VOL.
8,
NO.
8,
AUGUST
1969
Biosynthesis of Fatty Acids in Obese Mice in Vivo. 11. Studies Succinate-2,3-’HH-2,3-’“C, and with ~~-Malate-2-~H-3-’~C, E. Lamdin, W. W. Shreeve, R. H. Slavinski, and N. Ojit
ABSTRACT: Biosynthesis of fatty acids in the liver and in other tissues of the remaining carcass of obese hyperglycemic mice and their lean siblings has been investigated by isolation and counting of radioactivity in total fatty acids of mice sacrificed 90 min after intraperitoneal injection of trace amounts of malate, succinate, or isocitrate specifically labeled with tritium and carbon-14. The patterns of relative extent of transfer to liver and carcass fatty acids of 3H and 14Cwere similar for malate and succinate, and transfer for both compounds was of the same order as earlier observed with pair-labeled lactate and glycerol. The findings are compatible with a coupling of oxidation of all of these substrates with L-malate: oxidized nicotinamide-adenine dinucleotide phosphate oxidoreductase (malic enzyme) followed by subsequent transfer of tritium from reduced nicotinamide-adenine dinucleotide phosphate to fatty acids. This coupling appears to have particularly high activity in the liver. Theoretical considerations of different intracellular sites of metabolism of malate and succinate suggest a compartmental advantage of mitochondrially formed malate over exogenous malate in the transfer of metabolic reducing hydrogen. In contrast to findings with ~ ~ - m a l a t e - 2 -
A
previous publication from this laboratory (Shreeve et al.. 1967) reported that tritium transferred during the oxidation of labeled lactate and glycerol in c i w is utilized for reduction of intermediates formed during biosynthesis of fatty acids. As found with the obese hyperglycemic mouse and its lean siblings, this was particularly true for the liver and to a lesser extent for the other tissues included collectively in the headless carcass. Comparisons of isotopic yields from DL-lactate-2glycer01-2-~H, g l ~ c o s e - l - ~ Hand , gluc0se-6-~H and from corresponding IC-labeled carbohydrates suggested that in the liver transfer of protons from the three-carbon precursors is more “eficient” than that from glucose. These studies were consistent with earlier reports showing biosynthesis of fatty acids from ~ ~ - l a c t a t e - 2 -(Lowenstein, ~H 1961a; Foster and Bloom, 1961) and glycer01-2-~H(Foster and Bloom, 1961) in rat liver slices, from ~-lactate-2-~H in perfused rat liver (D’Adamo et a/., 1961), and from ~ ~ - l a c t a t e - 2 -in~ H human subjects in cico (Ghose et a/., 1964). The above-cited evi* From the Division of Biochemistry, Medical Research Center, Brookhaven National Laboratory, Upton, New York, and from the Departments of Medicine, University of Pittsburgh, School of Medicine, and Veterans Administration Hospital, Pittsburgh, Pennsylvania. Receiced Febriiar.i, 21, 1969. t Present address: First Department of Medicine, Osaka University Medical School, Dojima, Fukushima-ku, Osaka, Japan.
and s~ccinate-2,3-~H transfer of 3H from ~~Gsocitrate-23H was more extensive for the carcass than for the liver fatty acids, as was found previously with g l ~ c o s e - l - ~ H A .significant contribution of reducing equivalents cia D-rhreo-isocitrate: oxidized nicotinamide-adenine dinucleotide phosphate oxidoreductase in some nonhepatic tissues is indicated. On the other hand, the conversion of 14C from ~L-isocitrate-5,6-’~C (cia aconitase and citrate lyase) was more extensive in liver than in other tissues. All 3H- and 14C-labeled carbohydrates in the present study, like those previously tested, were converted into hepatic fatty acids of obese mice in severalfold higher extent than to those of lean mice, whereas conversion into total fatty acids of the carcass was only moderately higher in the obese. However, the range of differences among labeled carbohydrates for labeling of fatty acids of the liver of obese L‘S. lean mice was greater than previously found. Thus, the obese:lean ratio was only 3-fold for ~ ~ - m a l a t e - 2 -but ~ H as high as 12-fold in the case of s~ccinate-2,3-~~C. Unlike findings with labeled carbohydrates related to the glycolytic sequence, the formation of 14C02from 14C-labeled malate, succinate, and isocitrate was not lower in obese mice than in lean mice.
dence for major substrate sources of reducing hydrogen other than glucose is in accord with calculations that under certain conditions the pentose cycle can provide only one-half to three-fourths of the reducing equivalents necessary for biosynthesis of fatty acids in adipose tissue in citro (Flatt and Ball, 1964; Katz et al., 1966). Marked preferences of the various enzymes comprising the “fatty acid synthase” complex for the reducing coenzyme, NADPH, rather than NADH has been clearly shown in citro (Gibson et a/., 1958). Assuming that this finding is a reflection of in uico specificities, significant contribution of reducing hydrogen from NAD+-linked substrates, such as lactate and glycerol, must be somehow reconciled with the preference for NADPH demonstrated in citro. Of interest, therefore, are the reports (Young et a/., 1964; Pande et a[., 1964; Leveille and Hanson, 1966) of parallel changes in the activity of “malic enzyme” (L-malate:NADP+ oxidoreductase (decarboxylating), EC 1.1.1.40) and fatty acid synthesis in liver and adipose tissue. Such measurements support suggestions (Lowenstein, 1961a; Young et al., 1964; Pande et a/.,1964; Ball, 1966; Shreeve et al., 1967) of a possible key role of malate as a mediator of proton transfer to fatty acids via a coupling of various NAD+-linked reactions through NAD*-dependent L-malate dehydrogenase (EC 1.1.1.37) to NADP+-dependent “malic enzyme.”
BIOSYNTHESIS OF FATTY
ACIDS I N OBESE MICE
in Vice
3325
BIOCHEMISTRY
Substrates other than those mentioned above have also been suggested as possible contributors via pyridine nucleotides of reducing equivalents in the biosynthesis of fatty acids. Thus, isocitrate cia its cytoplasmic dehydrogenase (D-threeisocitrate:NADP+ oxidoreductase (decarboxylating), EC 1.1.1.42) could conceivably transfer protons directly to NADP+ and thence to fatty acids (Lowenstein, 1961b). Also, tritium from s~ccinate-2,3-~H added in oitro to mitochondria of rat aorta has been identified in fatty acids (Whereat, 1965). The relevance of this finding to fatty acid synthesis in oioo is uncertain. Furthermore, the relationship of the oxidation of succinate to the generation of reduced NAD+ remains controversial, particularly in its quantitative aspects (Gawron et al., 1964; Hoberman et al., 1964; Chance et a/., 1965; Griffiths and MacNeice, 1965; Krebs, 1967). In order to evaluate further the potential of these carbohydrates to donate protons for fatty acid synthesis in oivo, the studies reported herein extend our previous observations to results obtained with labeled malate, succinate, and isocitrate.
pound were comparable with respect to distribution of age, sex, and total body and liver weights. The range of blood glucose values was as reported earlier. The experimental protocol was essentially as described previously (Shreeve et al., 1967). Mice were fasted for 5 hr before intraperitoneal injection of a dilution of labeled carbohydrate (either 3H or I4C or both). No carrier was added to any of the original labeled materials. One milliliter, containing the radioactivity noted above, was administered to each obese mouse and 0.5 ml to each lean sibling. Each obese mouse or pair of lean mice was then immediately placed in a separate metabolism cage which enabled collection of 14C02 in the expired air for 90 min. Techniques of sacrifice and subsequent handling of blood and tissues as well as methods for saponification and extraction of fatty acids, analysis of 3HOH and glucose in blood and of l4COPin expired air, assay of radioactivity, and calculation of the conversion of 3H from labeled precursors into 3HOH were as described in earlier publications from this laboratory (Shigeta and Shreeve, 1964; Shreeve et a/., 1967).
Materials and Methods Succinic acid-2,3-3H (specific activity of 130-1 39 mCi/ mmole), m-malic acid-2- 3H (65 mCi/mmole), and m-isocitric a ~ i d - 2 - ~and H -5,6- 4C, containing approximately 50% DL-allo-isocitric acid by company assay (70 and 2.8 mCi per mmole, respectively), were obtained from New England Nuclear Corp., Boston, Mass. Succinic acid-2,3-14C (5.7 mCi/ mmole) and DL-malic acid-3-14C (12 mCi/mmole) were obtained from Volk Radiochemical Co., Chicago, Ill. Labeled compounds were diluted in sterile 0.9% NaC1.l Amounts given to each obese or pair of lean mice were as follows: succinate, 40-300 mCi of 3H and 5-10 pCi of 14C; malate, 4050 pCi of 3H and 4-5 pCi of 14C; isocitrate, 50 pCi of 3H and 5 pCi of 14C. No differences were found over the large range of succinate used. As in the preceding study (Shreeve et al., 1967) the animals used were the obese hyperglycemic mice of the strain, C57BL/ 65 obob, from the Jackson Laboratory, Bar Harbor, Me., and their lean siblings. Two lean mice were paired for comparison against a single obese litter mate. Obese mice ranged in weight from 37 to 64 g (mean = 49.2 g), and the range in weight per pair of lean mice was 38-63 g (mean = 46.6 g). Mice ranging in age from 8 to 24 weeks were used with approximately equal distribution between male and female animals. Weights of livers from obese mice ranged from 2.1 to 4.7 g (mean = 3.14 g) and those of paired livers from lean animals ranged from 1.4 to 2.5 g (mean = 1.90 g). Thus, the ratio of liver weight: body weight was approximately 50% greater in the obese mice than in the lean. Groups of animals studied with each com-
1 All labeled compounds were at least 98% radiochemically pure according to assay with paper or thin-layer chromatography by the commercial vendors. Tests in our laboratory have shown that
