Abstracts--Strategies , . cold Symposum 97

October 7, 1977, Afte rnoon

VOLUNTARY CONTRIBUTIONS ON "THERMOGENESIS

AND TEMPERATURE REGULATION"

C h a i r m a n : W i l l i a m R. D a w s o n ( U n i v e r s i t y of M i c h i g a n )

CENTRAL AND PERIPHERAL INTERACTIONS IN THE CONTROL OF TRER- MOGENESIS. E. Z e i a b e r g e r , Zentrum fur P h y s i o l o g i e , J u s t u s L l e b l g - U n i v e r s l t g t Gle~sen, Aulweg 129, D-63 Gtessen.

An e s s e n t i a l f e a t u r e of a c u t e co ld exposure i s the a c t i - v a t i o n of the p e r i p h e r a l sympathe t ic system. The no rad rena - l i n e (NA) l i b e r a t e d from t h i s system reduces heat l o s s and promotes heat p roduc t ion mechanisms. NA i s a l s o known to In- f luence the hypothalamic thermoregulatory centers. The mean body temperature at which guinea plgs start to shiver (~b-Sh) has been found to be increased after an intrahypothalamic (i.h.) mlcroinJection of NA and decreased a f t e r i . h . a p p l i - c a t i o n of an o-adrenerglc b locke r (Zeisberger & BrUck: PflU- gets Arch. 362, 113, 1976). Electrical stimulation of the lower brain stem at sites presumed to be parts of the as- cending catecholamlnerglc pathways (ACP) had the same effect as i . h . NA m i c r o i n J e c t i o n ( S z e l ~ n y l e t a Z . : Pf lUger s Arch. 364, 123, 1976). The s e n s i t i v i t y to i.h. NA microlnJection was h i g h e r in cold adapted (CA) and in newborn (NB) than in warm adapted (WA) guinea p i g s . The s e n s i t i v i t y to i . h . phen- to lamine i n j e c t i o n s was r e c i p r o c a l l y r e l a t e d . Th i s i n d i c a t e s t h a t the a c t i v i t y in the ACP Is h ighe r in WA than in CA or in NB an ima l s . By c o n t r a s t t he p e r i p h e r a l r e l e a s e of NA i s i n c r e a s e d in CA an ima l s exposed to co ld and i s g r e a t e r in NB an ima l s than in WA an imals even a t n e u t r a l t empera tu re . In o rde r to answer the q u e s t i o n whether the NA r e l e a s e d p e r i p h - e r a l l y i n f l u e n c e s the n e u r a l a c t i v i t y in the ACP, the p e r - i p h e r a l NA level in NA guinea pigs was increased by i.m. NA i n f u s i o n (1 .3 mE/ks • h) l a s t i n g 2 hrs and the e f f e c t on Tb-Shwas studied. The Tb-Sh was lowered by I"C as early as 15 minutes after the beginning of the infusion. After the infusion the Tb-Sh remained low for at least 3 hours. By contrast i.m. infusion of adrenaline increased the~b-Sh. It is concluded that the high peripheral NA level supresses the a c t i v i t y in the ACP by some negative feedback mechanism. Thlscould be the basic mechanism of the cold-adaptive shift of Tb-Sh.

BROWN ADIPOSE TISSUE AND NON-SHIVERING THERMOGENESIS Barbara Cannon, Jan Nedergaard, Ulf Sundin and Olv Ltndberg. The Wenner-Gren I n s t i t u t e , U n i v e r s i t y of Stockholm, N o r r t u l l s g a t a n 16, $-113 45 Stockholm, Sweden.

By mlcrocalorimetric measurements we have recently demonstra ted chat the h e a t i n g c a p a c i t y of brown f a t in hamsters i s low when c a l c u l a t e d on the b a s i s o f t he whole an imal . None the le s s , t h e r e i s an ex t remely h igh oxygen con- sumption in the t i s s u e and t h e r e f o r e c o n s i d e r a b l e l o c a l heat evolution. The high rate of oxygen utilisation re- quires either a high ATP synthesizing capacity Of the mlto- chondrla together with a high rate of ATP hydrolysis oP a loosening of coupling between oxygen consumption and ATP synthesis. We have demonstrated in isolated mitochondrla that an ATP syntheslsin K system which could parallel the r e s p i r a t o r y a c t i v i t y found dur ing thermogenes i s i s not p r e - sen t in brown f a t mi tochondr ia from hamster . I n s t e a d , a loosen ing of coup l ing is requ i red which in some manner seems to involve cy top lasmic pu r lne n u c l e o t i d e s (eg. GDP). When GDP i s bound to a s p e c i f i c membrane p r o t e i n of i s o l a - t e d , uncoupled m i t o c h o n d r i a , they become capab le of energy c o n s e r v a t i o n . In the thermogenic c e l l acyl-CoA w i l l be pro- duced and we have shown t h a t low c o n c e n t r a t i o n s of pa lmi - toyl-CoA c a n remove GDP from its mi tochond r i a l b ind ing s i t e . Th i s a c t i o n would f a c i l i t a t e a loosening of coup l i ng . The apparen t K m fo r o x i d a t i o n of acyl-CoA i s high so i t i s f e a - s i b l e to t h i n k t h a t e l e v a t e d acyl-CoA c o n c e n t r a t i o n s can be found a t l e a s t l o c a l l y . When e n t r y in to the c i t r i c a c i d c y c l e i s l i m i t i n g (eg. due to l a c k of condensing p a r t n e r ) acyl-CoA combustion w i l l on ly preceed through ~ - o x i d a t i o n to the a c e t y l l e v e l . High r a t e s of o x i d a t i o n demand t h a t the l e v e l of c i t r i c a c i d c y c l e i n t e r m e d i a t e s must be i n - c r e a s e d , and we have found t h a t brown f a t mi tochondr ia possess an e f f i c i e n t pyruva te ca rboxy lase which, du r ing the rmogenes l s , can s y n t h e s i z e oxa loace ta te as a condensing p a r t n e r for acety l -CoA. By t h i s r e a c t i o n it is thus p o s s i b l e to i n c r e a s e the r a t e of energy d i s s i p a t i o n in brown f a t .

TEMPERATURE-INDUCE~ CHANGES IN THE METABOLITE PATTERN UPON NOREPINEPHRINE STIMULATION OF HAMSTER BROWN ADIPOCYTES. Vlbeke S.M. Bernson, Per Lundberg and B e r t l l P e t t e r s s o n ,

The Wenner--Gren l o s t l t u t e , U n i v e r s i t y of Stockholm, Norr- tullsgatan 16, S-I13 45 Stockholm, Sweden.

At low t empera tu re a c e t a t e has been d e ~ n s t r a t e d to be a major end-product of f a t t y a c i d o x i d a t i o n in brown ad ipose t i s s u e ml tochondr ia from hamste r s . In i s o l a t e d brown ad ipo - c y t e s n o r e p i n e p h r i n e (NE) i s the n a t u r a l thermogenic e f f e c - t o r which s t i m u l a t e s l i p o l y s i s and i n c r e a s e oxygen consump- t i o n . Free f a t t y a c i d s can r e p l a c e NE as a s t i m u l a t o r of oxygen consumption. The aim of our i n v e s t i g a t i o n i s to study to what ex ten t t he f r e e f a t t y a c i d - i n d u c e d r e s p i r a t i o n in i s o l a t e d hamster brown s d i p o c y t e s r e p r e s e n t e d p roduc t ion of c i t r i c a c i d c y c l e i n t e r m e d i a t e s and a c e t a t e . As the hamster, a h i b e r n a t o r , can va ry i t s body t empera tu re between about 38"C and 5"C, the temperature dependence of these pathways was investigated. Isolated brown adipocytes were incubated in K r e b s - g l n g e r - p h o s p h a t e - b u f f e r (KRPB) in an oxygen e l e c - t rode chamber and oxygen consumption was measured a s a func- t i o n of added NE or palmitic acid. Halo,ate was used to test to what extent the rate of oxygen consumption was dependent on the citric acid cycle. At 370C in KRPB bubbled with 5Z CO 2 in air the maximal oxygen consumption upon NE stimula- tion was 490 nmol O2/mln x 106 cells, of which about 75% re- presents the citric acid cycle, and 23%, which cannot be in- hibited by ~alonate, represents fatty acid oxidation s topp- ing a t the acetyl -CoA l e v e l . At 30°C 50% r e p r e s e n t s the c i - t r i c acid cycle and 50% the acetyl-level, while at 2O°C as much as 77% of the maximal NE-inducible oxygen consumption r a t e r e p r e s e n t s end p roduc t s a t the a c e t y l - l e v e l . Ace t a t e l e v e l s in l i v i n g hamsters were measured by a n a l y s i n g blood samples wi th an enzymatic a s say in a spec t ropho tomete r . The acetate concentration in blood from warm-adapted, cold-adap- ted and h i b e r n a t i n g hamsters were in the mmolar r ange , wi th the highest values found in hibernating animals These da t a t o g e t h e r with old literature references reporting gQ-values lower than 0.7 measured during hibernation could indicate that acetate is an important "end" product in brown adipose tissue during hibernation and/or arousal from hlbernat Ion.

ULTKASTRUCTURAL DEMONSTRATION OF A UNILOCULAR TO MULTI- LOCULAR TRANSFORMATION OF ADIPOCYTES IN DEVELOPING HAMSTER INTERSCAPULAR ADIPOSE TISSUE. POSSIBLE ROLE OF THE SYMPATHETIC INNERVATION. Myriam N~chad. The Wenner-Gren Institute, University of Stockholm, Norrtullsgatan 16, S-I13 45 Stockholm, Sweden.

During the development of Interscapular adipose tissue in the hamster, the adipocytes are first not multi- but unilocular. The development of the multilocularity does not start before shout 4 days pos~ p~tt#~and is complete by 15 days post pa~-~um. We have investigated this process by e l e c t r o n microscopy, n o r e p l n e p h r i n e d e t e r m i n a t i o n s and light microscopic autoradlography. We observed two diff- erentiatlon pathways occurring simultaneously: (I) form- ation of m u l t i l o c u l a r ad ipocy t e p r e c u r s o r c e l l s from e n d o t h e l i a l c e l l s as p r e v i o u s l y d e s c r i b e d ; (2) d i r e c t t r a n s f o r m a t i o n of u n i - i n t o m u l t i l o c u l a r a d l p o c y t e s showing the same c h a r a c t e r i s t i c development of the chond- rlome as in pathway ( I ) and format ion of smal l f a t d r o p l e t s in the p e r i p h e r a l cytoplasm. By 12-15 d a y s , the u l t r a - structure of these cells has become indlstlnguishable from those formed via (1). Such direct transformation of white i n to brown f a t has been r e p o r t e d in a d u l t human p e r i r e n a l fat in association with e.g. seve re chronic hypoxemia and pheochromacytoma. In the developing hamster brown fat, i t occurs p a r a l l e l w i th a marked i n c r e a s e in t i s s u e n o r - e p i n e p h r i n e c o n c e n t r a t i o n . The d e n s i t y of sympathet ic nerve f i b r e s i s be ing i n v e s t i g a t e d by l i g h t microscopic su to r ad iog raphy .

SUPRACRIASMATIC DINUCLEAR LESION ALTERS CIRCADIAN TEMPERATURE RHYT~'S AMPLITUDE AND TIMING IN LIGHT-DARK SYNCHRONIZED RATS. Powel l , E .N. , J .N . P a s l e y , B. Brockway, L.E. Scheving, W. Lu- banovic and F. Halberg . U n i v e r s i t i e s of Minnesota , Minneapo l i s , Minn. , 55455, and Arkansas , L i t t l e Rock, 72201, USA

I n t r a p e r l t o n e a l t empera tu re was t e l e m e t e r e d (Space L i f e Sc ience 2 :437 , 1971) a t l O ' - I n t e r v a l s fo r 9 days from young a d u l t male inbred F i she r r a t s kept a t 24 ± l ' C envi ronmenta l t empera tu re wi th food and wate r f r e e l y a v a i l a b l e , s i n g l y in cages measuring 6 ' ~ x 5"H x 10'% in l i g h t ( approx lmate ly 25 lux ) and darkness a l t e r n a t i n g every 12-h (LD 1 2 : 1 2 ) . The r a t s had been opera ted upon under a n e s t h e s i a t w i c e , f i r s t fo r p l a c i n g s t e r e o t a x l c a l l y an e l e c t r o l y t i c l e s i o n of t he sup ra - c h l s s m a t l c nuc leus (SCL). S k u l l openings were made wi th a d e n t a l d r i l l in o rder to p o s i t i o n the e l e c t r o d e s in the sup ra - c h i a s m a t l c n u c l e i fo r t h e i r l e s l o n l n s (0 .5 mm a n t e r i o r to bregma; L-O, D-8 u below s k u l l s u r f a c e ) or fo r i n s e r t i o n of the e l e c t r o d e wi thou t pa s s ing c u r r a n t in o r d e r to produce c o n t r o l (SL) a n i m a l s , w l th same c o o r d i n a t e s as above, except d a p t h w a s 7 m below s k u l l s u r f a c e ; second, s e v e r a l weeks l a t e r , fo r l a p l a n t l n g senso r s fo r t empera tu re mon i to r ing (Aerospace Ned. 48 :3177 , 1969) . For c h r o n o b l o l o g l c a l s e r i a l s e c t i o n s ( I n t . J . o f Cbronoblology 1 : 3 1 , 1973) a 24-h cos ine curve was f i t t e d by l e a s t squares to 48-h da ta s e c t i o n s , the