, Koike M, Touchi A, Yochlmo A, Sugero L: Quantitative determination of cytochrome P-450 in rat liver homogenate. Anal Biochem 75: 596-603, 1976 Thomas PE, Lu AYH, Kawalek J, West SB, Levin W:
30_
31.
32.
33. 34. 35.
36. 37.
38.
39.
40.
Properties of electrophoreticaUy homogeneous phenobarbital inducible and ~-naphthoflavone-inducible forms of liver microsomal cytochrome P-450. J Biol Chem 251: 7929-7933, 1976 Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (Lond) 227: 680-685, 1970 Trazarkos JM, Bowen WD, Shafice A, Fischer RT, Gaylor JL: Cytochrome P-450 dependent oxidation of lanosterol in cholesterol biosynthesis. J Biol Chem 259: 13402-13412, 1984 Krishnaiah KV, Joshi VC, Ramasarma T: Effect of dietary cholesterol and coenzyme Q on the isoprene synthesis in rat liver. Arch Biochem Biophys 121: 147-153, 1967 Moore RW, Welton AF, Aust SD: Methods Enzymol 52: 324-331, 1978 Joel CD, Ball EG: The electron transmitter system of Brown Adipose Tissue. Biochemistry 1: 281-284, 1962 Sivaramakrishnan S, Ramasarma T: Activation of succihate dehydrogenase in Brown Adipose Tissue Mitochondria. Indian J Biochem Biophys 15: 14-18, 1978 Ernster L, Nordenbrand K: Microsomal lipid peroxidation. Methods Enzymol 10: 574-580, 1967 Orrenius S, Dallner G, Ernster L: Inhibition of the TPNHlinked lipid peroxidation of liver microsomes by drugs undergoing oxidative demethylation. Biochem Biophys Res Commun 14: 329-334, 1964 Kappus H: Lipid peroxidation; Mechanisms, Analysis, Enzymology and Biological Relevance in 'Oxidative stress' (Sies H ed) Academic Press, London, 1985, pp 273-310 Pederson TC, Buege JA, Aust SD: The role of reduced nicotinamide adenine dinucleotide phosphate-cytochrome c reductase in liver microsomal lipid peroxidation. J Biol Chem 248: 7134-7141, 1973 Ramasarma T, Seshadri Sekhar B, Ramakrishna Kurup CK: Cellular Thermogenesis: A New Approach in 'Bioenergetics: Structure and Function of Energy Transducing Systems (Tozawa T, Papa S, eds) Japan Sci Soc Press Tokyo/Springer-Verlag, Berlin, 1987, pp 225-233
Address for offprints: B. Seshadri Sekhar, Department of Biochemistry, Indian Institute of Science, Bangalore 560 012, India
