478

CAN. J. BIOCHEM. VQL. 53, 1975

(18) can be generalized to explain these appar-

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ently conflicting results. The pathway, as applied to diketopiprazine-like or pyrazine-like metabolites, would suggest the following sequence: (1)the appropriate linear dipeptide is formed on, and bound to, an enzyme complex; ( 2 ) this system may react further to form appropriate open-chain, enzyme-bound metabolites; (3) these metabolites cyclize and then are released by the enzyme, or are bound loosely enough to be replaced by appropriate lT-labeled compounds; and (4) the product of the last step may undergo further reactions. Stage 3 would be the first point at which intermediates could be demonstrated by the conventional method outlined above. If the product of stage 3 was either the final metabolite or a complex cyclic dipeptide (10): then a simple 14C-labeledcyclic dipeptide would not be incorporated into the metabolite (e.g., biosynthesis of cyclopenin and cyclopenol (lo), mycelianamide and gliotoxin, neoaspergillic and aspergillic acids(l1)). If the product of stage3 was the simple cyclic dipeptide, then the 14Clabeled compound would be incorporated into the metabolite (e.g., biosynthesis of pulcherrimhic acid (9), echindin (I), and brevianamide A (2)). The above working hypothesis is of obvious

interest in designing experiments to show how such metabolites may be biosynthesized. We thank Mr. G. Bishop, Mr. L. R. Hogge, and Mr. D. M. Tenaschuk for technical assistance. A culture of P. terlikowski and infrared and proton magnetic resonance spectra of gliotoxin were kindly supplied by Drs. D. Brewer and A. Taylor, Atlantic Regional Laboratory of the National Research Council of Canada, Halifax, Nova Scotia. 1. Slater, G . P., MacDonald, 9. C. & Nakashima, R. (1970) Biocftemistry 9, 2886-2889 2. Baldas, J., Birch, A. J. B6 Russell, R. A. (1974) J. Chem. Sac. Perkin Tram. 1, 50-52 3. Beecham, A. F., Fridrichsons, J. & Mathieson, A. McL. (1966) Tetruhedrotz Eeti. 27, 313 1-3138 4. Gallina, C., Romeo, A., Tarzia, 6 . & Tortorella, V. (1964) Gazz. Cltim. Ital. 94, 1301-1 380 5. Suhadolnik, R. J. (1967) in A~sibiorics(Gottleib, D. Bs Shaw, P. D,, eds), vol. 2, pp. 29-31, SgringerVerlag, New York, N.Y. 6. Bu'Lock, J. D. & Ryles, A. P. (1970) J. Clietn. Soc. D , 1404-1 406 7. Kirby, G. W. & Narayaraaswami, S. (1973) J. Cltem. Soc. Chern. Cornrnura. 322-323 $. Sckerrer, R., Louden, L. & Gerhardt, P. (1974) J. Bctcieriol. 118, 534-546 9. MacDonald, J. C. (1965) Biochem. /. 96, 533-538 10. Framm, J., Nova, L., Azzouny, A. E., Richter, H., Winter, K., Werner, S. & Luckner, M. (1973) Eur. J. Biochern. 37,?8-85 11. Misetich, R. G. & MacDonald, J. 6 . (1965) J . Biol. Chern. 240,1692-1695

The Incorporation of I4C from [l=14C]Palmitate into Glucose and Glycogen In Mice Biology and Agricir1fnp-eDivision, Bi~abhaAtomic Research Centre, Bombay 400085, 1tldii-i Received August 16, 1974

Pushpendran, C. K.Bs Eapen, J. (1975) The Incorporation of I 4 C from [I-14C]Palmitateinto Glucose and Glycogen in Mice. Catt. J. Biochem. 53,478-484 The incorporation of 14Cfrom [I-14C]palmitateinto blood glucose and liver and kidney glycogen in postnatal mice has been studied. Incorporation s f 14Cfrom [1-14C]palmitafeinto blood glucose and hepatic glycogen is relatively high in suckling mice. In contrast, the incorporation into kidney glycogen is low in suckling mice and high in adults. The study indicates the possible utilization of palmitate for glucose synthesis. Pushpndran, @. K. Lk Eapen, J. (1975) The Incorporation of 14@from [I-lCIPalmitate into Glucose and Glycogen in Mice. Curt. J. Biochem. 53, 478-484 Nous avons CtudiC l'incorporation du lC provenant du El-lC3palrnitate dans le glucose sanguin et le glycogkne hdpatique et renal chez ies souris aprbs la naissance. L'incorporation du "C dans le glucose sanguin et le glycogbne hCpatique est relativement 6levCe chez les souris allaities. En revanche, 19incorporationdans le glycogkne r6nal est faible chez les souris allaitdes et forte chez les adultes. Les resultats suggbrent I'utilisation possible du palmitate pour la synthkse du glucose. [Traduit par le journal]

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NOTES

Introduction The capacity of rat liver and kidneys for gluconeogenesis from sources such as amino acids, lactate, and pyruvate has been shown to be maximal during the suckling period (1-5). The availability of amino acids for gluconeogenesis, however, is restricted because of the greater requirement for protein synthesis in neonatal animals (6,7). It has been reported that during the suckling period, lipogenesis is low in rat liver and adipose tissue because the diet is rich in lipids (8-10). The diet of the adult animal contains relatively more carbohydrate than fat. There is evidence that ketone bodies are metabolized by the brain of the neonatal rat whereas the adult brain utilizes glucose as the predominant source of energy (I I). Even at the reduced levels of glucose utilization by the suckling rat, the dietary supply sf carbohydrates is insufficient to cover the glucose requirement (12). Thus, the abundance of lipids and the deficit of glucose in the diet suggest that Fdtty acids might be utilized for gluconeogenesis during neonatal development. In the present study, we have explored the possible utilization of 14@ from [l-14C]palmitate for the synthesis of glucose and glycogen, particularly in suckling mice. Materials and Methods Swiss albino mice, ranging in age from 1 to 75 days, were used in this study. The mice that were 20 days or older were all males. The I-day old animals were those used within 24 h of their normal birth. The I-, 5-, and 10-day old mice were used not later than 15 min after separation from the dams. The pups suckled until 20 days after parturition. The weaned animals were maintained on a balanced diet. The mice were injected intraperitoneally with 0.2 pCi [1:'4ClpaBmitate (specific activity 49.0 mCi/~nmol, obtained from the Isotope Division of the Bhabha Atomic Research Centre) in 0.9'A, NaCl per gram body weight. The animals were sacrificed by decapitation at various t h e intervals between IS rnin and 240 rnin, and blood, liver, and kidneys were collected. The blood and the tissues from six 1- and >day old and four 10-day old mice were pooled for each experiment. The tissues from older animals were not pooied. Blood was collected from the neck region in precooled, heparinized tubes soon after decapitation. The blood was deproteinized (6 3) and centrifuged at 1085 X g for 5 rnin. Aliquots of the supernatant were used for glucose estimation by the anthrone method (14). Similar samples were lyophilized, and aliquots, in duplicate, were streaked (20 mm long) on Whatman chromatography paper along with glucose standards, and run by descending chromatography at 25 "@ for 16 h in a solvent system containing

479

11-butanol:pyridine :water (6 :4 :3, v/v/v). The paper was dried after the run, and one of the two sample spots along with the giucose standard spot was stained with aniline phthalate (15) and heated at 105 "C for I0rnin. The corresponding unstained area of the duplicate sample was cut out and eluted with distilled water (0.5 mi). One part of the eluate was used for glucose estimation by the anthrone method and another part for radioassay in a Becknlan LS-100 liquid sciiltiliation spectrometer. The scintillation fluid contained 4 g BBOT in 1 1 methanol: toluene (1 :I, v/v). The counting efficiency for was 92-952,. Liver and kidney samples were solubiIized in 30:j KOH by heating at 100 "C for 113 rnin. Glycogen was precipitated by addition of 907; ethanol and the tubes were centrifuged at 1085 X g for 5 man. The precipitate was dissolved in 105, trichloroacetic acid, the solution cleared by centrifi~gation,and diluted with l(MB'X ethanol. The precipitate obtained by centrifugation was washed thrice with ethanol:ether (1:1, v/v) and twice with ether alone to ensure removal of lipids. The purified precipitate was dissolved in distilled water, and aliquots were used for glycogen estimation by the anthrone method (14). Identicai aliquots of the material were used for counting in the scintillation spectrometer. In another set of experiments, 5-, lo-, and 75-day old mice were injected intraperitoneally with nonradioactive glucose (1 mg /g body weight) along with [I- lC]palmitate (specific activity and dose as described above). The animals were sacrificed at 15, 36, and 60 min after injection. Liver was collected, and analyses for glycogen levels and radioactivity were carried out as described earlier. Incorporation of 14Cfrom [l-ilC]paImitate into amino acids was also investigated. Five- and ten-day old mice were injected with [I-14C]palmitate(specific activity and dose as mentioned earlier). The animals were sacrificed 15 min after administration of [14C]yalmitate.Blood from six mice was pooled for each experiment. An aliquot (0.5 ml) of heparinized blood was deproteinized (13) and centrifuged. The supernatant was mixed with 3 volumes of chloroform, and the'aqueous (upper) layer containing free amino acids was passed through a 0.9 X 10cm column of Dowex 50 X 8 (Wsform), prepared as described by Moore and Stein (16). Sugars, organic acids, and other such substances were removed by washing the column repeatedly with water. The amino acids were eluted with 40 ml 2 N aimonia. The eluate was lyophilized and the residue dissolved in 0.5 ml 1 ,V HCB. Aliquots of this solution were radioassayed in the liquid scintillation spectrometer.

Results The weights of liver, kidneys, and whole body of 5-, 18-, and 75-day old mice are shown in Table 1. Blood glucose and hepatic dycogen increase as a function of age (Table 1). The lowest levels are found in 5-day old animals. Glycogen concentration of kidneys is rnaximal, unlike that of liver, in the suckIing mice. But the amount of glycogen present in kidneys is relatively low in comparison with that in liver in all

480

CAN. J. BIOCWEM.

VOL. 53.

1975

TABLE 1. Weight of whole body, liver, and kidneys; levels of glycogen in liver and kidneys and blood glucose of mice as a function of agea Weight

Glucose (rngj100 ml)

Glycogen (mgjg tissue)

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Age (days)

Whole body

&I

&Eachvakue is the mean

_+

Liver bang>

Kidneys (mg)

Liver

Kidneys

Blood

S.E.from four experiments.

TABLE 2. Levels of liver glycogen and incorporation of I4C from [l-~4Ce]palmitate into iiver glycogen after injection of non-radioactive glucosea Time after injection (rnin) Glycogen, mg jg tissue Age (days)

Radioactivity, c.p.m. jmg glycogen

15

30

60

15

30

8.92k1.18 11.35+0.87 32.17k1.80

6.90L1.27 10.86k0.61 33.20k1.92

4.51 1 0 . 7 3 7.6550.62 32.30k1.72

2684 i- 326 32102 359 Trace

1685 227 2040k189 Trace

+

60 1016 k 220 1832k153 Trace

aErpcA value is the mean 9 S.E. From four experiments. Glucose, 1 rng /g body weight.

0

20

30

75

AGE IN BAYS FIG.1. Incorporation of l4G from [I-14@]palmitateinto blood glucose (-A-), sand liver (-8-1 and kidney (--0glycogen 1 as a function of age. Incorporation was for 15 min in the case of glucose and 2 h in the case of glycogen. Values are means f S.E. from five experiments.

the age groups. Levels of hepatic glycogen in 5-, 10-, and 75-day old mice after glucose adrninistration are shown in Table 2. Injection of glucose enhances the amount of hepatic glycogen in both 5- and 10-day old mice but not in 75-day old animals. The amount of I4C incorporated into blood glucose from 61 -P4C]galmitate(Fig. 1) is highest in

the 5-day old mice. The specific activity of glucose is nearly 10 times higher than that observed in the case of 75-day old animals, and over two times more than that in the case of 1-day old animals. The I4Cincorporation into glucose as a function of time has been studied, and the results show that at 15, 30, 60, and 120 rnin after [14C6]palrnitatateinjection, the specific activity of

48 1

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NOTES

TIME IN MINUTES FIG.2. Incorporation of l4C from [1-14C]palmitateinto blood glucose in 5-day old (-a-), 10-day old (--CJ-), and 75-day old (-A,--) mice as a function of time. Values are means S.E. from five experiments. The point without bars includes S.E.

+

blood glucose is high in 5- and 10-day old animals and low in 75-day old animals (Fig. 2). The incorporation of 14Cfrom labelled palmitate into glucose reaches a peak and then declines within 2 h in 5- and 10-day old mice, whereas in the 75-day old animals it remains more or less unvarying. The incorporation of 14Cfrom labelled palmitate into liver glycogen, 2 h after injection, is highest in 5-day old animals (Fig. 1). There is a considerable amount of 14C incorporated into hepatic glycogen of 10-day old mice also. Incorporation by 30- and 75-day old animals is, in comparison, negligible. Incorporation of 14Cinto hepatic glycogen, studied as a function of time between 1 and 4 h, shows a steady increase in both 5- and 10-day old mice (Fig. 38). The incorporation of 14Cfrom labelled palmitate into kidney glycogen shows a steady increase between the ages of 1 day and 75 days (Fig. 1). The extent of incorporation into kidney glycogen between 2 and 4 h remains unchanged in 10-day old mice, unlike that in 5-day old animals, in which there is progressive increase in labelling (Fig. 3B). Incorporation of I4Cinto hepatic glycogen is increased considerably in suckling mice injected with un-

labelled glucose (Table 2). The maximum increase is discernible 15 min after glucose injection. The results (expressed as c.p.m. associated with amino acids per millilitre of blood) of incorporation of I4Cfrom [1-14C]palmitateinto amino acids in 5- and 10-day old mice (238 4 and 192 8 respectively, means S.E.) show that the amount of labelling of amino acids is small.

+

+

+

Discussion Our results show that blood glucose concentration rises during the suckling period. Adult levels of blood glucose are noticeable soon after weaning. A similar observation has been made in rabbits where there is a fall in blood glucose soon after birth followed by a rise (17). The dietary supply of glucose and other carbohydrates is inadequate during the suckling period and, hence, enhanced gluconeogenesis becomes a necessity. Liver glycogen remains low during the first 10 days of postnatal life and then rises, reaching adult values by the 30th day. This study shows that 14Cfrom C1-14C]palmitate is incorporated into both blood glucose and hepatic glycogen. This conversion, which may be

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CAN. J. BHOCHEM. VOL. 53, 8975

TIME IN HOURS FIG.3. (A) Incorporation of 14C from [I-l*@]palmitateinto liver glycogen in 5-day old (-@-) and 10-day old (--9mice ) as a function of time. (13) Incorporation of 1% frorn [l-lC]palmitate into kidney glycogen in 5-day old (-@-) and 10-day old (-O-) mice as a function of time. Values are means f S.E. from four experiments.

termed gluconeogenesis because the source is non-carbohydrate, is significantly more during the suckling period. The labelling of blood glucose of 10-day old animals reaches a peak 30 min after administration of ['"]palmitate. In the case of 5-day old animals, blood glucose labelling shows two p?aks, one 15 min and another (larger) peak 60 anin after injection. The incorporation of 14C from [lTCgpalmitate into the blood glucose of 75-day old animals is low and remains unvarying between 15 and 120 min after administration. en corpora ti om^ into hepatic glycogen of both 5- and 10-day old mice increases steadily up to 240 min after injection. The specific activity of the liver glycogen of 75-day old mice is relatively low. In renal glycogen, too, there is 14Clabelling

frorn [I, -l4C]palmitate. But unlike liver glycogen, kidney glycogen of 75-day old mice has higher specific activity than that of suckling mice. This is apparently not in conformity with the observation of Zorzolli el al. (4) that in developing rats renal gluconeogenesis is highest during the first 2 weeks after parturition; but apart from observations on the species difference, these authors also measured the levels of glucose-6-phosphatase and phosphoenolpyruvate carboxykianase as well as glucose synthesis from pyruvate and Lglutamate. The levels of kidmy glycogen are higher in suckling mice than in older animals, but on the whole, the concentration of renal glycogen is low in comparison with that of hepatic glycogen. Kidney cortex is reported to have a high capacity for gluconeogenesis in vitrs,

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potentially comparable to hepatic gluconeogenesis (I$), and keeping rats on a diet low in carbohydrates for 3-5 days enhances gluconeogenesis by kidney cortex itz vitro to the extent of 50-18070 (18). However, certain differences have been observed between renal and hepatic glucoweogenesis (19). It is interesting to note that the highest incorporation of from [I-14C]palmitateinto blood glucose as well as into liver glycogen is observed on the fifth day after parturition, in other words, after 5 days of milk feeding. Perhaps it takes that long to induce the full complement of gluconeogenic enzymes. Glucose-6-phosphatase, one of the enzymes involved in gluconeogenesis is highest in rat liver at about the third postnatal day (20). The hepatic metabolism of rats adapts to the nutritional changes that occur during development (3, 5, 9, 10, 21, 22). At birth, there is a sudden change from a predominantly carbohydrate nutrition (transplacental) to a diet (milk) that is rich in lipids. Again, at weaning, there is a sudden switchover from the milk diet to a diet high in carbohydrates and relatively low in lipids. The higher levels of gluconeogenesis ~bservedin liver of suckling animals in comparison with that of weaned or adult animals is likely to be a natural consequence sf coping with the demands for glucose. It has been suggested that nearly 50% of glucose required by the suckling rats is provided through gluconeogenesis (12). Several non-carbohydrate sources are known to serve as precursors for the synthesis of glucose and glycogen, especially during the preweaning period (1-3, 5, 23-25). Amino acids may not be readily available for gluconeogenesis during the neonatal period because of the large requirement for protein synthesis (6, 7). The abundance of lipids in the diet of the suckling animals makes them potential precursors of glucose synthesis. Both short- and long-chain fatty acids have been shown to be utilized for the synthesis of glucose. For example, isotopically labelled carboxyl carbans of and but~ricacids label glucose, predominantly in the 3 and 4 positions, as shown in studies with fasted rats (26). Incorporation of 14Cfrom [I-14C]palmitateinto glucose (carbons 3 and 4) has been demonstrated in diabeticpancreatectomized dogs It has been suggested that labelled carboxyl carbon of acetate, butyrerte, and palmitate label carbons 3 and 4 of

glucose through several steps, which involve the Krebs cycle also (28). The net yield of glucose is reported to be controlled by whether or not there is an influx of non-acetate metabolites into the Krebs cycle (28). from 11-14C]Our results suggest that palmitate is a possible source of glucose (in either mono- or polysaccharide form), especially in suckling mice. However, the contribution, if any, of palmitate towards net synthesis of glucose has not been assessed in this study. The incorporation of from [1-lCCJpalmitateinto glucose per unit volume of blood is nearly sixfold higher in suckling mice than in 75-day old mice. The labelling of hepatic glycogen is enhanced appreciably when unlabelled glucose is injected along with [ 1-14C]palrnitate.It has been reported that injection of unlabelled glucose along with labelled gluconeogenic precursors augments synthesis (labelling) of glycogen in starved-refed rats (29, 30). It seems reasonable to expect that the gIuconeogenic capacities of the livers of suckling and starved-refed adult animals are comparable. The amount of radioactivity associated with the amino acids of blood is comparatively low. Thus, amino acids which might be synthesized from [1-14C]palmitatedo not seem to contribute significantly to the observed synthesis of glucose. The salient feature of the present study is that the specific activity of blood glucose and hepatic glycogen is considerably higher in suckling mice than in weaned and older animals when [l-WCJpalmitate is administered as a precursor. This shows the greater potential of suckling mice to utilize the I4C from labelled palmitate for the synthesis of [14C]glucoseand [lC]glycogen. 1 . Ballard, F. J. & Oliver, I . T. (1963) Biochs'm. Biophys. Acta 71, 578-588 2, Vernon, R. G., Eakon, S. W. & Walker, B. G. (1967) Bischern. J . 105, 15P 3. Yeung, D. & Oliver, I. T. (1967) Biochem. J. 103,

744-748 4. Zorzoli, A., Turkenkopf, I. J. & Mueller, V. L. (1969)

Biochenz. J . 111, 181-185 j. Vernon, R. G e & Walker, D. G. (1972) Biochem. J . 127, 531-537 6. Hahn, P.?Koldovskp, O., Krecek, J., Martinek, J . & Vacek, Z . (1961) in Somatic Stability in the Newlyborrt (Wolskenholrne,G . E. W. & B'Conner, M., eds), pp. 131-155, J. & A. Churchill Ltd., London 7. Miller, S. A. (1969) in Mammalian Protein Metabulism (Munrs, H. N., ed.), vol. 3, pp. 183-233, Academic Press Inc., New Ysrk, N.Y.

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CAN, J. BIOCHEM. VOL. 53, 1975

8. Villee, C. A. & Hagerman, D. D. (1958) Am. J. Pltysiol. 194, 457-464 9. Ballard, F. J. & Hanson, W. W. (1967) Biockem. J . 102,952-958 10. Taylor, C. B., Bailey, E. & Bartley, W. (1967) Bioc h m . J. 105, 717-722 11. Hawkins, W. A., Williamson, D. H. Se Krebs, H. A. (1971) Biochem. J. 122, 13-18 12. Vernon, 8. G. & Walker, B. G. (1972) Biocllena. J . 127, 521-529 13. Somogyi, M. (1952) J . Biol. Clzsm. 195, 19-23 14. Van Handel, E. (1965) Anal. Biochsm. 11, 256-265 1 5. Waldi, D. (1965) in Tlrin-layer Cltrsmatograpl~y.A Laboratory Handbook. (Stahl, E., ed.), pp. 483-502, Academic Press Inc., New York, N.Y. 14. Moore, S. & Stein, W. H. (1951) J. Biol. Chem. 192, 663-48 1 17. Shelley, H. J. & Neligan, G. A. (1966) Br. ,%fed.BUN. 22,34-39 18. Krebs, H. A. (1964) Proc. R. Soc. Ser. B, 159,545-564 19. Weber, G. (1963) in Advaraces irt Enzyme Regalation. (Weber, G., ed.), vol. 1, pp. 1-35? Pergamon Press Ltd., Oxford, London, New York, Paris

20. Zsrzoli, A. (1962) J. GerantoC. 17, 359-362 21. Lockwood, E. A., Bailey, E. & Taylor, C. B. (1970) Bioehem. J . 118, 155-162 22. Vernon, R. G. & Walker, D. G. (1968) Biockem. J. 106, 321-329 23. Ballard, F. J. 8k Oliver, I. T. (1965) Biocllern. J. 85, 191-200 24. Yarnell, G. R.,NeIson, P. A. & Wagle, S. R. (1946) Arch. Biochem. Bioplys. 114, 539-542 25. Yeung, B. & Oliver, I. T. (1967) Biochsrn. J. 105, 1229-1233 26. Eifson, N., Lorber, V., Sakami, W. & Wood, H. G. (1948) J. Bisl. Clrem. 176, 1263-1284 27. Abraham, S., ChaikoR, I. L. & Hassid, W. 2.(1952) J . Biol. Clzetn. 195, 567-581 28. Weinman, E. O., Strisower, E. H. & Chaikoff, 1 . L. (1957) Pltysiol. Rev. 37, 252-272 29. Hems, D. A., Whitton, P. D. & Taylor, E. A. (1972) BiocEzeraz. J . 129, 529-538 30. Olavarria, J. M., Godeken, 8.G. R.,Sandruss, 8. & Flawia, M. (1968) Biochim. Bioplys. Acta 165, 185-188