BIOCHEMISTRY
Biosynthesis of Fatty Acids in Obese Mice in Vivo. I. Studies with Glucose-l-3H(l-"C), Gluc0se-6-~H( 6-"C), ~ ~ - L a c t a t e - 2 -2-I4C), ~ H ( and Glycer01-2-~H( 1,3-I4C)* W. W. Shreeve, E. Lamdin, N. Oji, a n d R . Slavinski
Rates and mechanisms of biosynthesis of fatty acids in the liver and in other tissues of the remaining carcass of obese hyperglycemic mice and their lean siblings have been investigated by isolation and counting of radioactivity in total fatty acids of mice sacrificed 90 min after intraperitoneal injection of trace amounts of carbohydrates labeled with tritium and carbon-14. The pairs of labeled carbohydrates injected were g l u ~ o s e - l - ~ H and g l ~ c o s e - l - ~ ~gluc0se-6-~H C, and glucose-6-14C, ~ ~ - l a c t a t e - % ~and H DL-lactate-214C, or glycerol-2-3H and glycerol-1,3-14C. All of the labeled carbohydrates (whether 3H or I4C) showed similar and highly significant increases of about fiveto eightfold greater incorporation of radioisotope into liver fatty acids of obese mice than into those of lean mice. Incorporation into carcass fatty acids of obese mice was only about 1.5- to 2-fold greater than in lean ABSTRACT:
E
arlier studies of transfer of 3H and 14C from position 1 of glucose to fatty acids of liver and carcass in lean and obese mice in vivo (Shigeta and Shreeve, 1964) suggested that hydrogen from this location was not a prominent source of reductive hydrogen via formation of NADPH.' Low labeling of fatty acids from g l u ~ o s e - l - ~might H be explained by a protonhydrogen exchange between water and that reductive hydrogen which contributes to the odd-carbon position of fatty acids (Foster and Bloom, 1963) and/or by isotope discrimination against the transfer to NADP+ from g l u ~ o s e - l - ~ H compared to glucose-3-3H (Katz et al., 1965). However, an additional reason for the finding of 3H: 14C ratios of 1.0 or less from glucose-l3H(l-I4C) in both liver and carcass fatty acids in uioo (in contrast to ratios of 6.0 or 7.0 found with rat liver slices; Foster and Bloom, 1961) could be the availability of other major substrate sources for providing reducing equivalents for fatty acid synthesis. Positive evidence for the existence of sources of
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* From the Division of Biochemistry, Medical Research Center, Brookhaven National Laboratory, Upton, New York 11973, and Departments of Physiology and Medicine, University of Cincinnati, Cincinnati, Ohio 45219. Received October 3, 1966. 1 Abbreviations used: NADP+ and NADPH, oxidized and reduced nicotinamide-adenine dinucleotide phosphates; CoA, coenzyme A; NAD+, oxidized nicotinamide-adenine dinucleotide.
SHREEVE,
LAMDIN,
OJI,
A N D
SLAVINSKI
mice. In the liver, tritium from lactate and glycerol was converted to fatty acids much more extensively than that from the first or the sixth carbons of glucose in regard to the percentage of administered amount and in relation to the amount of l4C converted to fatty acids from the same carbohydrates. In the liver, glucose-1-3H showed no more specificity of transfer to fatty acids relative to glucose-l-14Cthan did glucose-63H relative to gl~cose-6-'~C. For carcass fatty acids the 3H :l4C ratio was higher for 1-labeled glucose than for 6-labeled glucose or pair-labeled lactate or glycerol. Conversions to 3HOH from various 3H-labeled carbohydrates were either the same or greater in obese mice than in lean mice. On the other hand, conversions of l4C-labeled carbohydrates to I4CO2were either the same or lower in obese mice than in lean mice.
reducing hydrogen for the constitution of fatty acids other than the NADPH generated in the pentose cycle pathway has been indicated by the findings of conversion to fatty acids of ~ ~ - 1 a c t a t e - Z ~ (Foster H and Bloom, 1961; Lowenstein, 1961) and glycer01-2-~H (Foster and Bloom, 1961) in rat liver slices, L-lactate2-3H (D'Adamo et al.,1961) in perfused rat liver, DL-lactate-2-3H (Shreeve, 1965) in human subjects in ciuo,and succinate-2,2'-3H (Whereat, 1965) in aortic mitochondria. Combined radioisotopic and balance studies (Flatt and Ball, 1964; Katz et al., 1966) have further indicated that the reducing equivalents generated in the pentose cycle pathway could provide only about onehalf to three-fourths of those needed for synthesis of fatty acids in adipose tissue under conditions of stimulation by insulin or refeeding. It has been suggested (Lowenstein, 1961; Young et al., 1964; Leveille and Hanson, 1966) that a portion of metabolic NADPH may be derived by transhydrogenation through a coupling of NAD+-linked malic dehydrogenase and NADP+-linked "malic enzyme." The coupling of these two malic dehydrogenases from the supernatant fraction of liver homogenates has been demonstrated (Young et al., 1964). Also, measurement of changes of enzyme activities in both liver (Young et al., 1964) and adipose tissue (Young et a/., 1964; Leveille and Hanson, 1966) upon fasting and refeeding suggests involvement of this transhydrogenation mechanism in
VOL.
6,
NO.
4,
1967
A P R I L
LABELED CARBOHYDRATE
LIVER
7.0
RATIO
6.0
1
FOR
OBESE:
5.0
LEAN MICE
4.0
OF PER CENT
3.0
UPTAKG INTO
2.0
FATTY ACIDS
1.0
OF
’H OR “C DOSE
CARCASS
L
-n
FIGURE
1 : Incorporation of 14Cand 3H into fatty acids of mice.
biosynthesis of fatty acids. We have investigated isotopic yields of 3H and 14C in the liver and carcass fatty acids of lean and obese mice after intraperitoneal injection of various labeled carbohydrates in addition to g l u ~ o s e - l ( ~ H - l - *(Shi~C) geta and Shreeve, 1964) in order to assess incorporation of 3H by pathways other than the pentose cycle.2 Findings described in this paper indicate that in vivo incorporation into liver fatty acids of tritium from glucose-6-3H is similar to that from glu~ose-l-~H, while that from DL-lactate-2-3H or glycer01-2-~H is substantially greater than from either labeled glucose
2 Preliminary reports have appeared in Federation Proc. 23, 165 (1964), 24, 343 (1965), and Ann. N. Y. Acad. Sci. 131, 464(1965).
compound. On the other hand, for carcass fatty acids, glu~ose-l-~H shows the highest labeling. Ratios of differences in extent of isotope incorporation between lean and obese mice were similar for all of these 3Hor 4C-labeledcarbohydrates. Materials and Methods The methods used were essentially those previously described (Shigeta and Shreeve, 1964). The obese hyperglycemic mice of the Bar Harbor strain and their lean siblings used in the present studies ranged in age from 8 to 24 weeks. The weight of the obese mice varied from 34 to 64 g (mean of 52 g) and the weights of two (paired) lean mice varied from 30 to 55 g (mean of 44 g). Mice of both sexes were used, predominantly female. Neither age nor sex appeared to affect signifi-
BIOSYNTHESIS OF FATTY ACIDS IN OBESE M I C E
1161
in V i v o
BIOCHEMISTRY
TABLE I :
Comparative Transfers of Tritium or Carbon-14 from Various Carbohydrates (and Water) into Liver and
Carbohydrate
Glucose
Position and Kind of Label Type of Mouse
% dose in liver fatty acids (no. of expt)
% dose in carcass fatty acids (no. of expt)
7, dose in body water (no. of expt)
- 1 - 3 ~
0
L
0
0.025 0.004 (6)
0.176“ f 0.020 ( 5 )
0 027 f 0.003 (8)
0.228O f 0.074 ( 5 )
0.24* 0.04(4)
0.56* 0.13(5)
0.88* 0.07(8)
l.72a* 0.22(5)
44.64~ 2.0 ( 5 )
42.2 zk 2.1 ( 5 ) 39.8 f 1.8(8)
30.Rb zk 2 . 1 (4)
L
0
L
0
L
0.035 f 0.003c (8)
0.175& f 0.011 (6)
0.038 f 0.007 ( 5 )
0.193b f 0.060 ( 5 )
0.62 O.lO(8)
1.25”* 0.16(5)
1.2Of 0.30(5)
1.672~ 0.23(4)
62.7 f 3 . 4 (7)
78.8b 6.4 (5)
*
*
dose in respiratory COZ(no. of expt)
45.7 f 1.5 (3)
-6-14C
-6-3H
-1-14c
*
43.0 f 7 . 0 (2) -~
a
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P
