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Incorporation of 1, 3-C14-glycerol into triglycerides of adipose tissue from suckling rats
Life Sciences Vol . 7, pp . 187-190, 1968 . Printed in Great Britain.
Pergamon Press
INCORPORATION OF 1,3-C 14 -GLYCEROL INTO TRIGLYCERIDES OF ADIPOSE TISSUE FROM SUCKLING RATS P . Hahrt** and 8 . Greenberg*** Department of Pediatrics, and the Joseph P . Rennedy, Jr . Laboratories Stanford ûniversity School of Medicine
(Received 1 September 1967 ; in final form 11 December 1967)
It has been reported that brown adipose tissue from adult rate possesses
a glycerinase which could only be assayed after first passing the cell-free extract through a Sephadea G25 column l . According to Dawkins and Hu11 2 , the brown adipose tiasué of newborn rabbits is devoid of this enzyme but this fact is only mentioned in the discussion of their paper and, presumably, tissue eatracta were not first passed through Sephadex . McBride and Rorn 3 have recently demonstrated the presence of a glycerokinase in mammary glands using radioactive glycerol but could not demonstrate the enzyme by two other methods involving the oxidation of NADH4 ' S .
Adult
white adipose tissue has repeatedly been shown not to be equipped with a glycerokinase 6 , yet the activity of this enzyme is said to be increased by a factor of 3 in obese mice l .
Since fat is rapidly laid down during early postnatal development into
both types of adipose tisaue 8 and thus triglycerides are formed from glycerophosphate and free fatty acids to a greater extent than later in life, it seemed worthwhile to examine both adipose tissues during early postnatal development,
In order to avoid the possible effect of a glycerophosphate-
phosphatase6 and the uncertainties of the two enzyme assay methods mentioned above4 ' S the incorporation of 1,3-C14 glycerol into the triglyeerides of white and brown adipose tissue was studied . METHiDD6 Adipose tissue, cut into small pieces and weighing between 80 and 120 mg, was incubated in 1 ml Rrebs-Ringer bicarbonate buffer without calcium, contain#Supported by grants NB-05131, HO-02147 and HD-!,9 from the National Institutes of Health . ##pn leave of absence from Laboratory of Developmental Nutrition, Inatit.ute of Physiology, Czechoslovak Academy of Science, Prague, Caechoslovakie . ###Research Career Development Arrardee, National Institutes ~~f Health .
187
GLYCEROL . INCORPORATION
18K
Vol . 7,
No . 4
ing 3% bovine albumin and 0 .5 uC 1,3-C 14 glycerol (specific activity ; 0 .1 mC/1 .01 mg) for 2 hours at 37 0 .
Total lipids were extracted with chloroform9
and an aliquot of the chloroform extract was used to determine the radioactivity in the total lipids .
Another aliquot was hydrolysed with alcoholic
KOH10 and the radioactivity of the free
fatty acids was determined .
The com-
pleteness of the extraction and hydrolysis procedures was tested by separating . the isolated lipids on thin layer chromatography ll RESULTS Radioactive glycerol is incorporated to a much greater extent into both white and brown adipose tissue of suckling rats than into the smoe tissue of animals that are older (see Fig, 1, Table I),
The highest rate of incorpora
tion was found into the lipids of fetal brown adipose tissue .
Only about 1 to
57 of the total activity incorporated into triglycerides was found in free fatty acids,
i .e . moat of the labelled glycerol appears in the glycerol moiety
of triglycerides . TABLE I Rate of incorporation of labelled glycerol into triglycerides of white and brown adipose tissue
White adipose tissue
Brown adipose tissue
cpm/mg fat
cpm/mg wet wt .
cpm/mg fat
Age days,
5-12
(8)
1194 ± 87
590 ± 52
(6)
4000 ± 420
18-50
(7)
393 ± 50
190 ± 28
(4)
1900 ± 112
cpm/wet wt,
1103 ± 129 830 ±
45
All differences between suckling and older rats are significant for p 0.001 except for cpm/mg wet wt . in brown adipose tissue,
189
GLYCEROL INCORPORATION
Vol . 7, No . 4
F : 90 000
4000
0 Fig . 1 .
40 A
20
Activity of triglycerides isolated from adipose tissue incubated with
1,3-C14 glycerol in cpm/mg wet tissue (ordinate), days .
F = fetus, A ~ adult .
White :
white, black :
Abscissa :
age in postnatal
brown adipose tissue .
The rate of incorporation of labelled glycerol is suppressed by increasing concentrations of nonlabelled glycerol, thus showing that it is the glycerol label that is incorporated into triglycerides (see Fig. 2) . In order to make sure
that glycerophosphate is the intermediate formed
from glycerol, the supernatant from white and brown adipose tissue obtained from 10-day-old rats was incubated with labelled glycerol in the absence of CoA,
as described by P1cBride and Korn 3 ,
Amberlite IR-4B (formate form), with lr1 NH 40H .
After separation of the medium on
glycerophosphate was eluted from the column
The total amount of activity incorporated into glycerophosphate
by 0 .1 ml of the 20% centrifuged homogenate was 70,000 cpm for brawn and 20,000 cpm for white fat,
i,e . about 25 and 8% of the total radi~activi~y
respectively . DISCUSSION The evidence presented here strongly suggests that white adipose tissue of suckling rats possesses a glycerokinase,
the activity of which decreases
with age and that the reported absence of this enzyme 2 from brown adipose
190
GLYCEROL INCORPORATION
Vol . 7, No . 4
O
O
Fig. 2 .
Effect of addition of nonlabelled glycerol on the rate of incorpora-
tion of 1,3+C14 glycerol. into triglyceridea of white adipose tissue from 10-day-old rats . Ordinate :
Abscissa :
concentration of glycerol in umol/ml medium .
activity in X of that obtained when no cold glycerol was âdded .
tissue of newborn rabbits has been deduced from insufficient evidence . REFERENCES 1.
D, H, TREBLE and E . Ball, Fed . Proc . 22, 357 (1963) .
2.
M. J. DAi1RINS and D . Hull, J. Physiol . 172, 216 (1964) .
3. 4.
0. A. MC BRIDE and G. D. &orn, J. Lipid Res . 5~ 442 (1964) . 0, WISLAI~ and M. SUYTER, Biochem. Ztachr . 329, 329 (1957) .
5.
C . Bi1BLITZ and E . P . RSNHEDY, J . biol . Chem . 211,
7.
S. LOCHAYA, J, C, HAMILTON and J. MAYER, Nature 197,
8.
P, HAHN, J. SKALA, R, VIZER and M, NOVAR, Physiol . bohemoslov . 14, 546
6.
951 (1954) .
S . MARGOLIS and M. VAIIGHAN, J. biol . Chem . 237, 44 (1962) . 182 (1963) .
(1965) . 9.
J, FOLCH, M. LEES aad C. H. SLOANE-STANLEY, J. biol . Chem . 226, 497 (1957) .
10 .
M, ROBELL, J .biol . Ch~m . 329,
375 (1964) .
11 .
M. DOBIASOVA, Roavraw Cel . Akademia Vad 76, 10 (1966) .
Pergamon Press
INCORPORATION OF 1,3-C 14 -GLYCEROL INTO TRIGLYCERIDES OF ADIPOSE TISSUE FROM SUCKLING RATS P . Hahrt** and 8 . Greenberg*** Department of Pediatrics, and the Joseph P . Rennedy, Jr . Laboratories Stanford ûniversity School of Medicine
(Received 1 September 1967 ; in final form 11 December 1967)
It has been reported that brown adipose tissue from adult rate possesses
a glycerinase which could only be assayed after first passing the cell-free extract through a Sephadea G25 column l . According to Dawkins and Hu11 2 , the brown adipose tiasué of newborn rabbits is devoid of this enzyme but this fact is only mentioned in the discussion of their paper and, presumably, tissue eatracta were not first passed through Sephadex . McBride and Rorn 3 have recently demonstrated the presence of a glycerokinase in mammary glands using radioactive glycerol but could not demonstrate the enzyme by two other methods involving the oxidation of NADH4 ' S .
Adult
white adipose tissue has repeatedly been shown not to be equipped with a glycerokinase 6 , yet the activity of this enzyme is said to be increased by a factor of 3 in obese mice l .
Since fat is rapidly laid down during early postnatal development into
both types of adipose tisaue 8 and thus triglycerides are formed from glycerophosphate and free fatty acids to a greater extent than later in life, it seemed worthwhile to examine both adipose tissues during early postnatal development,
In order to avoid the possible effect of a glycerophosphate-
phosphatase6 and the uncertainties of the two enzyme assay methods mentioned above4 ' S the incorporation of 1,3-C14 glycerol into the triglyeerides of white and brown adipose tissue was studied . METHiDD6 Adipose tissue, cut into small pieces and weighing between 80 and 120 mg, was incubated in 1 ml Rrebs-Ringer bicarbonate buffer without calcium, contain#Supported by grants NB-05131, HO-02147 and HD-!,9 from the National Institutes of Health . ##pn leave of absence from Laboratory of Developmental Nutrition, Inatit.ute of Physiology, Czechoslovak Academy of Science, Prague, Caechoslovakie . ###Research Career Development Arrardee, National Institutes ~~f Health .
187
GLYCEROL . INCORPORATION
18K
Vol . 7,
No . 4
ing 3% bovine albumin and 0 .5 uC 1,3-C 14 glycerol (specific activity ; 0 .1 mC/1 .01 mg) for 2 hours at 37 0 .
Total lipids were extracted with chloroform9
and an aliquot of the chloroform extract was used to determine the radioactivity in the total lipids .
Another aliquot was hydrolysed with alcoholic
KOH10 and the radioactivity of the free
fatty acids was determined .
The com-
pleteness of the extraction and hydrolysis procedures was tested by separating . the isolated lipids on thin layer chromatography ll RESULTS Radioactive glycerol is incorporated to a much greater extent into both white and brown adipose tissue of suckling rats than into the smoe tissue of animals that are older (see Fig, 1, Table I),
The highest rate of incorpora
tion was found into the lipids of fetal brown adipose tissue .
Only about 1 to
57 of the total activity incorporated into triglycerides was found in free fatty acids,
i .e . moat of the labelled glycerol appears in the glycerol moiety
of triglycerides . TABLE I Rate of incorporation of labelled glycerol into triglycerides of white and brown adipose tissue
White adipose tissue
Brown adipose tissue
cpm/mg fat
cpm/mg wet wt .
cpm/mg fat
Age days,
5-12
(8)
1194 ± 87
590 ± 52
(6)
4000 ± 420
18-50
(7)
393 ± 50
190 ± 28
(4)
1900 ± 112
cpm/wet wt,
1103 ± 129 830 ±
45
All differences between suckling and older rats are significant for p 0.001 except for cpm/mg wet wt . in brown adipose tissue,
189
GLYCEROL INCORPORATION
Vol . 7, No . 4
F : 90 000
4000
0 Fig . 1 .
40 A
20
Activity of triglycerides isolated from adipose tissue incubated with
1,3-C14 glycerol in cpm/mg wet tissue (ordinate), days .
F = fetus, A ~ adult .
White :
white, black :
Abscissa :
age in postnatal
brown adipose tissue .
The rate of incorporation of labelled glycerol is suppressed by increasing concentrations of nonlabelled glycerol, thus showing that it is the glycerol label that is incorporated into triglycerides (see Fig. 2) . In order to make sure
that glycerophosphate is the intermediate formed
from glycerol, the supernatant from white and brown adipose tissue obtained from 10-day-old rats was incubated with labelled glycerol in the absence of CoA,
as described by P1cBride and Korn 3 ,
Amberlite IR-4B (formate form), with lr1 NH 40H .
After separation of the medium on
glycerophosphate was eluted from the column
The total amount of activity incorporated into glycerophosphate
by 0 .1 ml of the 20% centrifuged homogenate was 70,000 cpm for brawn and 20,000 cpm for white fat,
i,e . about 25 and 8% of the total radi~activi~y
respectively . DISCUSSION The evidence presented here strongly suggests that white adipose tissue of suckling rats possesses a glycerokinase,
the activity of which decreases
with age and that the reported absence of this enzyme 2 from brown adipose
190
GLYCEROL INCORPORATION
Vol . 7, No . 4
O
O
Fig. 2 .
Effect of addition of nonlabelled glycerol on the rate of incorpora-
tion of 1,3+C14 glycerol. into triglyceridea of white adipose tissue from 10-day-old rats . Ordinate :
Abscissa :
concentration of glycerol in umol/ml medium .
activity in X of that obtained when no cold glycerol was âdded .
tissue of newborn rabbits has been deduced from insufficient evidence . REFERENCES 1.
D, H, TREBLE and E . Ball, Fed . Proc . 22, 357 (1963) .
2.
M. J. DAi1RINS and D . Hull, J. Physiol . 172, 216 (1964) .
3. 4.
0. A. MC BRIDE and G. D. &orn, J. Lipid Res . 5~ 442 (1964) . 0, WISLAI~ and M. SUYTER, Biochem. Ztachr . 329, 329 (1957) .
5.
C . Bi1BLITZ and E . P . RSNHEDY, J . biol . Chem . 211,
7.
S. LOCHAYA, J, C, HAMILTON and J. MAYER, Nature 197,
8.
P, HAHN, J. SKALA, R, VIZER and M, NOVAR, Physiol . bohemoslov . 14, 546
6.
951 (1954) .
S . MARGOLIS and M. VAIIGHAN, J. biol . Chem . 237, 44 (1962) . 182 (1963) .
(1965) . 9.
J, FOLCH, M. LEES aad C. H. SLOANE-STANLEY, J. biol . Chem . 226, 497 (1957) .
10 .
M, ROBELL, J .biol . Ch~m . 329,
375 (1964) .
11 .
M. DOBIASOVA, Roavraw Cel . Akademia Vad 76, 10 (1966) .