Nonphosphorylating respiration of mitochondria from brown adipose tissue of rat

Hittelman, K. J.

Roberts, J. C.

Smith, R. E.

English

NASA Technical Reports Server

1 ' i 
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NON-PHOSPHORYLATING RESPIRATIOIT OF MITOCHONDRIA 
FRON BROWN ADIPOSE TISSUE OF RATS 
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$- 
Robert E. Smith CFSTl PRICE(S) $ 
Jane C. Roberts 
and 
 
Hard c2p-i (HC) /e 0-0 
Microfiche (MF) 
ff 853 July 85 
J. Hittelman 
Department of Physiology 
School of Medicine 
The Center for the Health Sciences 
University of California 
Los Angeles , California 90024 
e4 
I N66 30586 
' 
(ACCESSION NUMBER) 
https://ntrs.nasa.gov/search.jsp?R=19660021296 2020-03-16T20:50:56+00:00Z
f Smith 2 
ABSTRACT 
rats ( 6 0 ~ )  
L 
IIitochondria from brown adiuose tissue of cold-acclimated 
oxidize %-ketoglutarate at a rate twice that of controls (26OC). 
groups, however, the P/O ratio with a-ketoglutarate has never exceeded 
unity, and is essentially zero with either succinate or 
ATPase activity of these mitochondria is very l o w  and is not stimulated 
by 2 ,b-dinitrophenol (DNP) . 
phorylation are unaffected by ADP, DIP, bovine serum albumen or glutathione. 
Endogenous respiration of tissue slices is not stimulated by DNP. It is 
suggested that brown fat mitochondria are not capable of oxidative phos- 
phorylation, but do phosphorylate at the substrate level. As these find- 
ineprovide an unusual example of electron transport via an energetically 
non-conservative pathway, their significance to thermogenesis by brown 
adipose tissue is particularly emphasized. 
In both 
a-glycerophosphate. 
Additionally, both respiration and phos- 
Smith 3 
I n  clean, tightly-coupled hear t  o r  l i v e r  mitochondrial systems, the 
P/O r a t i o  i n  v i t r o  i s  between 3.0 and 4.0 when a-ketoglutarate  is  used as 
substrate .  then t h e  P/O r a t i o  is reduced e i t h e r  by t h e  presence of extra- 
loitochondrial mater ia l  (1) or by t h e  addi t ion of an uncoupling agent 
such as 2,4-dinitrophenol ( D I P )  (2)  t h e  proportion of energy u t i l i z e d  i n  
the  formation of ATP i s  decreased r e l a t i v e  t o  t h a t  d i r ec t ly  d iss ipa ted  as 
heat.  Thus i n  brown adipose t i s s u e ,  f o r  which t h e  primary known w c t i o n  
i s  thermogenesis ( 3  - 81, an idea l  metabolic state would appear t o  e x i s t  
i f  the mitochondria maintained a high rate of resp i ra t ion  and a r e l a t i v e l y  
l o w  r a t e  of phosphorylation. As it had been observed f o r  some time i n  
t h i s  laboratory t h a t  mitochondria from brown adipose t i s s u e  of rats 
phosphorylated only very poorly, it seemed of pa r t i cu la r  importance t o  
inves t iga te  t h i s  i n  more de t a i l .  
Mitochondria from brown adipose t i s s u e  of adult  male Long-Evans 
rats acclimated e i the r  at 26 or  6 O c  were i so l a t ed  by a modification of 
t he  method of Schneider ( 9 ) .  The t i s s u e  was homogenized i n  10 volumes 
of 0.25 M sucrose, and centrifuged a t  14,000 x g f o r  10 minutes. 
the  supernatant was careful ly  aspirated from beneath the  overlying l i p i d  
l aye r ,  the la t ter  was  removed and t h e  w a l l s  of t he  tube swabbed out t o  
eliminate as much l i p i d  as possible. 
o r ig ina l  volume of 0.25 M sucrose and treated according t o  Schneider ( 9 )  
f o r  the i so l a t ion  of the  mitochondrial f r ac t ion  (G). 
After 
The p e l l e t  w a s  resuspended i n  t he  
Oxygen consumption w a s  measured a t  37OC by t h e  d i r e c t  method of 
Warburg w i t h  air as t h e  gas phase. 
contained, i n  p o l e s :  eke tog lu ta ra t e ,  10; K F ,  10; PO4 as KH2PO4 buffer  
(pH 7.4) ,  15; ATP, 2; MgC12, 5; tris buffer  (pH 7.4), 25; glucose, 25 and 
25 KM un i t s  hexokinase (Sigma Type IV). 
added, the  glucose-hexokinase t r a p  was omitted. Total  react ion mixture 
volume w a s  1.0 m l ,  including 0.4 - 0.5 m l  mitochondria (ca.  0.3 mg N). 
The center wel l  contained 0.05 m l  105 KOH and folded filter paper wick. 
Following a 12 minute temperature equi l ibra t ion ,  t r a p  or  ADP ( 4  x lO-3hi 
f i n a l  concentration) w a s  tipped i n t o  the  vesse l  and readings were taken at  
5 minute i n t e r v a l s f o r 1 5  minutes. 
of 0.5 n i l  25% 
contents of each Warburg f lask  using the  method of Gomori (10). 
resp i ra t ion  of t i s s u e  s l i c e  w a s  measured by the  same method i n  a medium 
essen t i a l ly  the same as t h a t  used f o r  h, but omitting subs t ra te  and 
glucose-hexokinase t r a p  and subs t i tu t ing  KC1 f o r  ICF. DPIP ( 4  x 10-5M 
f i n a l  concentration) was  tipped i n  at the time of the  first reading. 
Readings were taken a t  5 minute in t e rva l s  fo r  20 minutes. Nitrogen w a s  
assayed by a standard micro-Kjeldahl method. 
The incubation medium f o r  mitochondria 
In  experiments where ADP was 
Reactions were stopped by t h e  addi t ion 
t r ichloroacet ic  acid and phosphate assays performed on the  
EndogenoB 
ATPase a c t i v i t i e s  were assayed i n  a medium containing, i n  p o l e s :  
ATP, 15; MgC12, 5; KF', 10; PO4 as KH?PO4 buffer (pH 7.41, 15; glucose, 25 
and tr is  buffer (pH 7.4), 25. 
0.4 m l  mitochondria. 
The f i n a l  volume w a s  1.0 m l ,  including 
Reactions were car r ied  out a t  37OC f o r  12 minutes. 
The results summarized i n  Table 1 show tha t  brown f a t  mitochondria 
from cold-acclimated rats oxidize a-ketoglutarate  at a r a t e  twice t h a t  of 
. -  
Smith 4 
the  control  rats. 
of t i s s u e  i n  the cold (5 ,  111, contributes s ign i f i can t ly  t o  the  t o t a l  
heat production ( 5 )  assignable t o  brown fat  i n  t h e  cold-acclimated animal. 
This increase i n  SO,, coupled with the  increased mass 
Also notable are  the  low P/O r a t i o s  observed i n  the  mitochondria 
from both the control  and cold-acclimated animals (Table 1). 
r a t i o s  do not appear t o  result from ATPase a c t i v i t i e s ,  which i n  f a c t  a r e  
very low and moreover a re  not stimulated i n  v i t r o  by addition of DNP 
(Table 2). However, it has been noted t h a t  oligomycin (2 ) ~ g / m g  prote in)  
e f fec ted  approximately 90% inhibi t ion of t h e  ATPase ac t iv i ty .  
These low 
Tnus far, a l l  attempts t o  r e s to re  phosphorylation i n  brown fat  
mitochondria have been unsuccessful. 
(6  o r  15 %/f lask)  nor glutathione (lO-3M) , when added t o  the  Warburg 
f l a sks ,  enhances t h e  P/O ra t io .  Moreover, the  presence of BSA i n  t he  homo- 
genization medium (6 %/&) has not affected the  P/O r a t i o s  of the  isolated 
mitochondria, suggesting t h a t  these l o w  r a t i o s  do not r e s u l t  from an un- 
coupling e f f e c t  of f a t t y  acids (12) released upon homogenization (Table 3 ) .  
Neither bovine serum albumin (BSA) 
Experiments with other  substrates  ind ica te  that mitochondria i so l a t ed  
from brown adipose t i s s u e  do not y i e ld  theo re t i ca l ,  o r  even near-theo- 
r e t i c a l ,  P/O r a t i o s .  
determined with a polarized platinum electrode have yielded results 
iden t i ca l  t o  those reported here (13). 
employing 
spect ively,  0.086 f 0.028 and 0.111 ? 0.023), t he  P/O r a t i o  of near un i ty  
observed with 
subs t ra te  leve l .  
f inding tha t  addition of DNP (2  or  4 x 10-5M) does not s ign i f i can t ly  affect  
t he  phosphorylation or  the  respirat ion (Table 3). It is known ( 1 4 )  t h a t  
the  subs t ra te  l e v e l  phosphorylation with a-ketoglutarate i s  insens i t ive  
t o  DNP. 
hexokinase t r a p  or  ADP, P/O r a t i o s  were zero; addition of ADP, however, 
did result i n  phosphorylation, but the  values only approached those ob- 
ta ined  i n  t he  presence of the t r ap .  
I n i t i a l  rates of resp i ra t ion  and r e su l t an t  P/O r a t i o s  
Since the  P/O r a t i o s  i n  systems 
a-glycerophosphate and succinate are es sen t i a l ly  zero (re- 
a-ketoglutarate may represent phosphorylation at t he  
This in te rpre ta t ion  i s  fu r the r  substant ia ted by t h e  
Moreover, with a-ketoglutarate i n  the  absence of e i the r  glucose- 
Although the  inh ib i tory  e f f e c t  of oligomycin on t h e  mitochondrial 
ATPase ac t iv i ty  m a y  be interpreted as evidence f o r  the  presence of a 
phosphorylating mechanism usually associated with the electron t ransport  
system (15), t h e  s tud ies  on t i s s u e  s l i c e s  f a i l  t o  show any stimulation of 
resp i ra t ion  by DNJ? e i t h e r  i n  control  or cold acclimated animals. 
r a t i o s  of resp i ra t ion  (+DNP/-DNP) were 0.86 and 1.09 i n  t he  control  and 
cold acclimated t i s s u e  s l i c e s ,  respectively.  These r e s u l t s  strongly 
suggest tha t  t he  lack of oxidative phosphorylation exhibited by t h e  mito- 
chondrial preparations may not be a r t i f a c t  of i so l a t ion ,  but r a the r  
may r e f l e c t  an absence of coupled oxidative phosphorylation -- i n  vivo. 
The 
If indeed the  usual  oxidative phosphorylation mechanism does not 
operate i n  t h i s  highly respir ing,  heat producing t i s s u e ,  requirements f o r  
ATP might s t i l l  be met by substrate  phosphorylations such as that  suggested 
here with respect t o  a-ketoglutarate, and those presumably occurring via 
Smith 5 
the  glycolyt ic  pathway. 
a -ketoglutarate oxidation were of the  same order as seen in v i t r o ,  t h i s  
would y i e ld ,  i n  the control  r a t ,  17.1 p o l e s  ATP/mg N hr  and i n  the  cold- 
acclinated r a t ,  29.1 p o l e s  ATP/% N hr .  In 350 - 400 p rats, t h e  t c t a l  
mitochondrial nitrogen of t he  brown fa t  i s  1.95 mg f o r  control  rats and 
8.67 mg f o r  cold-acclimated rats (16). The t o t a l  amount of ATP formed 
by the  brmm fat  would then be 33.5 ,moles/hr i n  the  control  and 251.9 
ymoleslhr i n  the cold-acclimated rat .  
sabs t ra te  l e v e l  phosphorylation with 
the  brown fat  of cold-acclimated rats could be increased 750% over t h a t  i n  
controls.  
Thus, i f  i n  vivo the  P/O r a t i o  and 902 of -- 
Hence, so le ly  on the basis of 
a-ketoglutarate,  ATP formation i n  
Signif icant  i n  the  present findings are a )  the apparent absence of a 
system coupling resp i ra t ion  with phosphorylation and b )  t h e  result inR 
lack  of resDiratory control  by ADP. 
of synthesizing a high energy in t emed ia t e  or  compound, which i f  t h i s  
were a coupled system, would occur i n  large amounts, while t h e  absence of 
ADP respiratory control would insure a high rate of subs t ra te  oxidation. 
The resu l t ing  e f f ec t  would therefore  maximize the rate of production and 
ava i l ab i l i t y  of heat evolved by the respir ing system. 
with the  increased q02 of the mitochondria of brown fat from cold- 
acclimated rats, i s  believed t o  bear primarily upon the  augmentation of 
heat production i n  t h i s  t i s sue .  Also, these findings are i n  concert w2th 
t h e  known physiological r o l e  of brown adipose t i s s u e  as a thermogenetic 
e f f ec to r  organ both i n  cooled o r  cold-exposed mammals and i n  hibernators 
during cold-induced arousal ( 3  - 8).  
The former eliminates the  necessity 
This, together 
, - 4 -  
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1. 
2. 
3. 
4. 
5. 
6. 
7. 
8. 
9. 
10. 
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13. 
1 4 .  
15 
16. 
17 
~~ - 
Smith 6 
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R.E. Smith, Federation Proc., 21, 221 (1962). -
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and J . C .  Roberts, Am. J. Physiol.,  206, 143 (1964). 
_L 
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P. Borst, J . A .  LOOS, E.J. Chr i s t ,  E.C. S l a t e r ,  Biochim. Biophys. 
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J.C. Roberts and R.E. Snith,  i n  preparation. 
Supported i n  pa r t  by NASA Research Grant NsG 721 and USPHS Research 
G r a n t  HD-01826. K.J .R.  i s  a predoctoral fellow of t h e  USPHS. 
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Non-phosphorylating Respiration of Mitochondria from Brown Adipose Tissue of Rats

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