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Life Sciences, Vol. 32, pp. 2123-2130 Printed in the U.S.A. Pergamon Press FATTY ACID UTILIZATION AND PURINE NUCLEOTIDE BINDING IN BROWN ADIPOSE TIS...

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Life Sciences, Vol. 32, pp. 2123-2130 Printed in the U.S.A.

Pergamon Press

FATTY ACID UTILIZATION AND PURINE NUCLEOTIDE BINDING IN BROWN ADIPOSE TISSUE OF GENETICALLY OBESE (ob/ob) MICE Sylvette Bas*, Elisaketh Imesch*, Daniel Ricquier++, Francoise Assimacopoulos-Jeannet , Josiane Seydoux** and Jean-Paul Giacobino*

* Departement de Biochimie Medicale and ** Departement de Physiologie, Centre Medical Universitaire t tt

9, avenue de Champel, 1211 Geneve (Switzerland) Laboratoire de Recherches Metaboliques 64, avenue de la Roseraie, 1205 Geneve (Switzerland) Laboratoire de Physiologie comparee (C.N.R.S./L.A. 307) Universite P.M. Curie, 4 place Jussieu, 75 230 Paris Cedex 05 (France) (Received in final form February 7, 1983) Summary

The activities of the main enzymes involved in fatty acid utilization i.e. palmitoyl CoA synthetase as well as peroxisomal and mitochondrial B-oxidation were measured in brown adipose tissue homogenates of lean and -ob/ob mice kept at 23°C or acclimated at 4°C. The proton conductance pathway, i.e. the number of purine nucleotide (GDP) binding sites and the percentage of 32,000 polypeptide in brown adipose tissue mitochondria were also measured. In the ob/ob mice at 23 C, the specific activities of the palmitoyl CoA synthetase and of the B-oxidation as well as the number of GDB binding sites were lower than in the lean mice by 26%, 43% and 37%, respectively. The percentage of 32,000 polypeptide, however, was the same in both groups. In the ob/ob mice at 23" C, the lower homogenate B-oxidation specific activityas due to the fact that the peroxisomal and mitochondrial specific activities were 44% and 37% lower, respectively. Cold acclimation at 4" C was found to cause an increase of the palmitoyl CoA synthetase specific activity, of the palmitoyl CoA synthetase and peroxisomal B-oxidation total activities and of the number of GDP binding sites, in both lean and ob/ob mice. Cold acclimation increased the percentage of 32,000 polypeptide in the ob/ob mice only. It has been suggested that brown adipose tissue plays a role in dietinduced thermogenesis and thus might contribute to whole body metabolic efficiency and resistance to obesity (l-3). It has also been shown that thermogenic capacity is reduced in pre-obese and obese ob/ob mice (4-6) and this could explain the high metabolic efficiency observed ixtEse mice (7). Recent studies have shown that in ob/ob mice, the metabolism of brown adipose tissue is impaired in the following ways : there is a marked decrease of sympathetic nervous system activity (B,9), of the metabolic response of brown adipose tissue to norepinephrine (9) and of its capacity to catabolize octanoate (10). It has also been reported that in ob/ob mice, there is a decrease of the number of mitochondrial purine nucleom (GDP) binding sites (ll-13), i.e. of the specific sites on a polypeptide of molecular weight 32,000 associated with the thermogenic proton conductance pathway of brown adipose tissue (14). This decrease was found not to be accompanied by any change in the percentage of the mitochondrial 30-33,000 polypeptide (12). For all of these parameters, cold 0024-3205/83/182123-08$03.00/O Copyright (c) 1983 Pergamon Press Ltd.

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acclimation at 4° C was found to restore the values partially~or to practically those obtained for lean mice (8-10,12). The aim of the present study was to further investigate the cause of the decreased octanoate catabolism observed in the brown adipose tissue of ob/ob mouse and of its partial restoration after cold acclimation (10). For this purpose, measurements were made of the a c t i v i t i e s , in brown adipose tissue homogenate, of the main enzymes involved in fatty acid u t i l i z a t i o n i.e. the enzyme catalyzing fatty acid activation, palmitoyl CoA synthetase (EC 6.2.1.3.) and those catalyzing palmitoyl CoA #-oxidation in the peroxisomes and mitochondria. The brown adipose tissue studied was obtained from lean and ob/ob mice kept at 23° C or acclimated at 4° C. Since both fatty acid catabolism and the mitochondrial proton conductance pathway are involved in brown adipose tissue heat production, the GDP binding value and the percentage of 32,000 polypeptide of mitochondria were also measured in the brown adipose tissue from the same animal.

M a t e r i a l s and Methods

11-13 week old male mice C57 BL/6J (ob/ob) and lean controls (+/+) were obtained from the Jackson Memorial Laboratories, Bar Harbor, Maine USA., and fed ad libitum with standard laboratory chow. They were maintained at either 28 or 23°C with a fixed 12 h light cycle. For cold acclimation, the mice were f i r s t transferred to 15°C for 2 weeks and then exposed to 4°C for 4 weeks. They were killed by cervical dislocation and the interscapular and cervical brown adipose tissue were removed and carefully separated from surrounding white fat. The tissue was then homogenized in a Potter-Elvejhem homogenizer in 5 volumes of sucrose 0.25 M, glycylglycine buffer 0.02 M at pH 7.5, EDTA 0.001 M, centrifuged at 1,300 x g for 10 min and the resulting nuclei-free homogenate was used for all subsequent enzymatic studies. The palmitoyl CoA synthetase a c t i v i t y was measured using the incubation medium described by Bar-Tana et al (15) but with the addition of bovine serum albumine 1.16 mg/ml. The homogenate palmitoyl CoA oxidation was measured according to the technique described by Shindo and Hashimoto (16) except that [1- C] palmitoyl CoA 40 ~M(sp~!fic a c t i v i t y 1.25 Ci per mmole) was used~s labelled substrate instead of [U- C] palmitoyl CoA and the CoASH and NAD concentrations were increased to 0.2 mM and I mM, respectively. The palmitoyl CoA p-oxidation a c t i v i t y was measured by the production of labeled acid-soluble acetyl CoA. The technique of labelled palmitoyl CoA oxidation allows for the measurement of both the peroxisomal and the mitochondrial ~-oxidations (16-18). Unlike the mitochondrial ~-oxidation system, the peroxisomal system was found to be independent of the mitochondrial respiratory chain and therefore insensitive to specific inhibitors of this chain in the l i v e r (16 19) as well as in brown adipose tissue (20, 21). In the present study, the homogenate was incubated in the absence or in the presence of KCN 1 mM. The results obtained in the absence of KCN represent the total (peroxisomal + mitochondrial) p-oxidation, whereas those obtained in the presence of KCN represent the CN insensitive peroxisomal ~-oxidation. The mitochondrial CN - sensitive ~-oxidation was calculated by subtracting the peroxisomal ~-oxidation from the total ~-oxidation value. The incubations were of 10 min. duration and at 37°C. The amounts of homogenate proteins used per assay were 10 ~g and 40 ~g for palmitoyl CoA synthetase and ~-oxidation, respectively. In both assays, the amounts of proteins chosen were in the range in which the enzymatic a c t i v i t i e s increased linearly with the protein concentration. Mitochondria were isolated by d i f f e r e n t i a l centrifugation of brown adipose tissue homogenate according to Ricquier et al. (22). Proteins were determined by

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the Coomassie B r i l l i a n t b l u e method, (23). The GDP binding to isolated mitochondria was measured using equilibrium dialysis with [8-~H]-GDP (specific activity 11.3 Ci per mmole) as described by Lin and Klingenberg (24). The amount of the 32,000 polypeptide in isolated mitochondria was calculated from the densitometric traces of sodium dodecylsulfate-polyacrylamide gel electrophoresis of mitochondria proteins as described by Ricquier and Kader (25). Student's significance.

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[1-14C]-palmitate and [l-14C]-palmit~yl CoA were purchased from New England Nuclear, Boston, Mass. and [8- H] GDP was obtained f r o m the Radiochemical Center, Amersham, England. All other chemicals (reagant grade) were obtained from Merck, Darmstadt, F.R.G. or from Sigma, St Louis, Miss. Results

The total amount of proteins in the brown adipose tissue~homogenate of on~ mouse was found to be similar in lean and ob/ob mice (4.5 mg ~ 0.4 and 4.9 mg 0.3, respectively. ; n = 11). It was increased by cold acclimation respectively 2.2 and 2.4 times in lean and ob/ob mice. Fig.1 shows the palmitoyl CoA synthetase activity values of brown adipose tissue homogenate. At 23°C, the specific activity was found to be slightly lower

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FIG. 1 Palmitoyl CoA synthetase a c t i v i t y in the homogenate of brown adipose tissue from lean a+nd ob/ob mice kept at 23°C or acclimated at 4°C. The columns represent the mean - SEM of the number of experiments indicated in parenthesis under each column :::::::::::::::::::::::: ob/ob mice. The results are expressed in specific a c t i v i t i e s ( l e f t ) or in the total a c t i v i t i e s in the brow~ adipose tissue~ of one mouse ( r i g h t ) . * : p < 0.05 ob/ob vs lean control ; : p < 0.01, ~ : p < 0.001 acclimated at 4°C vs 23~-C.-

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in ob/ob mice than in the c o n t r o l s w h i l e no d i f f e r e n c e in t o t a l a c t i v i t y could be observed f o r the two groups. Cold a c c l i m a t i o n caused a 1.7 and 2 . 0 time increase of s p e c i f i c a c t i v i t y in lean and ob/ob mice, r e s p e c t i v e l y . Since cold

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A. Palmitoyl CoA # - o x i d a t i o n s p e c i f i c a c t i v i t i e s in the homogenate of brown adipose t i s s u e from lean and 9b/ob mice kept at 23 ° C or acclimated at 4 ° C. The columns represent the mean SEM of the number of experiments i n d i c a t e d in parenthesis under each column. Total = the a c t i v i t y measured in the homogenate in the absence of KCN. Peroxisomal = the a c t i v i t y measured in the homogenate in the presence of K C N . M i t o c h o n d r i a l = the d i f f e r e n c e between t o t a l and peroxisomal activities;!iiiiiiiiiiiiii= ob/~_bmice * • p < 0 . 0 5 , * * : p < 0.005 and * * * : p < 0.001 ob/ob vs lean c o n t r o l ; : p < ' 0 . 0 5 acclimated at 4 ° C vs 23 ° C. B. Palmitoy-I CoA # - o x i d a t i o n t o t a l a c t i v i t i e s in the homogenate of br~wn adipose t i s s u e from lean and ob/ob mice kept at 23 ° C or acclimated at 4 ° C. : p < 0.001 acclimated at ~ - C vs 23 ° C.

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acclimation caused an approximately two-fold increase of the protein content of the brown adipose tissue homogenate, the calculated total a c t i v i t i e s were found to have increased by 3.6 and 4.2 times in lean and ob/ob mice, respectively. Fig.2 A and B shows the t o t a l , the peroxisomal and the mitochondrial palmitoyl CoA ~-oxidative a c t i v i t i e s of brown adipose tissue homogenate. The peroxisomal #-oxidation represents 59 % of the total 6-oxidation in lean mice at 23°C. In the ob/ob mice at 23°C the value obtained for the total #-oxidation specific activity was found to be 43% lower than in the controls due to the fact that the peroxisomal and mitochondrial a c t i v i t i e s were 44% and 37% lower, respectively. In the ob/ob mice at 23°C the value obtained for B-oxidation total a c t i v i t y per mouse was found to be 40% lower than the control value due to the 44% lower peroxisomal total a c t i v i t y value. No s i g n i f i c a n t difference, however, was found for the mitochondrial total a c t i v i t y value, which was calculated by subtraction. Fig. 2 also shows the effects of acclimation at 4°C on the #-oxidation values. Cold acclimation had no e f f e c t on the s p e c i f i c a c t i v i t y values, in e i t h e r lean or ob/ob mice except f o r a s l i g h t i n h i b i t i o n of the mitochondrial a c t i v i t y in lean mice. On the other hand, i t was found to cause an increase of the total a c t i v i t y of 2.0 and 2.2 times in lean and ob/ob mice, r e s p e c t i v e l y , due to a s i g n i f i c a n t increase of the peroxisomal total a c t i v i t y of 2.6 and 2.8 times in lean and ob/ob mice, respectively. TABLE I GDP Binding Sites and 32,000 Polypeptide in Brown Adipose Tissue Mitochondria Lean mice

4oc

23°C

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0.24

C 0.01 (9)

0.47

t

0.04 (4) a

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0.15

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6.0 C 0.2 (8) 6.7 t 0.I + The results represent the mean - SEM of the number of experiments parenthesis and are expressed in nmoles of bound GDP.mg- of proteins ( s p e c i f i c values). The 32,000 polypeptide results are percent ofbthe total mi~ochondrial proteins. **:p < 0.005 ob/ob vs control ; : p < 0.05, : p < 0.001 acclimated at 4°C vs 23°C.

(5) a (5) b

indicated in mitochondrial expressed in lean

Table I shows that in ob/ob mice, there were 37% fewer mitochondrial GDP binding sites than in the controls. Cold acclimation, however, was found to increase the number of GDP binding sites by 2.0 and 2.9 times in lean and ob/ob mice, respectively, suppressing the difference observed between the two groups at 23°C. No difference was found between the ob/ob and the lean control mice with regard to the amount of 32,000 polypeptide in brown adipose tissue mitochondria. Cold acclimation was found to cause a s l i g h t increase of this amount in the ob/ob mice but to have no e f f e c t in the lean controls. Acclimation at 28°C which is a temperature used in previous studies on GDP binding sites and 32,000 polypeptide (11,12) was found to r e s u l t in no s i g n i f i c a n t differences, as compared with acclimation at 23°C in any of the parameters studied, i . e . , palmitoyl CoA synthetase a c t i v i t y , ~-oxidation a c t i v i t y , number of GDP binding sites and amount of 32,000 polypeptide (data not shown).

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Discussion In an attempt to better understand the decreased capacity of the brown adipose tissue of ob/ob mice to u t i l i z e f a t t y acids as well as the partial restoration of this capacity by cold acclimation ( I 0 ) , measurements were made of the a c t i v i t i e s of palmitoyl CoA synthetase and of those of the peroxisomal and mitochondrial #-oxidation pathways. The specific palmitoyl CoA synthetase a c t i v i t y value obtained for cold-acclimated mice in the present study was very close to that found by Norman and Flatmark (26) in cold-acclimated guinea pig brown adipose tissue mitochondria, i f i t is assumed that one third of total homogenate proteins in brown adipose tissue are mitochondrial as found by Skala et al (27). In the ob/ob mice, this value was only s l i g h t l y lower than in the lean mice. A peroxisomal #-oxidation system (CN--insensitive) has been found in rat brown adipose tissue (20,21) by means of the palmitoyl CoA-dependant NAD reduction technique. In the present study # - o x i d a t i o n l ~ c t i v i t y was determined by measuring the release of the f i r s t acetyl unit of [ i - C] palmitoyl CoA (16). I f the peroxisomes of brown adipose tissue, like those of the l i v e r , catalyze only the degradation of palmitoyl CoA to octanoyl CoA (28), the peroxisomal #-oxidation a c t i v i t y measured by means of the technique used in this study would be overestimated by a factor of 2. With this factor taken into account, i t could be estimated that peroxisomes and mitochondria contribute about 40% and 60% respectively to the total brown adipose tissue palmitoyl CoA degradation. This relative contribution of peroxisomal and mitochondrial ~-oxidation a c t i v i t y is comparable to that which can be deduced from the results of Kramar et al. (20) and the peroxisomal ~-oxidation specific a c t i v i t y determined in the present study was p r a c t i c a l l y identical to that reported for the rat by Nedergaard et al (21). In brown adipose tissue from ob/ob mice, both peroxisoma] and mitochondrial #-oxidation specific a c t i v i t i e s were s i g n i f i c a n t l y lower than in control brown adipose tissue. This is in sharp contrast to the finding that in l i v e r of ob/ob mice, the peroxisomal #-oxidation specific a c t i v i t y was 3 times higher than that of the lean mice (29).In the l i g h t of the present findings, the decreased capacity for octanoate degradation in ob/ob mouse brown adipose tissue observed previously (10) should not be attributed to a decrease in f a t t y acid activation but rather, at least in part,to the 40 % decrease of the total #-oxidative capacity of the ob/ob mouse brown adipose tissue. Since peroxisome respiration is involved in heat production and energy dissipation (30) i t is possible that the decrease in brown adipose tissue peroxisomal #-oxidation a c t i v i t y plays a role in the reduced thermogenic capacity and increased metabolic efficiency observed in ob/ob mice (4-7). Cold acclimation was found to selectively stimulate palmitoyl CoA synthetase a c t i v i t y in the lean as well as in the ob/ob mice. Despite the high specific a c t i v i t y of palmitoyl CoA synthetase, i t has been suggested that this enzyme is rate-limiting in the sequence of reactions leading to fatty acid degradation in isolated mitochondria (26) and, consequently, the increase in its a c t i v i t y might yield an overall increase in ~-oxidation. Cold acclimation however did not affect the specific but increased the total ~-oxidation a c t i v i t i e s in lean or ob/ob mice. The present results indicate that i f cold acclimation in ob/ob mice p a r t i a l l y restores octanoate degradation (10) i t would do so by increasing both acyl CoA synthetase and ~-oxidative capacities. The observed decrease in the number of GDP binding sites in brown adipose tissue mitochondria of ob/ob mice and the cold-induced increase of this number in that of both lean and ob/ob mice are in agreement with previous findings (11-13). A similar decrease of the number of GDP binding sites was also observed in db/db mice (31). Although a slight increase has been reported of the percentage of 30-33,000 protein after cold acclimation in both lean and ob/ob mice (12), in the present study, an increase of the 32,000 protein was foun---d

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only in the ob/ob mice. This discrepancy is d i f f i c u l t to explain but may be due to the fact that the effect of cold acclimation on the 32,000 protein in mice is very s l i g h t as found both in the present study and by Hogan and Himms-Hagen (12). I t is of interest to note that this s l i g h t effect in mice is in contrast with the considerably more marked effect reported for rats (25, 32). In the ob/ob mice at 23° C, the specific a c t i v i t i e s of the palmitoyl CoA synthetase and of the ~-oxidation as well as the number of GDP binding sites were lower than in the lean mice. The fact that the differences between lean and ob/ob mice in ~-oxidative capacities and in the number of GDP binding sites are diminished or suppressed by cold acclimation may indicate that they are not due to an i r r e v e r s i b l e primary defect of brown adipose tissue but rather to i n s u f f i c i e n t sympathetic stimulation of brown adipose tissue which is restored to normal by cold acclimation. Acknowledgements We wish to thank Miss C. Colomb and Miss B. Pastori for t h e i r excellent technical assistance and Mrs Judith Noebels for her invaluable help in the editing of the manuscript. This work was supported by the Swiss National Science Foundation grant no. 3.385.78 and by the French Fonds National de la Recherche Scientifique. References 1. 2. 3. 4. 5. 6. 7. 8. 9. I0. ii. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.

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